VOL. XXI 2019 Issue

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Dartmouth Undergraduate Journal of Science FA L L 2 0 1 7

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ASSEMBLY

Cooperating for a Common Cause

Cilia and Flagella

Original Research

ISEC 2017 Winner

From Discovery to Disease

The Mouhefanggai and Cavalieri's Principle

Feeding the World with Die Rolls

p. 3

p. 29

p. 34

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About this Special Issue Dear Readers, Neuroscience continues to be one of the most rapidly evolving fields of scientific study, with new methodological advances and discoveries literally being made on a daily basis.

The fast pace of discovery can make it challenging to expose students to the breadth of recent breakthroughs and cutting-edge methods in a traditional course format. To address this, I teach an innovative seminar, PSYC 86: Advanced Neuroscience Seminar and Annual Meeting, to explore the topics and issues that are on the cutting edge in the field of neuroscience.

This past fall, as in prior years, the vehicle for this exploration was the program for the Annual Meeting of the Society for Neuroscience. At the beginning of the term, students selected thirteen topics to focus on during term, choosing from among those that were the subject of special lectures, panels, and keynote addresses scheduled for the Annual Meeting (held from November 3-7, 2018 in San Diego, CA). We read several articles on each topic (including at least one authored by the individual who was to speak on that topic at the Annual Meeting) and discussed the articles together during class. Two students were responsible for leading each class discussion. By November 3, we were intimately familiar with the content of the scheduled presentations.

The Dartmouth Undergraduate Journal of Science aims to increase scientific awareness within the Dartmouth community by providing an interdisciplinary forum for sharing undergraduate research and enriching scientific knowledge.

EDITORIAL BOARD President: John Kerin '20 Editor-in-Chief: Shivesh Shah '19, Anders Limstrom '20 Chief Copy Editor: Nishi Jain '21 Managing Editors: Anna Brinks ‘21, Meghan Zhou ‘21, Sam Neff ‘21 Assistant Editors: Raniyan Zaman ‘22, Liam Locke ‘21, Ed Buckser ‘21, Kristal Wong ‘22, Sonal Butala ‘22 Layout & Design Editor: Gunjan Gaur ’20, Shivesh Shah ‘19 Operations Director: Josephine Nyugen ‘22

STAFF WRITERS

We then travelled together to the Annual Meeting in San Diego, which is attended by over 35,000 neuroscientists from around the world and includes over 20,000 data presentations (posters and talks). Students attended the presentations we covered in class and selected and attended presentations of personal interest. Upon on return, we discussed the assigned presentations and the meeting in general.

Students prepared an in-depth final paper on a topic of personal interest primarily using information and research obtained at the Annual Meeting, written in the form of a Current Opinions in Neurobiology review article.

I am delighted that many students took the opportunity to share their articles with you in this special issue. I hope that you will find their articles of interest, and perhaps they will also ignite a spark of neuroscience interest in you as well.

David J Bucci, PhD Professor of Psychological and Brain Sciences Dartmouth College

Kevin Chao ‘19 Josephina Lin ‘19 Samuel Reed ‘19 Shivesh Shah ‘19 Jack Kerin ‘20 Anders Limstrom ‘20 Armin Taakkoli ‘20 Anna Brinks ‘21 Ed Buckser ‘21 Liam Locke ‘21 Megan Zhou ‘21 Nishi Jain ‘21 Sahaj Shah ‘21 Sam Neff ‘21 Chengzi Guo ‘22 Isabella Chao ‘22 Jason Wang ‘22 Mien Nyugen ‘22 Raniyan Zaman ‘22 Sonal Butala ‘22

ADVISORY BOARD Alex Barnett – Mathematics David Bucci – Neuroscience Marcelo Gleiser – Physics/Astronomy David Glueck – Chemistry Carey Heckman – Philosophy David Kotz – Computer Science Richard Kremer – History William Lotko – Engineering Jane Quigley – Kresge Physical Sciences Library Roger Sloboda – Biological Sciences Leslie Sonder – Earth Sciences DUJS Hinman Box 6225 Dartmouth College Hanover, NH 03755 (603) 646-8714 http://dujs.dartmouth.edu dujs@dartmouth.edu Copyright © 2017 The Trustees of Dartmouth College

SPECIAL THANKS Dean of Faculty Associate Dean of Sciences Thayer School of Engineering Office of the Provost Office of the President Undergraduate Admissions R.C. Brayshaw & Company

In Loving Memory of David Bucci Dr. Bucci passed away on the evening of October 15th, 2019 at his home in Vermont. To us, his students, a world-class professor and a distinguished scientist are the least of who David Bucci was. Dave was a loving friend, an unwavering advisor, a talented musician, and one hell of a father. His energy lit up every room he entered, and inspired every soul that was fortunate enough to know him. Words simply cannot describe how incredible David Bucci was.

Dave truly hoped that this series of articles would spark your interest in neuroscience. In his honor, we hope that you read this issue with care, and should you find yourself intrigued, we ask that you act on it. If you are a student, reach out to an advisor and let your curiosity lead the way.

Likewise, if you are an expert, we ask that you inspire and cultivate the curious minds around you, and that you do so wholeheartedly. That is the legacy of David Bucci, and that is what gives us strength in his absence.

Dave may no longer walk the halls of the Moore Psychology Building at Dartmouth, but his legacy, love, and passion lives within every single one of us. Dr. Bucci: We love you, miss you, and will never forget you.

P.S. SFN will never be the same without you.

Psychology 86 Class

Table of Contents Have No Fear: The Past, Present, and Future of Fear Extinction and Renewal

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4ve No Feat, Present, and

Armin Tavakkoli '20 Oxidative Stress’s Role in Aging and Neurodegenerative Disease

11ve No Feat, Pres

Gabrielle Helton '19 Implications of Adolescent Stress on Adulthood Behavior

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19ve No Feat, Pres

Shivesh Shah '19 Spatial Cognition in Disease and Normal Aging: A Perspective on Grid Cells and Theta Oscillations

27ve No Feat, Pres

Pelin Ozel '19

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Emerging Treatments for Amyotrophic Lateral Sclerosis

34ve No Feat, Pres

Grace Herron '19 Early-Life Stress and Adolescent Depression: Mechanisms and Models

42ve No Feat, Pres

Andrew Boghossian '19

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A Body of Work in Progress: the State of Stroke Recovery

48ve No Feat, Pres

Huy Dang '19 From Emotional Contagion to Altruism: Toward Animal Models of Empathy

57ve No Feat, Pres

Mia Drury '20

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Music and the Brain: A Call for a Therapeutic Future

65ve NFeat

Caroline Puskas '19 The Influence of Stressors and Adverse Life Experiences on Brain Development

71ve No Feat, Pres

Chloë Conacher '19

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DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

Have No Fear: The Past, Present, and Future of Fear Extinction and Renewal

BY ARMIN TAVAKKOLI '20

ABSTRACT Exposure therapy is the first line treatment for many anxiety and trauma-related disorders that affect more than 18% of the U.S population alone. During exposure therapy, patients receive repeated exposure to the feared stimuli in order to extinguish their fear response. Extinction learning, however, is context dependent; removal from the extinction context results in renewal of the extinguished fear. Past studies report conflicting results on the contribution of the fornix and the hippocampus to extinction and renewal learning. We attribute these difference to differential use of conditioned freezing and conditioned suppression paradigms to measure fear. Here, we review the past and current studies of extinction and renewal, looking for potential explanations for these differences. Furthermore, we seek to develop a unifying picture of our current understanding of extinction and renewal circuitry. We report that based on recent findings, the hippocampusFALL 2019

prefrontal-amygdala model of extinction and renewal circuitry fails to capture the breadth of interconnectivity seen both within these regions as well as with the sensory cortices. As a result, we point out some prominent gaps in our knowledge that require further investigation.

Figure 1a: A salient and fear-evoking encounter with a german shepherd may lead to the development of an acute fear of all dogs Source: Wikimedia Commons

INTRODUCTION Imagine that on a glorious Saturday morning hike, just as you’re about to reach the summit, you are attacked by another hiker’s german shepherd. If this interaction is salient enough, you will develop an acute fear of german shepherds, if not of all dogs. You survive this experience, and a few weeks later on the way to work, you stumble upon your friend who also happens to have a german shepherd.

Although you may initially be scared, you come to learn that your friend’s dog is friendly. Hence, you interact with the dog and you no longer fear your friend’s dog (i.e your fear is extinguished).

Finally, a month later, as you are basking in the 4

“Although exposure therapy is extremely effective in decreasing the patient’s fear in the extinction context (e.g. the therapist’s office), the individual’s fear often returns if the patients faces the feared stimuli in a different context.”

Figure 1b: A Fear Renewal Paradigm. (A) An aversive encounter (US) with the dog (CS) while hiking (Context A) results in forming an aversive CS-US association (Fear response). (B) Being exposed to another dog (CS) without the US in the street (Context B) will result in extinction of fear response. (C) How do you respond if you face yet another dog in a at the beach (Novel Context C)?

Source: Armin Tavakkoli

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sun at the beach, you see yet another german shepherd running toward you. The question that has motivated over two decades of research is the following: how do you respond to the sight of the dog approaching you, and why?

Do you respond with fear remembering your painful hiking experience, or will you remember that your friend’s dog on the street was friendly, and thus the dog at the beach must be safe as well? (Figure 1) Early experiments by Bouton and colleagues found that facing the feared stimuli (e.g. the German shepherd) in a new environment (e.g. the beach) would result in a return of previously extinguished fear — a renewal of fear [1]. This finding was remarkable because for the first time it shed light on why exposure therapy, the first-line psychological treatment for many anxiety and trauma-related disorders, was not always effective and generalizable beyond the context in which the fear was extinguished (i.e the extinction context). Exposure therapy relies on repeated exposure to feared stimuli (CS) in a controlled setting to extinguish the patients’ fear. Although exposure therapy is extremely effective in decreasing the patient’s fear in the extinction context (e.g. the therapist’s office), the individual’s fear often returns if the patients faces the feared stimuli in a different context.

For some time, it was thought that in order for fear to be renewed following extinction, the threatening stimuli must be faced again in the context where it was originally associated with an aversive event (US). However, Bouton and colleagues later showed that mere removal from the extinction context is sufficient for renewal of fear [2]. Taking these findings together, Bouton developed the context-dependent theory of extinction, where extinction is thought to form a context dependent inhibitory CS-US association (Figure 2). This inhibitory association overrides the previous CS-US association, but only when the current context matches the extinction context.

This was especially problematic for those undergoing exposure therapy because it meant that facing their feared stimuli anywhere beyond where extinction had taken place would result in a return of their anxiety and fear response.

Knowing that nearly 18% of the U.S population (nearly 1 in 5) suffers from an anxiety disorder,

and given the impairing side effects of antianxiety medication, Bouton’s discovery fueled the desire to identify the neural representation of renewal, hoping that an understanding of this circuitry would allow for development of effective treatments. The goal of this review is to provide an overview of the current state of our understanding of extinction and renewal, as well as a brief synthesis about the implications of our understanding. First we will briefly consider some strengths and weaknesses of the past studies of extinction and renewal that inform the current state of the field. Then, we will consider some of the most prominent current — and unpublished— studies of extinction and renewal, many of which were presented at the Society for Neuroscience’s (SFN) 2018 meeting for the first time. Lastly, we will conclude with a new model to shape a unifying picture of the current state of our understanding, as well as the future of the field.

THE PAST

The Fornix and the Dorsal Hippocampus (DH). Renewal can be thought of as a contextual retrieval of fear memory, where contextual learning plays a crucial rule in the animal’s ability to distinguish various context and their associations from each other. The hippocampus, and specifically the DH has been implicated in contextual learning and contextual fear retrieval [3,4]. Hence, an early claim was that if the hippocampus is indeed necessary for contextual learning, inactivating the fornix or the DH must then abolish renewal which requires forming robust contextual associations. In response to this claim, early studies showed that neither pre-training DH and fornix lesions, nor posttraining DH inactivation affected renewal in ABA paradigms [5,6].

Although later studies by Maren and colleagues claimed that both pre and posttraining electrolytic DH lesions disrupt renewal [7], hence contradicting the work of Bouton and their own earlier findings, these contradictions are most likely attributed to their procedural differences. Two potentially confounding measures exists in Maren and colleagues methodology. First, in their 2005 study, Maren & Ji used electrolytic lesions and measured fear in a conditioned freezing

DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

E X T I N CT I O N A N D R E N E WA L paradigm [7]. Electrolytic lesions of the DH result in a hyperactivity phenotype, which can confound measures of freezing behavior [8].

In contrast, when electrolytic lesions of the DH were done and fear was measured using a conditioned suppression paradigm (where fear is measured by suppression of lever pressing), no effect of post-training DH lesions in renewal were seen [9]. Second, and more importantly, Maren’s methodology has traditionally used a 10 second tone CS during both conditioning and extinction, while a drastically longer 8 minute tone CS is used during renewal testing, and freezing is measured during the first 4 minutes.

It is known that rats can readily distinguish stimuli based on duration, and hence a longer duration can be interpreted as an entirely new CS [10]. It is also known that rats exhibit a natural startle response when they face a stimuli for the first time, and that this startle response is attenuated by hippocampal lesions [11]. Thus, it is possible that the freezing observed during their renewal testing is merely a startle response to the new significantly elongated CS, which is simply attenuated in the lesioned animals, and is being interpreted as “renewal” of fear. Indeed, Zelikowsky and colleagues have shown that pre-training DH lesions only attenuate renewal when the CS duration is violated at renewal testing (e.g when a longer CS is used) [12].

This can therefore at least partially explain the contradictory findings of Frohardt and Maren when it comes to pre-training ABA effects, although the same cannot be said for posttraining effects.

The Retrosplenial Cortex (RSC).

Although much less work has been done to investigate the role of the RSC in renewal, an argument can be made for its potential necessity for renewal. One possible explanation for the difference seen between the pre-training lesion effects in conditioned suppression and freezing paradigms stems from the facts that conditioned suppression involves lengthy and frequent training trials. Extended training can result in another brain region taking over the role of the DH, and RSC is a suitable candidate for such role. Being a poly modal association area, RSC is thought to be involved in forming multi-facet representations of the environment, and may thus be involved in context learning that occurs in renewal.

Despite the plausibility of its potential role, pre and post training lesions of the RSC failed to impact renewal in a conditioned suppression paradigm [9]. Furthermore, since post-training DH lesions also failed to impact renewal in a conditioned suppression paradigm, it is unlikely that functional compensation by another brain region can explain the differences observed between conditioned suppression and freezing.

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Figure 2: Model of Extinction Learning. Extinction consists of forming a new context-dependent inhibitory association between the CS and the US. In the extinction context, the inhibitory association prevails, and hence responding to CS is low.

Outside the extinction context, the inhibitory association is no longer “active”, resulting in an increase in responding to CS. Source: Armin Tavakkoli

A final hypothesis that could potentially explain the difference between the two paradigms is that perhaps extended exposure to contexts in conditioned suppression forms a context representation that is not dependent on the hippocampus. Although this remains untested, we will return to this point in our discussion, drawing evidence from some of the current work to evaluate this claim.

The Prefrontal, Amygdala, Hippocampus Circuitry. One hypothesis of extinction learning suggests that extinction and renewal of fear can be explained by a prefrontal, amygdala, hippocampus circuit [13]. The hypothesis claims that when a subject experiences the extinguished CS within the extinction context, the hippocampus signals the prefrontal cortex to inhibit the amygdala and suppress fear. Similarly, if the extinguished CS is faced outside of the extinction context, the hippocampus no longer directs the prefrontal cortex to inhibit fear, and hence fear is renewed (Figure 3). Further evidence also exists that hippocampus may directly activate the amygdala when exposed to the extinguished CS in absence of the extinction context, therefore contributing to renewal [14]. Studies evaluating this theory have only gained momentum recently, and will be discussed shortly.

However, taking together the findings perviously discussed, it is apparent dorsal hippocampus does not fit the above hypothesis.

This can be explained by a few possibilities. First, it is possible that simply other hippocampal regions, such as the ventral CA1 and CA3 regions, are responsible for extinction learning and renewal. Indeed, several c-fos studies show that ventral segments of CA1 and CA3 regions of the hippocampus are preferentially activated during renewal. However, the caveat here is that these regions also seem to be active during extinction, which poses a challenge to the aforementioned hypothesis. Second, as previously mentioned, it is also possible that utilizing a conditioned suppression paradigm creates a type of context representation that is independent of the hippocampus. Therefore, a

“One hypothesis of extinction learning suggests that extinction and renewal of fear can be explained by a prefrontal, amygdala, hippocampus circuit.”

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“ It can be concluded that context dependence is gated by cue specific sensory projections into the amygdala.”

brain-wide c-fos analysis using a conditioned suppression paradigm is necessary to answer this questions.

The studies discussed here, inclusive of their strengths and shortcomings, have given us the basis for our current understanding of extinction learning and renewal. With new cutting edge recording, lesioning, and reversible inactivation tools such as fiber photometry, NMDA, and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), we now have the capability to take a more refined approach to studying the renewal circuitry. This is reflected in the following section, where we take a look at the recent work being done on extinction and renewal.

THE PRESENT

The Amygdala. As previously mentioned, although amygdala has been shown time and time to play a role in fear learning, it has remained largely unstudied in the context of fear extinction and renewal. This is perhaps because amygdala’s traditional role as a “fear region” has been taken for granted, and aiming to understand extinction and renewal from an amygdala standpoint is deemed uninformative.

However, a look at current studies looking at amygdala’s role in extinction and renewal as a part of a larger circuitry proves otherwise.

In support of amygdala control theory of extinction and renewal, Wu et al. obtained electrophysiological recordings of excitatory synaptic inputs from the auditory cortex in the lateral amygdala (LA) in fear extinguished mice (note that a tone CS was used here). They report that renewal of fear upon returning to the conditioning context (ABA design) is associated with hyperexcitability of these synapses [15]. Additionally, optogenetic activation of pre-synaptic GABAB receptors decreases the renewal-induced hyper-excitability of the excitatory auditory inputs into the LA, attenuating renewal. Furthermore, activation of the excitatory inputs into the LA during extinction attenuates extinction, keeping fear

Figure 3: The Prefrontal, Amygdala, Hippocampus Circuitry Model. The hypothesized connectivity between the Hippocampus, PFC, and Amygdala is shown.

The model presents that CA1 and CA3 regions within the hippocampus innervate the IL, which in turn active the BLA within the renewal context .

Source: Marek et al., 2018

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at high levels. Similar findings are reported by Zhang and colleagues who study the role of GABAergic interneurons in the BLA [16]. They report that extinction activates GABAergic interneurons in the BLA, and that inhibition of these neurons blocks fear extinction.

Taking these two studies together, several conclusions can be made. First, it can be concluded that context dependence is gated by cue specific sensory projections into the amygdala. This provides another piece of the puzzle that can further inform the model presented in Figure 3, where in addition to hippocampal inputs, potential sensory (cue) specific inputs into the amygdala are necessary for extinction and renewal. Second, it can be concluded that extinction involves activation of GABAergic interneurons in the BLA, which provides a potential mechanism for hippocampal-prefrontal control of fear expression. Lastly, these findings also present a potential explanation for the difference observed between conditioned freezing and suppression paradigm. Perhaps, due to amount of training given in conditioned suppression paradigms, the cue specific projections into the amygdala are salient enough that can independently induce renewal in absence of hippocampal input. While this is yet to be tested, it will be rewarding to evaluate whether fear renewal using visual or olfactory CS also depend on cue specific projections into the amygdala.

If so, it’ll be interesting to evaluate whether the strength of these projections change as a function of training, and if they reach a point where hippocampal input is no longer necessary to modulate fear levels.

The Striatum. The main procedural difference between the methodology used by Maren and colleagues and that of Todd and colleagues is the use of a conditioned freezing paradigm (Maren) versus a conditioned suppression paradigm (Todd) to measure fear.

This difference noteworthy because although freezing is a natural response to threatening stimuli, lever pressing is a motivated behavior in food deprived rats. Hence, when rats freeze in response to an aversively conditioned CS, they are exhibiting a natural —perhaps involuntary— response, in contrast to cessation of lever pressing, which involves voluntary inhibition a motivated behavior. Given that the striatum is implicated in both voluntary movement and motivated behavior, it is hypothesized to be involved in extinction and renewal. Indeed, extinction recruits D1-expressing neurons in the dorsal striatum, and activation of nigrostriatal dopamine (DA) pathways during extinction enhance extinction and attenuate renewal [17].

To further elucidate the role of the striatum in fear extinction and renewal, Jaime and DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

E X T I N CT I O N A N D R E N E WA L associates set out to selectively inhibit the dorsal medial striatum (DMS) and dorsal lateral striatum (DLS) using a GABAA/GABAB agonist during extinction to determine the role of each subregion in extinction and renewal [18].

They report that when DMS is inactivated, fear extinction supported by the DLS is resistant to renewal (i.e extinction is no longer context dependent). In contrast, when the DLS is inactivated, fear extinction supported by the DMS is strengthened, but renewal is unaffected (i.e extinction remains context dependent).

Lastly, they report that D1 receptor signaling in the DMS, but not the DLS, contributes to extinction learning.

A less studied function of the striatum is its role in voluntary movement. Of special interest is the circuitry connecting the DLS and the substantia nigra (SN) which is thought to be imperative in formation of habitual behavior. It can be argued that if habit forming pathways are activated during extinction, the fear extinction memory could become context independent, since habitual behaviors are more resistant to relapse. Using optogenetics, Wiseman and colleagues activate the SN terminals in the DLS during extinction (i.e activate the habit forming pathway), and report that renewal is significantly reduced [19].

Taking these two studies together, we can draw two intriguing conclusions. First, approaching the striatum from a motivation aspect, we can conclude that temporary inactivation of the DMS during extinction can produce an extinction memory that is resistant to renewal. Although further studies are needed that evaluate the role of DMS and DLS in extinction and renewal using a conditioned suppression paradigm, this could potentially explainthedifferencesobservedbetweenthetwo paradigms. It is possible that since conditioned freezing does not involve a motivated behavior, it does not engage D1 signaling in the DMS that is sensitive to renewal. In contrast, lever pressing for food, which is a motivated behavior, can likely engage the DMS pathway, which would make it susceptible to renewal. Another possible conclusion is that since freezing is somewhat of an innate fear response, it can potentially engage the SN-DLS habitual circuitry, which would make it resistant to renewal. Finding an answer to both of these hypotheses requires replicating Jamie and Wiseman’s studies using a conditioned suppression paradigm, to see if striatal pathways are differentially activated when a different paradigm is used.

The Prefrontal Cortex. Also relating to the prefrontal-hippocampus-amygdala circuitry theory of extinction and renewal, the prefrontal cortex, as well as its inputs and outputs, are of interest to current research. Specifically, the FALL 2019

ventral hippocampus (VH) has been found to have several projections to the prelimbic cortex (PL), which is thought to in turn modulate the fear expression in amygdala. Using DREADDs, Vasquez and colleagues report that activation of VH cells projecting to the PL before renewal testing attenuated renewal of fear [20]. Yet again, this finding further enriches the model presented in Figure 3, and makes it ever more apparent that although hippocampusprefrontal-amygdala do most likely govern extinction and renewal, the interconnectivity of these regions are more complex than initially thought.

Two downsides exists in almost the entirety of literature on extinction and renewal. First, majority of such studies use rats as their model organism. Second, almost the entirety of work on extinction and renewal has been done in aversive learning paradigms, despite the fact that extinction and renewal are also observed in appetitive paradigms. As a result, our understanding of the evolutionary conservation of extinction and renewal, as well as why they would be adaptive in other non-fear related paradigms, remains limited. In an insightful study, Packheiser and colleagues investigated the role of the avian “prefrontal cortex” equivalent, the nidopallium caudolaterate (NCL), in an appetitive conditioning task [21].

By obtaining electrophysiological recordings from the NCL, Packheiser identified a subset of the neural populations, namely archetype 5, whose responding increased during acquisition, decreased during extinction, and was reinstated in renewal. Although the archetypes were chosen based on the data using a mathematical model, and hence their relationship may be purely incidental, it’d be informative to perform a retro-analysis of archetype 5 by identifying the positioning of electrodes that conform to this archetype. This would allow us to determine the spatial configuration of these specific neurons, and evaluate whether a specific region within the NCL consist of neurons that respond in this fashion. If so, this would provide a valuable insight into the conservation of renewal circuitry, and would further inform the more advanced models.

The Perirhinal, Postrhinal, and Posterior Parietal Cortices. The posterior parietal cortex (PPC) has been implicated in several “higher order” functions, including attention, planned movement, and spatial reasoning. Temporary inactivation of the using mucimol before renewal testing attenuates renewal [22]. It should, however, be noted that this particular study lacks several crucial controls, and hence further work is needed to definitively evaluate the role of the PPC in renewal.

“It is possible that since conditioned freezing does not involve a motivated behavior, it does not engage D1 signaling in the DMS that is sensitive to renewal."

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"The perirhinal cortex (PER) is known for its role for its interconnecting role between the hippocampus, the limbic system, and the lateral temporal and occipitotemproal association cortices."

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The perirhinal cortex (PER) is known for its role for its interconnecting role between the hippocampus, the limbic system, and the lateral temporal and occipitotemproal association cortices (i.e most sensory regions). This pattern of connectivity implicate the PER in acquisition and processing of declarative memory. The postrhinal cortex (POR), on the other hand, has been specifically implicated in associative memory of the position of an object, including it’s presence within a context [23]. As a result, both regions can potentially have a plausible role in extinction and renewal.

Potter and colleagues report that temporary inactivation of the PER prior to extinction training results in a temporary loss of fear memory during extinction trials (i.e consistently low fear throughout extinction) when a discontinuous CS is used [24]. In addition, when the extinction recall was tested a day after, subjects whose PER was inactivated prior to extinction showed an impaired recall of extinction memory, evident by high freezing (high fear), in contrast to control animals who successfully recalled their extinction memory and showed low freezing. Interestingly, when a continuous CS is used, consistently low levels of fear are seen across extinction trials similar to that of a discontinuous CS. However, in contrast to the discontinuous CS, extinction recall was not affected when a continuous CS was used.

Hence, it can be concluded that the PER is necessary for successful extinction learning, at least when a discontinuous CS is used.

Unfortunately, no renewal testing was done in this study. It’d be instructive to see how PER inactivation would affect renewal of fear.

Interesting findings are also reported by DeAngeli and colleagues on the role of POR in extinction and renewal of remotely acquired conditioning [25]. Using DREADDs to inactivate the POR during extinction and renewal testing, they report that POR inactivation significantly enhances extinction, while paradoxically attenuating renewal at the same time. In addition, in a separate experiment, they also report that POR inactivation during renewal testing alone reduces, but does not attenuate renewal. While this set of observations remain somewhat of a mystery, it is possible that inactivating the POR induces a partial retrograde amnesia, or perhaps opens up a window of enhanced plasticity. This would mean that during extinction, the CS-US association is more easily lost, and if so, renewal is attenuated. Similarly, it’d also explain the results of the second experiment, where if the POR is only activated during renewal testing, the retrograde amnesia would make the extinction and conditioning memory less salient, resulting in a less pronounced renewal. Further studies that seek to “rescue” this deficits by re-training

can provide further insight into the underlying mechanism of POR’s role in extinction and renewal.

A Curious Case of Music. In an interesting deviation from the traditional paradigms of studying the role of brain regions in extinction and renewal, Oliveira and colleagues investigated the role of music on extinction [26]. They report that playing classical music significantly enhances extinction learning. Unfortunately, how and whether renewal might be affected by classical music remains yet to be studied, as does the underlying mechanism that could contribute to enhancing extinction learning.

One possible explanation for how music could enhance extinction comes from a study by the findings that vagus nerve stimulation during extinction enhances extinction of auditory and olfactory fear conditioning [27]. It has been shown that classical music increases vagus nerve activity [28], and hence if classical music stimulates the vagus nerve, it can consequently enhance extinction.

CONCLUSION (THE FUTURE)

Taking all the studies discussed here together, only one definitive conclusion can be made: extinction and renewal circuitry is much more complex than what was initially laid out in the hippocampus-prefrontal-amygdala model.

That is not to say that the model was inaccurate; indeed, much of the studies discussed here keep returning to the observation that this circuitry is crucial for extinction learning and renewal.

Instead, the current studies show us that the it is the interconnectivity of these regions are much more complex than those presented in the initial model. In fact, in an informative talk by Hennings and colleagues, a new addition to the model was proposed [29]. Hennings proposes that each of the three regions described in the model from their own associations with the CS throughout acquisition, extinction, and renewal.

The model further proposes that higher-order processing then plays a role in resolving the “competition” between the fear and extinction memories among the three regions, ultimately modulating the fear response.

Although the above model requires further support before it can be widely accepted, the model presents a new perspective toward extinction and renewal that is instructive. The competition-resolving aspect of the model challenges the idea that we either do or do not see extinction or renewal. The truth is that fear response is not a categorical response; fear can have various levels, and a reduction of fear can be sufficiently —and clinically— significant to not require total attenuation. We must keep in mind that neurological factors are seldom considered in isolation in the context of psychopathology DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

E X T I N CT I O N A N D R E N E WA L and treatment, and even mild reductions in fear renewal can have meaningful implications for therapy.

Much work has been done to identify the basis of extinction and renewal, and there is still much to be done. Here we present some of the current work being done in the field, their strengths, and how they can be further improved and investigated to bring together the missing pieces of our knowledge. D

CONTACT ARMIN TAVAKKOLI AT ARMIN.TAVAKKOLI.20@DARTMOUTH.EDU

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8. Godsil, B. P., Stefanacci, L., & Fanselow, M. S. (2005). Bright light suppresses hyperactivity induced by excitotoxic dorsal hippocampuslesionsintherat.Behavioralneuroscience,119(5), 1339.

9. Todd, T. P., Jiang, M. Y., DeAngeli, N. E., & Bucci, D. J. (2017).

Intact renewal after extinction of conditioned suppression with lesions of either the retrosplenial cortex or dorsal hippocampus. Behavioural brain research, 320, 143-153.

10. Todd, T. P., Winterbauer, N. E., & Bouton, M. E. (2010).

Interstimulus interval as a discriminative stimulus: Evidence of the generality of a novel asymmetry in temporal discrimination learning. Behavioural Processes, 84(1), 412-420.

11. Fanselow, M. S. (2000). Contextual fear, gestalt memories, and the hippocampus. Behavioural brain research, 110(1-2), 73-81.

12. Zelikowsky, M., Pham, D. L., & Fanselow, M. S. (2012).

Temporal factors control hippocampal contributions to fear renewal after extinction. Hippocampus, 22(5), 1096-1106.

13. Hobin, J. A., Goosens, K. A., & Maren, S. (2003). Contextdependent neuronal activity in the lateral amygdala represents fear memories after extinction. Journal of Neuroscience, 23(23), 8410-8416.

14. Herry, C., Ciocchi, S., Senn, V., Demmou, L., MĂźller, C., & LĂźthi, A. (2008). Switching on and off fear by distinct neuronal circuits. Nature, 454(7204), 600.

15. Y. Wu; Discipline of Neurosci. and Dept. of Anat. and Physiol., Shanghai Jiao Tong Univ. Sch. of Med., Shanghai City, China. Auditory cortex-driven disinhibition of GABABR signaling in amygdala mediates fear renewal. Program No. 078.10. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

16. X. Zhang, Y. Zhou, W. Li; Shanghai Jiao Tong Univ., Shanghai City, China. The specific role of GABAergic FALL 2019

interneurons in fear extinction. Program No. 509.07. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

17. Bouchet, C. A., Miner, M. A., Loetz, E. C., Rosberg, A. J., Hake, H. S., Farmer, C. E., ... & Greenwood, B. N. (2018). Activation of nigrostriatal dopamine neurons during fear extinction prevents the renewal of fear.

Neuropsychopharmacology, 43(3), 665.

18. J. Jaime, M. K. Tanner, N. A. Moya, J. Davis, E. C. Loetz, B. N. Greenwood. Role of the dorsal striatum in fear extinction and relapse. Program No. 415.18. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018.

Online.

19. J. Wiseman, E. C. Loetz, E. B. Oleson, B. N. Greenwood.

Effects of optogenetic activation on nucleus accumbens projecting ventral tegmental neurons during fear extinction on fear extinction memory and relapse. Program No. 415.16.

2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

20. K.C. Leong, J. H. Vasquez, I. A. Muzzio. Selective manipulation of ventral hippocampus projections to the prelimbic cortex selectively facilitates fear extinction generalization. Program No. 415.05. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

21. J. Packheister, R. Puschi, O. Gunturkun, J. Donoso, S. Cheng. The neuronal mechanism of extinction learning and the renewal effect. Program No. 244.17. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

22. B. Joo, S. Lee, J. Koo. Role of the posterior parietal cortex in fear recovery after extinction. Program No. 327.14. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

23. Eacott, M. J., & Gaffan, E. A. (2005). The roles of perirhinal cortex, postrhinal cortex, and the fornix in memory for objects, contexts, and events in the rat. The Quarterly Journal of Experimental Psychology Section B, 58(3-4), 202-217.

24. N. Potter, C. A. Calub, S. C. Furtak. A possible role for perirhinal cortex in fear extinction learning. Program No. 415.13. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

25. N.E. Deangeli, D. J. Bucci, T. P. Todd. Renewal following extinction of remotely acquired conditioning depends on the postrhinal cortex. Program No. 329.11. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

26. P.H. Oliviera, D.H. Pietroboni, L.S. Lemos, A.C. De Andrade. Effect of classical music on the extinction of fear memory in rats. Program No. 327.16. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

27. C.R. Oleksiak, M.N. Tabet, S. Arora, M. P. Srivastav, R.R. Souza, C.K. McIntyre. Extinction-paired vagus nerve stimulation reduces avoidance of a conditioned odor. Program No. 415.06. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

28. Ellis, R. J., & Thayer, J. F. (2010). Music and autonomic nervous system (dys) function. Music Perception: An Interdisciplinary Journal, 27(4), 317-326.

29. A.C. Hennings, J.A. Lewis-Peacock, J.E. Dunsmoor. Mental context tagging reveals deficits of extinction learning in PTSD. Program No. 272.07. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

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Oxidative Stress’s Role in Aging and Neurodegenerative Disease

BY GABRIELLE HELTON '19 Figure 1a: Degeneration of the substantia nigra in Parkinson's Disease patients Source: Zhang et al. (1999)

"One commonality between aging and neurodegenerative disease is oxidative stress, which occurs when the balance of pro-oxidant to antioxidant is disrupted favoring the former."

ABSTRACT The imbalance of oxygen free radicals and antioxidants, known as oxidative stress, results in undesirable effects escalating from cognitive impairments seen in aging to the debilitating motor difficulties of Parkinson’s disease. The mechanisms of how oxidative stress can lead to such disastrous outcomes are not fully understood, and unfortunately, there are many direct and indirect ways reactive oxygen species (ROS) can lead to cognitive impairment, dopaminergic cell loss, amyloid beta plaques, and more. This paper will address a few methods of how oxidative stress can provoke overexpression of cell cycles, neuronal cell loss, and alterations in gene expression. Since there is not one defined pathway in which ROS precipitatesdamage,treatingneurodegenerative disorders like Parkinson’s and Alzheimer’s disease (AD) and neurodevelopmental disorders such as autism and schizophrenia is more difficult. Instead, targeting oxidative stress imbalance, the root of the problem, through antioxidants and diet has a more rewarding outcome.

INTRODUCTION In the United States, it is estimated that by the year 2030, twelve million people will suffer from a neurodegenerative disease [1].

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Although many have noted the severity of the problem, a cure or treatment is not much closer. One commonality between aging and neurodegenerative disease is oxidative stress, which occurs when the balance of pro-oxidant to anti-oxidant is disrupted favoring the former [2]. Any chemicals that induce oxidative stress are coined pro-oxidants. This imbalance often occurs in aging and under high oxygen intake, which can ultimately lead to DNA damage and lipid oxidation. Mitochondria dysfunction is thought to have a key role in oxidative stress. Mitochondria, the powerhouse of cells, play a vital role in apoptosis, calcium homeostasis, many cellular processes, and the generation of reactive oxygen species [3].

Reactive oxygen species (ROS), the byproduct of aerobic metabolism, are composed of hydrogen peroxide (H2O2), O2-, and hydroxyl (OH) radicals and are capable of attacking living tissues and inducing cell death [4, 5]. Figure 1 illustrates the ramifications of increased hydrogen peroxide, O2-, OH, and mitochondrial disruption on protein signaling, lipid oxidation, DNA damage, and more [4].

Given the importance of mitochondria in everyday cellular function, any impairment or damage can lead to crippling results, including neurodegenerative disease.

Organisms have developed an antioxidant defense system to prevent the destructiveness DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

AGING of ROS. However, overtime, the defense slowly weakens with senescence [5]. After neurons receive enough damage from free radicals, they will undergo apoptosis. Although the exact mechanisms are not completely understood, oxidative stress is believed to lead to microtubule destabilization, membrane oxidation, and autophagy impairment. In the presence of hydrogen peroxide – a model of reactive oxygen species—the autophagy marker microtubuleassociated protein-light chain 3 II is increased in the hippocampal CA1 region [5]. Axonal and dendritic degeneration also occur in hydrogen peroxide treated primary neurons and N1E-115 cells. These effects are even more pronounced when vitamin E is deficient.

As lipid peroxidation occurs, more cognitive impairments like memory loss are observed. In addition to the direct effects of oxidative stress, the free radicals also have an effect on other signaling pathways, which can intensify cognitive impairments [5]. For example, when neuroblastoma cells are exposed to hydrogen peroxide there is a significant level of de-methylation of PP2A [6]. Because PP2A is an important isoform responsible for de-phosphorylating tau, oxidative stress is indirectly causing an enhanced level of hyper phosphorylated tau, a common aggregate found in Alzheimer’s disease. This paper will address the direct and indirect role of oxidative stress in normal aging, Parkinson’s disease, Alzheimer’s disease, and neurodevelopmental disorders such as schizophrenia and autism. To conclude, the efficacy of pharmacological drugs and diets on preventing decline and restoring cognitive ability will be examined.

NORMAL AGING Increased forgetfulness, confusion, and reaction times are commonly seen as individuals age. One theory for the cause of increased cognitive impairments is oxidative stress and the impairment of the ROS system. More generally, as we age, the ongoing transcription and metabolic process can lead to a buildup of DNA damage [7]. DNA repair is crucial, and when this is disrupted – as it often is in normal aging – it can lead to minor cognitive impairments like a fading memory or more serious consequences such as Alzheimer’s and Parkinson’s disease.

Experimentally, gamma H2Ax, a biomarker for DNA double strand breaks, exhibited increased levels when stress was induced. Both replication stress and transcriptional stress were observed, illustrating a stronger effect from transcription stress [7]. Although there are many differences between the processes of replication and transcriptional stress, a key difference is p53 – a tumor suppressor gene. In a normal double FALL 2019

strand break, p53 is activated so that the damaged DNA does not continue to replicate, resulting in fewer neurons. However, without a functional p53, double strand breaks are never fixed, and the result is more impaired neurons present.

Similarly, the oxidative stress evoked DNA damage can lead to cellular senescence – an irreversible pathway that activates protein kinase A, increases ROS, and increases the expression of tumor suppressor genes p16 and p53 [9]. The over activation of protein kinase A has many effects encompassing increased lipid metabolism via the cAMP-signaling pathway.

As aging occurs and the immune system becomes less effective, the senescent cells are not efficiently cleared and chronic inflammatory molecules are secreted, resulting in the agerelated pathologies [10]. The senescent pathway, triggered by DNA-double strand breaks, has many detrimental effects to our cognitive ability including the acceleration of telomere shortening [9].

When comparing cultures of ‘young’ primary cortical neurons of embryonic mice to ‘old’ neurons, the old have an enriched and up-regulated oxidative stress and inflammatory response. However, the genes responsible for DNA-protein complex and nucleosome organization are down regulated in old neurons [11]. Furthermore, stressors related to aging – including oxidative stress – have been shown to enhance histone 3 lysine 9 trimethylation (H3K9me3) levels [12]. H3K9me3 is a marker of

Figure 1b: When oxygen is in excessive quantities, the mitochondria are overworked and produce 02- as depicted.

The NADPH oxidase also contributes to the level of O2-, which ultimately leads to the production of hydrogen peroxide. Lipid oxidation, DNA damage, and deficits in protein signaling occur when this combination is compounded with OH radicals.

Source: NeuroImage [4]

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Figure 2: After a double-strand DNA break, two pathways can occur. When p53 is activated, apoptosis occurs and no further transcription will occur. However, the signaling proteins can also activate repair mechanisms that may eventually lead to impaired neurons formed (8).

Source: Solov'yov et al. (2018)

“SUV39H1 can be used as an inhibitor of H3K9me3, resulting in increased synaptic density and brain-derived neurotropic factor in hippocampal neurons and overall cognitive improvement on behavioral tasks.”

oppressed transcription and is present at high levels in the hippocampus of aged mice [12, 13]. In addition to aged individuals, this marker is commonly present in Alzheimer’s disease [13]. When hippocampal neurons are cultured in 3 amino triazole, a medium that produces a high intensity of oxidative damage, H3K9me3 levels are enhanced. SUV39H1 can be used as an inhibitor of H3K9me3, resulting in increased synaptic density and brain-derived neurotropic factor in hippocampal neurons and overall cognitive improvement on behavioral tasks.

This experiment could have been taken a step further by also measuring H3K9me3 levels in the presence of H2O2, a more common method for modeling ROS. Another difference noticed in young versus old mice was that in aged microglia, Cathepsin B was noted to have leaked into the cytosol, causing degradation of mitochondrial transcription factor A [14]. This degradation led to higher levels of mitochondria-derived ROS and inflammatory byproducts. The accumulation of ROS and neuroinflammation was greatly reduced by genetically ablating Cathepsin B (CatB) through DNA modification.

Additionally, the previously elevated staining of lipid peroxidation and DNA oxidation in aged animals, marked by 4HNE and 8OHdG in immunohistochemistry stains respectively, was significantly improved when CatB was deficient.

The pharmacological method of inhibiting CatB could be one treatment to improve cognitive impairments caused by mitochondrial dysfunction and lysosome destabilization [14].

Since CatB is also found to be up regulated in premalignant lesions and cancers, the inhibition of CatB could reduce malignancy and agerelated degeneration.

PARKINSON'S DISEASE The brain demands a high abundance 13

of lipids and fatty acids and consumes a large amount of oxygen [15]. This makes the brain highly susceptible to oxidative damage.

Additionally, dopamine has a high potential to oxidize and generate reactive oxygen species.

Parkinson’s disease is commonly associated with motor symptoms consisting of tremor and bradykinesia and non-motor symptoms including mood changes and cognitive decline [16]. Although the mechanisms of Parkinson’s are not well understood, it is clear that oxidative stress has a role in Parkinson’s and the associated symptoms [17]. Figure 3 shows the effects of oxidative stress in the brain using immunohistochemistry staining [18].

Manganese and other metals are thought to play a vital role in ROS [15]. To examine Parkinson’s and oxidative stress, one study evaluated the motor effects of animals that inhaled high concentrations of manganese.

Inhalation of manganese produced behavioral motor deficits mimicking Parkinson’s disease, but the levels of lipid peroxidation – determined by thiobarbituric acid assay – were not affected in the motor cortex and hippocampus.

Contrarily, lipid peroxidation was significantly higher in the substantia nigra, striatum, and globus pallidus. This study showed a striking difference from other labs that have shown the hippocampus to be the most vulnerable to ROS [15]. Not only did this study find no effect in the hippocampus, but there was also no significant difference from wild type in the motor cortex, which behaviorally exhibited effects. Because the direct correlation of manganese and oxidative stress is not known, it would be interesting to see if this experiment could be replicated using other models of oxidative stress like hydrogen peroxide. Furthermore, the assay results would be more convincing if there was increased lipid peroxidation in the motor cortex to explain the behaviorally exhibited motor difficulties.

A more supported theory is that the overproduction of oxygen free radicals can lead to dopaminergic cell loss in the ventral tegmental area and substantia nigra [17]. Additionally, selective loss of noradrenergic cells in the locus coeruleus also cause a heightened degeneration of the dopamine system. This is characterized by the up regulation of microglia, astroglia, proinflammatory proteins, and enhanced levels of oxidative stress [17]. By simply attacking the noradrenergic system, the levels of 80HdG (a marker for DNA oxidative damage) increase.

A further study would be needed to see if oxidative stress could lead to similar deficits in the noradrenergic system as it produces in the dopaminergic system. If oxidative stress targets both the noradrenergic and dopaminergic system, this could explain the detrimental effects seen in Parkinson’s patients.

DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

AGING As previously seen, the mitochondrial impairment and oxidative stress dependent inflammatory response are most commonly associated with Parkinson’s disease through the degeneration of dopamine [19, 20].

However, oxidative stress is also known to activate the integrated stress response (ISR) [20]. The ISR functions to maintain homeostasis during times of cellular stress, but when it is perpetually activated, it can lead to cell death. ATF4, a transcription factor and key mediator of ISR, can cause the expression of pro-apoptotic target genes when expression is prolonged. Parkinson’s neurotoxins – 6 hydroxydopamine (6-OHDA) and methl-1-4 phenylpridinium (MPP+) – increase elevation of ATF4 and consequently pro-apoptotic genes. The result of oxidative stress activating ISR, which maintains activation of ATF4, is neurotoxin-induced neuronal cell death. This apoptotic characteristic can lead to many of the symptoms present in Parkinson’s patients, demonstrating yet another way oxidative stress triggers Parkinson’s disease.

ALZHEIMER'S DISEASE The causation between aging and neurodegenerative disease is not well understood, but one theory relies on the decreasedactivityofproteasomeandautophagy over time to explain the accumulation of protein aggregates like amyloid-beta and tau [11]. Amyloid-beta proteins, the main contributor in the formation of plaques, are fragments of an amyloid precursor protein, whereas tau is a microtubule-associated protein that can form neurofibrillary tangles when hyper phosphorylated [21]. Without tau, microtubule stabilization and axonal transport is distorted, and when amyloid-beta is present in large quantities, plaques can assemble [22].

The two hallmarks of Alzheimer’s, a disease characterized by dementia, are the accumulation of amyloid beta plaques and neurofibrillary tangles of hyper phosphorylated tau (ptau) [24]. However, the attention is beginning to shift towards oxidative stress and inflammatory responses as they have the potential to exacerbate both plaques and tangles. These aggregations form when proteases such as ubiquitin cannot efficiently degrade [25]. In drosophila, lower proteasome activity and lower ATP steady state levels were present at young ages compared to the higher levels detected in older animals. This demonstrates how the increase of debris as we age provokes up-regulated proteasome activity, eventually fostering over-worked and impaired ubiquitin. Additionally, SDS page showed that higher levels of ubiquitinated protein aggregates are formed under oxidative FALL 2019

stress conditions [25]. By decreasing oxygen consumption through calorie restriction, drosophila lifespan increased, but no staining was performed to see if the diet had a visible neurobiological effect.

Another type of protease and lysosome is peroxisome that breaks down toxic materials using oxidative enzymes [26]. Alterations in peroxisomes expression have been observed in relation to the onset of Alzheimer’s disease.

When observing mice at pre-symptomatic stages, two peroxisomal membrane proteins – PMP70 and PPARα – exhibited stronger staining. In addition, the enhanced 8OHdG staining revealed disrupted redox homeostasis. In younger mice, these results were not apparent. The increased levels of PPARα and oxidative stress were most noticeable in the hippocampal regions.

Targeting PPARα may be one way to enhance anti-inflammatory and anti-oxidant responses in Alzheimer’s patients; however, it may also be an effective treatment to target oxidative stress directly, minimizing the reactive oxygen species present. Since Alzheimer’s disease is notably associated with mitochondrial dysfunction and excessive reactive oxygen species present in the brain, the PTEN induced pyruvate kinase 1 (PINK1) gene may be altered in AD [27]. PINK1 is involved in instructing the maintenance and removal of defective mitochondria. Enhancing and restoring levels of PINK1 reduce amyloidbeta levels, oxidative stress, and mitochondrial dysfunction, consequently improving cognitive decline. These effects are primarily seen in the hippocampus and lead to a significant decrease in errors on the Morris Water maze and increased time spent in the target area. PINK1 also significantly reduced ROS accumulation in the hippocampus as observed using ER spectrum.

On the contrary, by lowering levels of PINK1, mitochondrial abnormalities, impairments in learning and memory, and debilitated synaptic plasticity are heightened. This highlights a

Figure 3: The figure illuminates oxidative DNA damage in the substantia nigra of a Parkinson’s patient by using the marker 80 Hdg (18).

Source: Zhang et al. (1999)

“ATF4, a transcription factor and key mediator of ISR, can cause the expression of pro-apoptotic target genes when expression is prolonged."

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possible treatment for Alzheimer’s disease.

By genetically activating PINK1, one could promote the rescues of amyloid beta pathology and clearance of oxidative stress, preserving cognitive abilities.

AUTISM AND SCHIZOPHRENIA

"Resveratrol, another type of antioxidant, has been shown to protect animals from deleterious effects caused by oxidative stress."

The effects of oxidative stress go beyond the cognitive impairments seen in normal aging and neurodegenerative disorders. The dysfunction of this system can also result in neurodevelopmental disorders, including autism and schizophrenia [28]. Oligodendrocyte progenitor cells have shown a vulnerability to oxidative stress, especially in the absence of glutathione [29]. In the presence of oxidative stress, oligodendrocytes get stuck in a premyelinated state, blocking the development of myelin.

The resulting hypomyelination leads to pre-frontal cognitive dysfunctions observed in schizophrenia. Furthermore, cellular stress – including oxidative stress and availability of nutrients – can play a role in the regulation of mTOR [28]. mTOR is a kinase important in regulating cell growth, survival, and proliferation [31]. It also plays a role in autophagy and transcription. In a LgDel mice model that is +Tsc2/-E14 and believed to have autism, the levels of oxidative stress and dysfunctional mitochondrial morphology were elevated. Oxidative stress is thought to impair cortical interneuron migration through the Cxcr4/mTOR pathway. This hypothesis is supported by the restoration of the cortical interneuron migration when mice are treated with an antioxidant, N-acteylcysteine. When the Cxcr4/mTOR pathway is disrupted, it can lead to several neurodevelopmental disorders such as autism and schizophrenia. Therefore, targeting mitochondrial oxidative damage is a promising solution.

POTENTIAL TREATMENTS As demonstrated throughout this paper, elevated oxidative stress has devastating effects including Alzheimer’s, Parkinson’s, schizophrenia, and autism. Methods for combatting reactive oxygen species are vital, and there are a few that have received increasing popularity: antioxidants and diet.

Since oxidative stress is the imbalance of prooxidants and anti-oxidants, it seems obvious to intake more anti-oxidants to reduce oxidative stress. Curcumin, a popular Indian spice with antioxidant powers, was shown to have time dependent effects reducing neurodegeneration in the hippocampus induced by ozone [32].

However, since the effects were most prevalent at eight hours, there should be some skepticism that curcumin can be used as an effective treatment. Additionally, the inhalation of ozone may not be an adequate model of the natural reactive oxygen species produced as we age.

From my previous research, curcumin had some effect on lipid peroxidation exhibited by a 4HNE immunohistochemistry stain. However, no significant behavioral changes were observed in the monkeys. Therefore, the ozone-curcumin study could be improved by adding behavioral testing to reveal if the staining is showing any real correlation to the cognitive abilities in mice.

Resveratrol, another type of antioxidant, has been shown to protect animals from deleterious effects caused by oxidative stress [33]. This compound was able to protect drosophila from oxidative stress at the neuromuscular junction induced by a hydrogen peroxide media. Interestingly, resveratrol was more neuro-protective at low doses. The acetyl group on the planar molecule was an important scavenger of free radicals. Another scavenger of reactive oxygen species is SWNT, an antioxidant carbon nanoparticle. SWNT’s role has primarily been studied in secondary injury cascades after

Figure 4: Immunohistochemistry stain for a β-amyloid deposit in plaque form on the left and neurofibrillary tangles on the right in the cortex of an Alzheimer’s patient. [23] Source: Armstrong (2010)

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DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

AGING Figure 5: Displays the process from oligodendrocyte progenitor cell to a myelinating oligodendrocyte.

With ROS dysfunction, the oligodendrocyte may never reach full development [30].

Source: Fields (2015)

spinal cord injuries [34]. It is known that after spinal cord injury, the overproduction of ROS leading to oxidative stress can cause persistent pain – both inflammatory and neuropathic.

Administering SWNT one and three hours after injury helps alleviate pain. Because this study was done in mice, pain was measured with a paw withdrawal task. In the contraption, mice were placed in an aversive light box, but they were able to travel to the more favorable dark side if they walked through a platform with pins sticking up [34]. The treated mice were more likely to travel to the dark side. However, the issue with this model of detecting pain is that since the mice are undergoing spinal cord injuries, the injury could cause issues with walking that are not necessarily attributable to the pain. There is a need to develop a more accurate model to detect pain before and after SWNT administration. In addition, since ROS are known to spike twenty-four hours after injury, SWNT treatment should be tested at different time intervals to see if its effect is timedependent.

Many studies that test the efficacy of antioxidants induce oxidative stress through diets.

Because the oxygen consumption is greater in high fat diets, this increases the risk of cognitive dysfunction via oxidative stress and ROS [35]. One lab tested the anti-oxidant effects of doctrineless, a vitamin that fights free radical damage [35, 36]. When mice consumed high fat diets and were treated with tocotrienols (T3), they showed decreased brain oxidation and improvements on the Morris water maze compared to mice that were not treated with T3 [34]. T3 is thought to up-regulate the antioxidant defense system. Since the risk of oxidative stress dysfunction is elevated in high fat diets, this makes calorie restriction or other diets optimal treatments.

The ketogenic diet is high in fat, low in carbohydrates, and believed to increase hippocampal levels of NAD+ [37]. Previously, higher levels of NAD+ have been shown to decrease inflammation and increase anti-aging.

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In a further study, rats fed with a ketogenic diet showed a rapid increase of NAD+ and a rapid decrease of 8OHdG after two days and even further reduction after three weeks. In support of this, when mice were fed high fat diets for either three days, 1 week, or two weeks, the hippocampus showed altered protein expression in metabolism, increased inflammation, and increased cellular stress after only three days [38]. If the mice then consumed a low fat diet, there was rapid recovery. These studies emphasize the importance of diet on cognitive abilities especially as we age.

Another more interesting choice of diet is high supply of lactate and pyruvate to improve the reduction in energy metabolism efficiency [39]. When nematodes were given a high pyruvate and lactate diet, SH-SY5Y cells were protected against hydrogen peroxide induced oxidative stress. The relationship between humans and nematodes may seem far off, but Dr. Rothman from University of California Santa Barbara argues a vast similarity between the number and identity of genes between the two species [40]. Ultimately, the nematodes consuming a high pyruvate and lactate diet had an increased lifespan, reduced sensitivity to cellular stress, and a delay in neuronal dysfunction [39]. Additionally, lactate and pyruvate can be used as an anti-oxidant mediating the ROS signaling pathway.

Future treatments could take a combination of the proposed treatments. With respect to diet, restricting carbohydrates and total caloric intake will decrease oxygen consumption, while adding wild blueberries, strawberries, and other high antioxidant power foods may restore the pro-oxidant / antioxidant imbalance.

Additionally, taking a vitamin E supplement may be useful. On a pharmacological level, the development of a free radical scavenger that could prevent formation or safely remove highly reactive compounds would make a huge impact on the treatment of neurodegenerative and neurodevelopment disorders. Since part

“With respect to diet, restricting carbohydrates and total caloric intake will decrease oxygen consumption, while adding wild blueberries, strawberries, and other high antioxidant power foods may restore the pro-oxidant / antioxidant imbalance.”

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of what makes free radicals so reactive is their unpaired electron, the designed scavenger must find a way to stabilize this charge [41]. Likewise, the scavenger must be capable of transferring hydrogen to the identified reactive oxygen species. By eliminating the reactants involved in initiation of the free radical chain, we could begin to treat the source of the problem and not the symptoms.

CONCLUSION

“In order to combat the many outcomes of enhanced oxidative stress, it is best to address the imbalance of antioxidants or limit the intake of oxygen species to begin with through diet."

Although we have yet to define an apparent mechanism, it is clear that oxidative stress plays a key role in the development of cognitive impairments in age, neurodegenerative disorders, and neurodevelopmental disorders.

This paper has addressed only a few of the ways oxidative stress can affect cellular function and process. Since mitochondria play a vital role in cellular function, their disruption leading to the release of O2- could produce a cascade of debilitating events. Oxidative stress has commonlybeenseentodecreaseproteasomeand autophagy activity as well as lead to a buildup of DNA damage. In relation to Parkinson’s disease, the free radicals are able to target and kill off dopaminergic cells in the ventral tegmental area.

ROS can also increase apoptotic transcription factors through pathways such as the integrated stress response and mTOR, while the decrease in protease activity and other genes responsible for removing defective mitochondria may result in Alzheimer’s. In order to combat the many outcomes of enhanced oxidative stress, it is best to address the imbalance of antioxidants or limit the intake of oxygen species to begin with through diet. Not only does diet and antioxidants minimize side effects, but they also focus on treating the problem instead of the dozens of symptoms provoked by oxidative stress. D CONTACT GABRIELLE HELTON AT GABRIELLE.N.HELTON.19@DARTMOUTH.EDU

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Implications of Adolescent Stress on Adulthood Behavior

BY SHIVESH SHAH '19

Figure 1a: The process of hippocampal neurogenesis, shown above, is heavily reliant on the molecule doublecortin (green).

Source: Flickr

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ABSTRACT

INTRODUCTION

Stressful early life experiences can induce behavioral dysfunctions that have been linked to mental illness and may extend well into adulthood. These early life stressors (ESLs) can be broadly categorized as either social stressors or non-social (but behavior-changing) stressors. Adolescent stress during the critical period of development has been associated with adulthood changes in both fear expression and safety learning, and these findings have been corroborated by neurophysiological models, the two most prominent of which are the GABAergic BLA model and the glutamatergic VTA model. Similarly, various paradigms and models have been developed to learn more about adolescent social stress, the most notable of which are chronic social defeat models (CSDS) and adolescent social isolation (aSI) models. These models have been crucial in understanding the role played by ELS in adulthood vulnerability to addiction, which is largely mediated by the nucleus accumbens (NAcc). Recent methodological innovation has advanced our ability to understand the neurocircuitry surrounding motivated and social behavior following ELS, although research is becoming increasingly focused on the neural underpinnings of ELS-induced behavioral changes. This review aims to summarize the current state of this field of research, present successful paradigms, and highlight significant findings.

Adolescence is a unique period of development marking an individual’s transition from childhood to adulthood. Widely considered to range from 10 to 25 years of age in humans and postnatal days 28 to 42 in mice, adolescence is characterized by increased cortical plasticity and development, both of which are critical to social maturation. Maturation during this critical period manifests as characteristically high levels of risk taking, exploration, sensation seeking, and social interaction. Many of these behavioral changes have been suggested to prepare individuals for adulthood maturation.

For example, increased adolescent social interactions in rodent models have been implicated in the development of social skill sets that enable them to become senior adult members of their respective social groups [1]. While many of these novelty-seeking behaviors do successfully prepare adolescents for adulthood, they oftentimes lead to the occurrence of unintended stressful situations [2].

There are a great variety of early life stressors (ELS) that can impact cortical development during the adolescence period, although psychosocial stress is particularly interesting. Given that individuals typically rely on social interactions to maintain health, emotional well-being, and fitness, compromised social environments can have deleterious effects on mental health – especially during the developmental period. Bullying by peers, DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S T R E S S A N D D E V E LO P M E N T for example, presents a social stressor that has been implicated in a variety of mental health disorders such as depression, low self-esteem, social withdrawal, and various mood disorders [3]. Similar results have been obtained from models of inadequate parenting in adolescent mice, and longitudinal studies have shown that these effects can last well into adulthood [4, 5].

These life-long changes are often marked by behavioral changes and can manifest as atypical fear expression, safety learning, and motivated behavior.

STRESS AND MOTIVATED BEHAVIOR Fear expression and safety learning. hrough conditioning, stimuli that are paired with specific threats (negative outcomes) or rewards (positive outcomes) can be assigned a predictive value and consequently induce fear or reward seeking respectively. It is widely known that both of these behaviors are modulated by safety cues, which signal the absence of a threat. Many studies have found that stressful adolescent experiences can have an impact on adult fear and reward learning, although the role of conditioned emotion has not been well-studied. One can therefore hypothesize that adolescent stress can have a “priming” effect on fear and safety responses in adulthood – and this is precisely the case.

Mueller et al (2018) conducted an exploratory study to determine how adolescent stress conditioning affects fear expression and the rate of safety learning in adulthood [6].

Rats were initially assigned to either adolescent (postnatal day 30) fear or reward conditioning groups. On postnatal day 70, both groups were given a discriminative conditioning (DC) task, where they were taught to distinguish fear, reward, and safety cues. Results suggested that reward preconditioning (during adolescence) did not contribute to later reward learning.

However, adolescent stress accelerated fear learning but surprisingly delayed safety learning, indicating that emotional experiences during adolescence such as stress can have longlasting effects on behavior [6].

To induce stress, the aforementioned study (Mueller et al. 2018) conditioned fear in the rodent subjects by pairing stimuli with foot shocks. Although studies have repeatedly shown these physical stressors to induce behavioral changes in adulthood, similar experiments have been carried out using environmental stressors (ES). A study conducted by Li et al.

(2018) investigated the effects of ES on behavior throughout adulthood, using an endocrine approach. Imposing sedentary behavior, FALL 2019

irregular sleep schedules, and eating times on a population of adolescent rats led to depressive symptoms in adulthood [7]. Endocrine changes were notable, with significantly diminished plasma leptin levels (p = 0.021) in both genders as well as increased CORT and decreased NYP in females. These results not only support the notion of adolescent emotional priming, but also illustrate the wide-reaching impact of adolescent stress across multiple physiological systems [7].

Figure 1: The dopamine pathway within the brain has been implicated in depressive symptoms resulting from ELS. Affected areas include the VTA, frontal lobe, and amygdala.

Source: Wikimedia Commons

Neural underpinnings: the glutamatergic model and critical period.

While many studies have found ELS to lead to depressive symptoms throughout adulthood, others have observed the emergence of deficits resembling schizophrenia. The dopaminergic system has been implicated in the onset of these symptoms of ELS (Figure 1). A strongly supported model proposes that the impact of stress on the dopamine system can only occur during the critical period of development.

Gome, Zhu, and Grace explored this model, hypothesizing that re-opening an organism’s critical period (and consequently “reverting” the organism to its adolescent state) would heighten its susceptibility to stressors. A select group of rats were administered HDAC inhibitors (VPA and SAHA), which are known to reinstate the critical period in adults by preventing gene deacetylation [8]. Stressors were administered in the forms of both foot shocks and restraints.

After 5-6 weeks of induced stress, activity of dopaminergic neurons in the ventral tegmental area (VTA) and pyramidal cells in the ventral hippocampus was measured. In adolescent rats, both hippocampal and VTA dopaminergic activity increased. In adult rats treated with VPA, dopamine activity within the VTA was

“There are a great variety of early life stressors (ELS) that can impact cortical development during the adolescence period, although psychosocial stress is particularly interesting."

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stress, proposing that PNNs emerge across adolescent development and mirror the closure of critical period plasticity. In addition to correlating the rate of PNN maturation with the extent of ELS in rat models, Johnson attributed the hyperactive symptoms resulting from ELS to a change in the structure of PNNs rather than the quantity of PNNs. Analysis of PNN structures in ELS rats supported this hypothesis, and imaging demonstrated PNNs that had significantly larger and far-reaching structures [11]. Although various models had proposed alternate explanations for the involvement of GABAergic systems in ELS, actual symptoms are likely a combination of explanations provided by multiple models.

SOCIAL STRESS

Figure 2: PNNs (red) encapsulate PV inhibitory neurons (blue) and modulate their activity. ELS increases PNN activity, increasing amygdala function.

Source: Wikimedia Commons

“PV inhibitory neurons are encapsulated by extracellular matrix structures known as perineuronal nets (PNNs), which have been extensively studied and linked to ELS."

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Models and paradigms: chronic social defeat stress (CSDS) further elevated (in comparison to adult control rats), demonstrating the same vulnerability commonly associated with adolescent stress [9].

Neural underpinnings: the GABAergic Model, BLA & PNNs Researchers have long been aware of ELSdependent changes in the basolateral amygdala (BLA), known for its regulation of the fear response, which manifest as elevated threat responses and hyperactivity. However, little is understood regarding the neural substrates and inhibitory circuitry that regulates BLA activity. Growing evidence is now supporting the hypothesis that ELS can directly alter the inhibitory neurocircuitry of the BLA. Research has primarily focused on fast-spiking parvalbumin positive (PV) inhibitory neurons, which play a central role in the BLA fear response by inhibiting its activity through the secretion of GABA [4].

PV inhibitory neurons are encapsulated by extracellular matrix structures known as perineuronal nets (PNNs), which have been extensively studied and linked to ELS. Lim et al. (2017) studied the effects of ELS on the perineuronal nets of the BLA, concluding that the quantity of PNNs plays a primary role in the effects of early life abuse. After artificially inducing ELS, Lim et al. discovered that the number of PNNs significantly increased. Fewer PNNs were needed to sufficiently regulate (ie. inhibit) BLA function, and reducing the inhibitory effects of PNNs led to an increase in hyperactivity [10]. Although these results have been replicated, other models of BLA have been proposed.

Johnson (2018) generated a slightly different model of BLA-dependent adolescent

Chronic social defeat stress (CSDS) models in rodents have long been used to conduct research on stress-related psychiatric disorders such as depression. In this paradigm, rodents are placed into situations that generate emotional and psychological stress. Oftentimes, this consists of placing the rodent of interest into the cage of an older, aggressive and dominant rodent. The induced behavioral changes are studied. Although CSDS has been invaluable tool in determining the impacts of adolescent stress, there are notable drawbacks, the most prominent of which is the reduced ability to distinguish whether the emotional or physical aspects of the induced stress are producing behavioral changes. Nevertheless, similar models have been established that focus solely on emotional stress via witnessing the defeat of a conspecific [12]. In such models, mice are placed into the home cages of male aggressive ICR mice, where they are attacked. The mice undergoing social stress are placed into an adjacent (but separated) chamber, where they can observe the attacks taking place.

Models and paradigms: adolescent social isolation stress (aSI)) Adolescent social isolation stress (aSI) is another commonly implemented paradigm used to study behaviors that are linked to increased vulnerability. This model is selfexplanatory: rodents are socially isolated and placed into chambers without any social contact or communication. Control group rodents are randomly housed in groups. Following social isolation, rodents undergo tail suspension tests (TSTs) and open field tests (OFTs), which are accurate assessments of anxious and depressivelike behaviors respectively [13]. A further DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S T R E S S A N D D E V E LO P M E N T measurement of stress-induced vulnerability tests whether rodents having undergone aSI exhibit greater EtOH (alcohol) intake. Results of this test provide insight into the development of social stress-induced vulnerability and be used to study addictive byproducts of adolescent social stress such as alcohol use disorder (AUD) [14].

Psychosocial vulnerability to addiction: the NAc Adolescent social stress plays a crucial role in the development of adulthood vulnerability to addiction. As previously mentioned, adolescence is marked by frontal cortical development, which contributes to the refining of reasoning and priority setting [15]. The implications of research in this field are significant, with social ELS such as social abuse, emotional abuse, and neglect being highly correlated with adulthood drug and alcohol abuse [16]. Many researchers have noted that psychosocial vulnerability is correlated with activity of the nucleus Accumbens (nAC), an area of the brain central to the neurobiology of reward-seeking behavior.

In 2002, Schramm et al. discovered that longterm potentiation was found more frequently in the nAC of adolescent mice exposed to ELS than control mice (adult and adolescents lacking exposure to ELS) [16]. Over the past decade, strides have been made in this field, further investigating the role played by the NAc. Ewin, Jones, and Karkhanis (2018) investigated the role played by NAc kappa opioid receptors (KORs) in ELS-induced adulthood vulnerability [14]. It is widely known that acute social stress elevates levels of dynorphin, an endogenous KOR agonist, within the NAc. Ewin et al. investigated aSI-induced changes in KOR activation. As expected, results indicated an aSIinduced increase in KOR function at dopamine and glutamate synapses within the NAc [14]. A follow up study was conducted by Ewin, where an exogenous KOR agonist, U50488, was injected into the NAc to mimic the endogenous increase in KOR function observed in aSI rats (Figure 3).

A two-bottle choice intermittent EtOH access paradigm was used to assess vulnerability. As predicted, this paradigm revealed a greater EtOH intake in U50488 rats and aSI rats in comparison to controls [14].

Interestingly, recent findings have also led to the development of a purinergic signaling model that is likely involved in the development of adulthood vulnerability to addiction after social ELS. Chiavegatto, Ulrich, and Correa-Velloso (2018) have pioneered this field, exploring purinergic receptor gene expression in the brains of male adolescent mice submitted to chronic social defeat stress [17]. Chiavegatto et al. employed a CSDS paradigm to study expression patterns of PFC purinergic receptors (P2x and P2y) that have been implicated in psychiatric disorders such as depression. After inducing social stress, rats were categorized as either “resilient” (showing no signs of avoidance behavior) or “susceptible” (showing signs of avoidance behavior); their PFC was analyzed for purinergic receptors via reverse transcriptase followed by qPCR. The “susceptible” group demonstrated an increase in P2y receptor expression in comparison to the “resilient” group, which showed no difference in expression. Nevertheless, both groups demonstrated signs of ELS through a consistent decrease in P2X5 purinergic receptor expression.

Results suggest that specific purinergic receptor subtypes may link ELS and depressive behaviors during adulthood.

“Interestingly, recent findings have also led to the development of a purinergic signaling model that is likely involved in the development of adulthood vulnerability to addiction after social ELS."

Neural underpinnings and molecular signaling pathways The psychiatric symptoms resulting from social ELS have been studied extensively, although little is known regarding underlying molecular signaling pathways. Many proposed mechanisms rely on glutamatergic models, although a newly proposed molecular mechanisms involves dopamine receptor 3 (Drd3)-expressing lateral septum (LS) neurons.

Shin et al. (2018) employed an early social deprivation (ESD) paradigm (analogous to Figure 3: Genes coding for kappa opioid receptors in the nucleus accumbens are located on Chromosome 8. Manipulations to this gene result in a decreased vulnerability response. [14] Source: Ewin et al. (2018)

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“The psychiatric symptoms resulting from social ELS have been studied extensively, although little is known regarding underlying molecular signaling pathways."

Figure 4: The process of hippocampal neurogenesis, shown above, is heavily reliant on the molecule doublecortin (green).

Source: Flickr

aSI) and measured LS Drd3 signaling activity after social deprivation. As hypothesized, Drd3 signaling in the LS was significantly down regulated following ESD, with severe communication deficiencies noted in adulthood [18]. Further manipulations of this system have yielded similar results, with in vivo calcium imaging being utilized to observe deficits in this Drd3 system. Notably, Shin et al. performed a follow up study, during which Drd3 activity (and corresponding behaviors) were rescued via two techniques: optogenetic activation of Drd3 neurons and pharmacological treatment with the Drd3 agonist, PD128907. It was concluded that the Drd3 signaling pathway within the lateral septum plays a key role as a mediator of ELS-induced social impairments throughout adulthood. Furthermore, the ability to rescue pre-ESD behavior provides hope for future therapeutic and pharmacological treatments for social impairments induced during early childhood [18].

Hippocampal neurognesis. Literature haslongdocumentedtheroleofthehippocampus in memory processes, including the encoding and recall of information as well as contextual scene recognition [19]. In particular, contextual memory has been an area of interest over the past decade, with many studies suggesting that aSI-induced deficits in contextual memory could lead to adulthood impairments in social interaction and fear learning. John, Kubat, Hicks et al. (2018) conducted an fMRI-based study, determining the relationship between cumulative adolescent stress exposure and hippocampal activation during contextual memory tasks (Context Separation and Completion (CSC) Tasks). Participants reported

the total number and severity of stressful events throughout their early life by filling out the Life Events Checklist (LEC), a measure of social ELS, and were asked to complete a contextual memory task while in an fMRI scanner. Results indicated a significant positive relationship between the number of stressful events in one’s life and hippocampal activation during the CSC task.

Furthermore, the development of the hippocampus during adolescence is brought on by a period of rapid neurogenesis, which itself plays a critical role in stress regulation, emotional behavior, and learning and memory (Figure 4).

Baglot, Morgan, Ubi, et al. (2018) determined that social isolation altered the expression of hippocampal neurogenesis in adolescent animals exposed to alcohol during their critical period of prenatal development. Notably, Baglot et al. investigated the combinatorial effects of social stress and prenatal alcohol intake on hippocampal neurogenesis. Subsequent to aSI, rat hippocampi were sectioned and processed via immunohistochemistry for the endogenous protein doublecortin (DCX), which is a marker for neuronal immaturity (Figure 5). As expected, aSI induced a decreased density of DCX within the dorsal dentate gyrus, a region of the hippocampus that is thought to contribute to the formation of episodic memory. However, these effects of SI-induced stress were not present in rats exposed to both prenatal alcohol and aSI. In fact, DCX levels of rats in this group increased, suggesting that prenatal alcohol exposure can severely alter the way that animals respond to social stress during adolescence [20].

Nevertheless, prenatal alcohol exposure has been proven to be one of many variables that can influence the impact of adolescent stress on behavioral and neural development.

VARIABLES Genetic predisposition. Although the neural underpinnings provide strong support for the impact of adolescent stress on cortical development and adult behavior, many argue that there is also a genetic component that must not be neglected. Researchers have recently identified a relevant gene, Orthodenticle homeobox 2 (Otx2), which increases adolescents’ susceptibility to stress and depressive-like behaviors in mice after induced ELS. The gene is predominantly expressed in dopaminergic VTA neurons, where it regulates the proliferation and differentiation of dopaminergic neurons [21].

Studies have validated this finding, observing fluctuations in mania and depressive states with overexpression of Otx2.

Furthermore, genes have begun to play an increasingly important role in the study of 23

DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S T R E S S A N D D E V E LO P M E N T sexually dimorphic gene expression. A study conducted by Walker et al. (2018) found that the expression of sexually dimorphic genes was disproportionately affected by social isolation stressors. Interestingly, social isolation stressors were found to result in the loss of sexually dimorphic gene co-expression the medial amygdala. Although further study regarding the manipulation of driver genes in the medial amygdala and other cortical regions must be conducted, significant advancements have been made within the past decade supporting the notion of a sexually dimorphic stress response in adolescents [22].

Gender differences. Increasing evidence has begun to suggest that early life stressors alter neurological functions in a sexually dimorphic manner. A basic study conducted by McQuinn et al. (2018) examined the differential effects of ELS on cortical thickness in a group of healthy men and women using the Computational Anatomy Toolbox (CAT12). Results revealed that females exhibited greater cortical thickness than males with respect to visual and auditory association areas, the ventral temporal cortex, premotor cortex, and superior parietal lobe. Similarly, males exhibited greater cortical thickness with respect to the middle occipital gyrus and extrastriate cortex [23]. Although this study does indicate that ELS has an effect on cortical development and that sex plays a role, the types and varieties of stressors were not controlled and age ranges varied considerably amongst participants (ages 18-45, n = 48).

Eck, Salvatore, Kirkland, et al. (2018) used a more standardized approach in determining that ELS results in sex-specific effects on cognition in rat models. Eck et al. utilized a limited bedding and nesting model (LBN), which parallels the low-resource environment that is characteristic of poverty. Pups (postnatal days 2 through 9) were given only a single paper towel as nesting material and were separated from bedding by grated flooring. After inducing stress through this model, cortical development was assessed in pups by monitoring activity of the hypothalamus-pituitary-adrenal (HPA) axis. Multiple findings emerged from this study.

First, as expected, LBN pups displayed slower cortical development. In addition, when put through a second round of stress exposure during adulthood, male LBN rats performed poorly in novel object recognition (NOR) tests in comparison to female counterparts. These results may suggest that males may express increased vulnerability as a result of ELS [24].

CONCLUSION Although the vast majority of research in the field of adolescent stress has converged on a few key findings, disagreement on neural FALL 2019

Figure 5: Doublecortin (molecular structure shown above) is a marker of neuronal underdevelopment and is present in greater quantities after aSI is induced.

Source: Wikimedia Commons

mechanisms and translational models have arisen. For example, while a majority of the field has come to the common conclusion that adolescent social stress has a negative impact on both brain development and behavior in adulthood, Buwalda et al. (2013) hypothesized and concluded that adolescent social stress does not necessarily lead to a compromised adaptive capacity during adulthood. Male adolescent rats were repeatedly subjected to a slightly modified CSDS stress paradigm on postnatal days 28, 31, and 34. As adults, these rats were housed with males that were either highly aggressive or non-aggressive, and it was found that these CSDS-induced rats more frequently initiated play behaviors but also adopted submissive postures during play rights, indicative of a heightened adaptive capacity. Following CSDS, a few acute but minor changes in hippocampal brain-derived neurotrophic factors (BDNF) were found, though they were transient and no physiological effects lasted into adulthood [5].

While results such as these may have not have been congruent with many of the previously-discussed findings, they support the “match-mismatch hypothesis,” which claims that the final consequence of childhood adversity depends on the extent to which an individual’s stressful early life environment resembles the challenges faced later in life [25].

Some studies have taken a step further and proposed that chronic adolescent stress exposure confers a protective effect on experimental traumatic brain injury acquired as an adult.

Barra De La Tremblaye, Wellcome, Wiley, et al. (2018) examined the long-term effects of adult emotional and cognitive behavior post traumatic brain injury (TBI) in animals having suffered chronic unpredictable stress (CUS) [26]. After induced TBI, rats were assessed for anxiety-like behavior via OFTs (open field tests) and learning memory via NOR tests (Figure 6).

Surprisingly, adolescent CUS exposure lead to better outcomes post TBI than with controls; CUS exposure was positively correlated with an increase in both NOR and OFT scores [26].

In light of such conclusions, considering

“Evidence has begun to suggest that early life stressors alter neurological functions in a sexually dimorphic manner."

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Figure 6: Traumatic brain injuries induced both cognitive deficits and anxiety-like symptoms during adulthood.

play a role in cortical development, one might question whether stress plays a significant intermediary role. Are the neurological changes thought to be a result of SES inherently rooted in stress? This is clearly an area of future study, although obtaining physiological (rather than structural) data in response to variations in SES may be difficult due to ethical concerns.

The field of adolescent stress itself is still in an early stage of development, and further research will be necessary to fully understand the complex interplay of behavioral, physiological, and neurological factors that constitute the behavioral effects of adolescent stress. D

Source: Wikimedia Commons

CONTACT SHIVESH SHAH AT SHIVESH.H.SHAH.19@DARTMOUTH.EDU

“Human-based clinical studies that correlate fMRI activity with selfreported levels of early life stress are inherently flawed, as the representations of stressful situations can vary widely from individual to individual."

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the variability between studies of adolescent stress is important, although several factors must be taken into account. First, discrepancies have often been noted between studies using different models and subjects (ie. human fMRI studies vs. rodent immunohistochemistry).

Even when conducting studies on rodents, there exists tremendous variability, such as genetic variation and breeding methods.

Secondly, human-based clinical studies that correlate fMRI activity with self-reported levels of early life stress are inherently flawed, as the representations of stressful situations can vary widely from individual to individual. Lastly, it is crucial to recognize the differences that exist between different neurological models of adolescent stress. Although thousands of genes and receptor subtypes have been individually studied and correlated with stress-induced behavioral changes, actual behavioral changes are likely a combination of these factors. And, although blame assignment is difficult in determining the molecular basis of adolescent stress, research in this field has been both highly convergent from a behavioral standpoint and relevant to many current-day sociological dilemmas.

It has long been suggested that indices of socioeconomic status (SES) such as family income and parental education are associated with brain development, although little is known regarding the role of SES in adolescent stress and its impact on cortical development.

Gonzalez et al (2018) conducted a study across 4,500 adolescent children 9-10 years of age. The Data Analytics Exploration Portal (DEAP) was used to tests the effects of SES on cortical surface structure. Interestingly, Gonzalez et al. found that higher parental income was correlated with a greater surface area of the prefrontal cortex [27]. Although SES is now known to

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8. Morishita, H., & Hensch, T. K. (2008). Critical period revisited: Impact on vision. Current Opinion in Neurobiology,18(1), 101-107. doi:10.1016/j.conb.2008.05.009 9. F. V. Gomes, X. Zhu, A. A. Grace; Univ. of Pittsburgh, Pittsburgh, PA; Univ. of Pittsburgh Dept. of Neurosci., Pittsburgh, PA. The impact of stress on the dopamine system is dependent on the state of the critical period of plasticity.

Program No. 499.07. 2018 Neuroscience Meeting Planner. San DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S T R E S S A N D D E V E LO P M E N T Diego, CA: Society for Neuroscience, 2018. Online.

10. K. Y. Lim, A. N. Santiago, M. Opendak, R. M. Sullivan, C. J. Aoki; Ctr. for Neural Sci., Child and Adolescent Psychiatry, New York Univ., New York, NY; Emotional Brain Inst., NKI & NYU Sch. of Med., New York, NY. Early life abuse alters GABAergic synaptic contacts in the basolateral amygdala of juvenile rats in a sexually dimorphic manner. Program No. 154.17. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

11. C. Johnson; Molecular and Cellular Biology, Harvard University, Cambridge, MA.. Early life stress alters neural processing of reward and punishment. Program No. 007.07.

2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

12. Y. Nakatake, M. Yamada, H. Furuie, H. Kuniishi, M. Ukezono, K. Yoshizawa, M. Yamada; Tokyo Univ. of Sci., Noda, Chiba, Japan; Dept. of Neuropsychopharm., Natl. Ctr. of Neurol. and Psychiatry, Kodaira, Tokyo, Japan; RIKEN, Kizugawa, Kyoto, Japan. Chronic social defeat stress induces social avoidance and changes the plasma cytokines levels in mice. Program No. 322.12. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018.

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13. Ieraci, A., Mallei, A., & Popoli, M. (2016). Social Isolation Stress Induces Anxious-Depressive-Like Behavior and Alterations of Neuroplasticity-Related Genes in Adult Male Mice. Neural Plasticity,2016, 1-13. doi:10.1155/2016/6212983 14. S. Ewin, S. Albertson, S. Jones, J. Weiner, A. Karkhanis; Physiol. and Pharmacol., Wake Forest Sch. of Med., Winston Salem, NC. Early-life stress augments kappa opioid receptor function selectively on glutamate and dopamine terminals in caudal nucleus accumbens of rats. Program No. 159.30. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

15. D. G. Gee; 2 Hillhouse Ave, Yale University, New Haven, CT.. Dynamic changes in threat and safety learning across human development. Program No. 007.03. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

16. Schramm, N. L., Egli, R. E., & Winder, D. G. (2002). LTP in the mouse nucleus accumbens is developmentally regulated.

Synapse, 45(4), 213-219. doi:10.1002/syn.10104 17. S. Chiavegatto, H. Ulrich, J. C. CorrĂŞa-Velloso; Biomed. Sci. Inst. - Univ. of Sao Paulo, Sao Paulo, Brazil; Dept. of Biochem., Chem. Inst., Sao Paulo, Brazil. Purinergic receptors gene expression in the brain of male adolescent mice submitted to chronic social defeat stress. Program No. 322.06. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

18. S. Shin, H. Pribiag, V. Lilascharoen, D. Knowland, X.-Y. Wang, B. Lim; Dept. of Biol. Sciences, Neurobio., Biol. Sci., UCSD, La Jolla, CA; UCSD, San Diego, CA. Drd3 signaling in the lateral septum mediates early life stress-induced social dysfunction. Program No. 499.05. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018.

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19. R. A. John, B. J. Kubat, A. Hicks, S. A. Joshi J. L. Abelson, I. Liberzon, E. R. Duval; Psychiatry, Univ. of Michigan, Ann Arbor, MI. The relationship between cumulative stress exposure and hippocampal activation during contextual memory. Program No. 227.09. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018.

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20. S. L. Baglot P. Ubi, E. Morgan, S. E. Lieblich, W. Yu, J. Weinberg, L. A. M. Galea; Social isolation stress alters the expression of hippocampal neurogenesis in adolescent animals prenatally exposed to alcohol. Program No. 365.08. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

21. A. Mundorf, N. Freund; Exptl. and Mol. Psychiatry, LWL Univ. Hospital, Ruhr-University Bochum, Bochum, Germany.

Otx2 as a possible mediator for depressive-like behavior.

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Program No. 233.06. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

22. D. M. Walker, X. Zhou, A. Ramakrishnan, M. E. Cahill, C. K. Lardner, et al.; Neurosci., Fishberg Dept. of Neurosci. and Friedman Brain Inst., Friedman Brain Inst., Genet. and Genomics, Icahn Sch. of Med. at Mount Sinai, New York, NY; Genet. and Genomics, Icahn Sch. of Med., New York, NY; Pediatric Endocrinol., Johns Hopkins Univ. Sch. of Med., Baltimore, MD. Adolescent stress reprograms the medial amygdala transcriptome and sex differences in reward.

Program No. 161.02. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

23. S. McQuinn; Univ. of Utah, Salt Lake City, UT. The effects of early childhood trauma and gender on brain structure.

Program No. 281.03. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

24. S. Eck, M. Salvatore, J. Kirkland, A. Hall, S. Famularo, D. Bangasser; Temple Univ., Philadelphia, PA. Early life stress has lasting effects on development and sex-specific effects on cognition in rats. Program No. 227.25. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

25. Santarelli, S., Lesuis, S. L., Wang, X., Wagner, K. V., Hartmann, J., Labermaier, C., . . . Schmidt, M. V. (2014). Evidence supporting the match/mismatch hypothesis of psychiatric disorders. European Neuropsychopharmacology,24(6), 907-918. doi:10.1016/j. euroneuro.2014.02.002 26. P. Barra De La Tremblaye, J. L. Wellcome, K. M. Wiley, I. H. Bleimeister, M. S. Helkowski, J. P. Cheng, C. O. Bondi, A. E. Kline; Physical Med. & Rehabil., Univ. of Pittsburgh, Pittsburgh, PA. Chronic stress exposure provided during adolescence confers protective effects on experimental traumatic brain injury acquired as an adult. Program No. 295.16. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

27. M. R. Gonzales, K. A. Uban, M. M. Herting, E. Kan, E. R. Sowell; Children's Hosp. Los Angeles, Los Angeles, CA; USC, Los Angeles, CA; Pediatrics, USC/CHLA, Los Angeles, CA. Associations between socioeconomic factors and brain structure in preadolescence. Program No. 281.04. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

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Spatial Cognition in Disease and Normal Aging: A Perspective on Grid Cells and Theta Oscillations

BY PELIN OZEL'19 Figure 1: Spatial cognition is highly reliant on the proper functioning of grid cells.

Source: Wikimedia Commons

“Spatial learning and memory, which engendered the classic Morris Water Maze task, has long inspired researchers to chart the human spatial cognitive map."

ABSTRACT For decades, modern spatial cognition research overlooked the intersection of spatial memory and human disease literature. This has recently changed as growing questions of psychopathologies highlight important navigational, orienting and locating abilities in mapping an environment. The clinical application of cognitive decline relies on recognizing the scope of cognition and higherorder processes to analyze the mechanisms at risk. Although literature reviews have effectively summarized innovative approaches to study neurons that respond to spatial memory such as grid cells and local field theta oscillations while investigating pathologies related to cognition such as Alzheimer’s, a new review must update the current direction between the fields of study.

In particular, grid cells and theta oscillations are excellent candidates for understanding sensory integration in the scope of spatial orienting to an environment in normal aging populations and in pathologies that affect these areas. By focusing our attention on these properties of cognitive spatial mapping, we will examine grid cells and theta oscillations in neurotypical conditions as well as cases of dysfunction to realize local correlates in spatial cognitive behaviors across psychopathologies.

INTRODUCTION Spatial learning and memory, which engendered the classic Morris Water Maze task, has long inspired researchers to chart the human spatial cognitive map. This spatial cognitive map gained vast recognition after 27

decades of O’Keefe’s place cell research in the rat hippocampus in conjunction with May-Britt and Edvard I [1]. Moser’s grid cell studies— which fire in a hexagonal grid pattern—in the rat entorhinal cortex. Together with these cell properties, O’Keefe and Nadel (1978) propose that humans rely on internal cognitive maps to navigate, orient, and locate objects in the realworld environment [2]. Yet, spatial cognition struggles to find a final definition in this schema of research due to the unknown limits of these cell properties.

Decades of electrophysiology research indicate that head direction, place, border, and grid cells exist in a circuit to encode these internal cognitive maps. Head direction (HD) cells, are found mainly spread across the Papez circuit in regions such as the postsubiculum, the retrosplenial cortex, and the medial entorhinal cortex. Taube et al. (1990) proposed that these cells fire in foundational orienting patterns to a preferential firing direction (PFD) when an animal orients itself to a specific direction in an environment [3].

In conjunction with this activity pattern, place cells—normally found in hippocampal regions (such as CA1)—fire specifically to a location in the environment acting as a representation of defined space [4]. These signals together bolster grid cell and place cell signals (found mainly in the entorhinal cortex) for environmental and higher-order navigation. These grid cells exist further upstream than place and HD cells. The sensory integration that leads to the ability to navigate from one location to another is path integration, which is necessary for a grid cell response. Yet, the way path integration DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S PAT I A L C O G N I T I O N develops to a more complex cognitive map from the rudiments of spatial memory is still being investigated. Notwithstanding, certain research points to rhythmic activity recorded from local field potential (LFP) from cell populations in thalamic and hippocampal regions called theta oscillations as a modulatory tool to update HD signals to form map-like signaling in grid cells [5]. Such research even points to HD units in the anterior thalamic nucleus, that fire rhythmically in the theta frequency range, to synchronize with hippocampal theta rhythms [3].

Although limits on our understanding of the spatial cognition circuit obscure conclusions to be made on cognitive maps, the field moves to elucidate cognitive markers in pathologies associated with the entorhinal cortex, such as Alzheimer’s Disease, and normal deterioration in aging. Patients revealing dysfunction in this region exhibit wandering behaviors and the inability to orient themselves back to their original locations. Thus, there is a need to further study the underlying system causing this behavior. I will emphasize current work on the characterization and rational of grid cell and theta oscillations signals in the role of higher-order cognition (such as navigating the real-world environment) in pathologies and normal aging. These works will depart from the plentiful research on protein tangling and plaque accumulation and instead focus on the spatial anatomical regions related to early cognitive decline. Although specific wandering behaviors in cognitive decline may not be attributed to spatial cognition deterioration, such disease research is useful to understand spatial cognition. These populations of individuals aid our understanding of this system due to the early signs of dysfunction seen in areas such as the entorhinal cortex. Studies identifying wholebrain differences in functional connectivity effectively explain the greater schema of genetic associates to pathology. However, these studies do not expound upon the mechanism of action that can be attributed to a specified cognitive facility such as spatial memory in navigation [6]. Under these conditions of pathology related to spatial cognition circuitry, grid cell dysfunction could predict pathology susceptibility in spatial memory tasks as a potential cognitive biomarker. Additionally, today’s research reveals localized molecular detriments of protein tangling and plaque accumulation. To parcel out the effects of aging from pathology, such as normal accumulation of these plaques, we also study normal aging and its consequences on the same system. We aim to realize the issues in orienting integration in cognitive map precursors and recommend methods to maintain the navigational system.

Though the momentum of disease and normal FALL 2019

aging research highlights the tribulations in the spatial system, current research may spread beyond the gamut of spatial cognition in understanding the cognitive properties that can be decoded from neural cells.

THE FUNCTION OF SPATIAL COGNITION Grid cells in the functioning animal spatial cognitive system. In the medial entorhinal cortex (MEC), an associational cortex, these grid cells that represent position and orientation present an alluring perspective into other association cortices that may reveal a downstream foundation to sensory memory transformation. Despite our understanding of the rudimentary areas feeding information into this cortical area, it is often difficult to ascertain higher-order information in the hierarchy since cells can have many correlates to neuronal firing. For example, certain HD cells can be velocity modulated and fired in relation to multiple sensory stimuli [1]. On the other hand, consistency remains—an HD cell cannot alter and become a grid cell. The grid cell, however, is the perfect candidate for studying pathologies related to the entorhinal cortex because we recognize the driving information from lowerorder cells such as place cells, HD cells, and velocity-encoding cells.

Some of these lower-order cells, such as place cells, rely on environmental cues to control firing locations or an allocentric basis for orienting cell activity. In contrast, HD cells rely on self-motion and other egocentric factors to presumably send information to place cells in later regions of the hierarchy (such as the medial entorhinal cortex) so that place cells can act stably in cue-deprived environments. This contrast between allocentric and egocentric information is essential for researchers to direct their attention to acknowledging which cues are vital in grid cell dysfunction [1,2]. O’Keefe maintained that sensory path integration must be examined along with environmental cues in clever behavioral models [2]. Together, these two inputs could reveal how grid cells vary in the size and shape of firing fields depending on the context of the environment. Sizing and shapes of hexagonal firing patterns in grid cells can be a biological advantage due to the ability to map onto any environment and for the brain to maximize sensory spatial information.

At the cellular level, grid cells were assumed to have unique morphology crucial for cell targeting. Canto et al. (2012) argued that the anatomical structure of these cells is stellate cells, corroborated by studies in MEC layer II consisting of only stellate cells and grid cells are most extant [7]. Domnisoru et al. (2013) found

“Lower-order cells, such as place cells, rely on environmental cues to control firing locations or an allocentric basis for orienting cell activity."

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that grid cells there are pyramidal cells that act as grid cells as well as the accepted stellate cell type [8]. This only further proves the multiplicity of the cells in the entorhinal associational cortex.

Studies today use cortical calcium imaging techniques to bridge morphological information with topographical data.

“In animal research, theta rhythms play a role in movement and location behaviors such as running and free movement."

Human Research. Although most of the previous behavioral research on grid cells has been limited to animal studies in rats, mice, bats, and monkeys, grid cells have also been identified in humans [1,10]. Functional magnetic resonance imaging in conjunction with virtual reality navigating tasks presents a region of interest analysis on the entorhinal cortex and finds sinusoidal modulation of activation with a 6-fold rotational symmetry oriented with the virtual running direction (9). Patients with electrode therapy for drug-resistant epilepsy in the entorhinal cortex region delineate grid-like spatial neuronal spiking (average waveform of 48 ÎźV, and activity at 28-32 kHz) during a spatial learning task [10]. Consistent with rodent literature, these grid cells behave in an allocentric manner to navigational tasks in the presence of visual cue information even without proprioceptive, self-movement cues.

However, it is important to note that these cells in epilepsy patients observe noisier firing maps than electrophysiology recordings in rodents, possibly due to electrode placement or task differences. Jacobs et al. (2013) question the significance of eye position in location searching tasks such as the one in their experiment, due to previous monkey research on grid cell-like correlates with eye movement [10]. Nonetheless, such neural activity patterns show significant coding schemes that strongly resemble animal literature which bolsters possible applications in the clinical setting for pathologies like Alzheimer’s.

Compounding on spatial cognition research, grid cells also fire outside the domain of physical space. For example, grid cells structures exist in eye movement patterns in monkeys [10]. In human research, Horner et al. (2016) use fMRI in the entorhinal cortex during a virtual navigation task and an imagined navigation task to suggest that grid cell-like patterns emerge in planning and rumination [11, 12]. Therefore, grid cell research in relation to disease likely expands the field of navigational cognitive abilities.

Local field theta oscillations in the functioning animal spatial cognitive system. The spatial cognitive system that needs a modulatory signal and computational integrator that unites sensory and memory driven activity.

Theta oscillations, which received their name from Hans Berger when separating frequency

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bands, are likely actors for this function and can illustrate the relationship between neurons (local field potential) that is obfuscated in single cell responses. These network patterns, specifically in the slow, theta-alpha frequency band, exist across mammals. The most consistent theta oscillations are present in REM sleep and navigational orienting tasks [13]. They generate in the CA3, the entorhinal input, along with Ca2+ currents in the dendrites of voltagedependent pyramidal cells [13]. The signal depends on dendritic organization; parallel dendrites in cortices engender high amplitude field potentials, while haphazard organization offers closed fields and smaller amplitudes [13].

Whether these theta waves are limited to the entorhinal cortex and hippocampus has yet to be determined since some evidence suggests that cortical regions demonstrate similar signaling.

Thalamic oscillatory signaling may synchronize cortical areas for faster sensory integration.

Inceptions of the rhythmic activity have yet to be defined, but strong connections between the medial septum-diagonal band of Broca and the hippocampus are necessary for higherorder theta oscillatory activity. The mechanism of action between the two areas involves cholinergic neurons in layer III of the entorhinal cortex that depolarize onto pyramidal neurons and interneurons. These interneurons also receive a rhythmic hyperpolarization signal from MS-DBB GABAergic neurons. The inhibitory potential in the pyramidal cell creates inhibitory theta signals, while excitatory potentials from the entorhinal cortex generate the excitatory theta signals. Together, these signals act in theta waves constantly processing. This classic model is currently at odds with recent research that debates the strength of the entorhinal cortex discharge overpowering the inhibitory signal, the intrinsic rhythmic nature of certain pyramidal neurons, and the recurrent circuitry of the CA3 [13].

In animal research, theta rhythms play a role in movement and location behaviors such as running and free movement. For example, electrophysiology exemplifies how place cells lock and spike at later phases in a theta oscillation when entering a place field and spikes move earlier in phase as it exits the place field. This notion is termed phase coding, which may be the code behind cognitive mapping.

Theta cycles remain in grid cell field responses and spiking begins later in the phase and shifts earlier during animal locomotion through a firing field [13]. Since phase encoding most strongly correlates to both grid cells and theta oscillations (& gamma oscillations at a different frequency band), most current research focuses on the dysfunction of this feature of theta waves.

Together, cellular grid responses and local field DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S PAT I A L C O G N I T I O N potential of theta oscillations strengthen spatial signals to regulate orienting abilities and other spatial cognitive behaviors [14].

Human cognitive research. Human research displays similar biological correlates to the hippocampus and entorhinal cortex. Kahana makes a point to move towards bridging single cell and local field potential recording in animals and EEG studies in humans through this type of recording. Intracranial electroencephalographic (iEEG) recordings from surgical patients reveal oscillations in frequency bands, theta (4-8 Hz) and gamma (30-80 Hz) [15]. These patients present increased theta activity during locomotion tasks, along with episodic memory cognitive processing tasks. As a result, theta cannot be solely linked to spatial cognition in humans, but it is an indispensable tool in understanding path integrating and orienting.

Currently, Chrastil et al. (2018) investigate with EEG during decision making tasks to redefine theta power properties and present increases in theta power during exploration rather than movement or speed [16].

Human research and intracranial recordings are revealing more about virtual navigation tasks and the relationship between human grid cells and theta oscillations. Maidenbaum et al. (2018) identified entorhinal theta oscillations with a six-fold modulation and symmetry attributed to grid cells [17]. New electrophysiological methods in humans allow for new studies of path integration in humans. Spatial phase dynamics of gamma oscillation, but not theta oscillation, have been argued to explain the emergence of grid cell-like patterns in entorhinal cortical neurons in humans in local field potential from electrophysiological data. Such gamma oscillations uncover phase coherence similar to theta phase locking in spike generation [18]. Still, phase encoding interactions cannot be made with these techniques. Consequently, the technology for phase encoding research in humans must develop to continue to link theta research to spatial cognition literature in animals.

The field, however, gains momentum in understanding drug mechanisms in theta oscillations. Theta wave research highlights the key receptors and transmitters that readily interfere with the oscillatory population potential. As BuzsĂĄki (2002) explains, theta diverges into two categories in pharmacological sensitivity including atropine-sensitive and atropine-resistant [11]. On one hand, atropinesensitive theta becomes silent in the presence of muscarinic blockers. On the other, atropineresistant waves retain oscillatory patterns.

Such localization of muscarinic effects may be substantial in patients who take muscarinic FALL 2019

Figure 2: An example of theta oscillation EEG data taken from a normal mouse using cortical electrodes closer to the hippocampus. Waking I describes behavior from an animal freely navigating and exploring the environment.

Waking II describes an animal with automatic movement or immobility during the awake state. Waking II theta oscillation are characterized as slower with larger amplitudes than Waking I. Source: Wikimedia Commons

agonists due to increase in theta oscillation [19]. Thus, overly active or not sufficiently tonic potential activity associates with pathology and may be a candidate for drug mechanism depending on oscillation pattern properties.

Before this, pathologies must be characterized in the schema of spatial patterns such as grid cells, theta oscillations, and phase encoding.

PATHOLOGY OF SPATIAL COGNITIVE DYSFUNCTION Alzheimer's disease and other dementias. Animal models of disease permit researchers to study the biology of disease in the schema of spatial cognitive properties.

These animal models are essential in connecting genetic and biological differences at the cellular and molecular level to study possible drug mechanisms to reinstate lost functions in the spatial memory system. The Tg2576 mouse model of AD prototypes the disease due to its mutant form of the APP protein. The mutant gene shows its effects through the decline in long-term potentiation (LTP) and synaptic plasticity in the CA1 of older mice [20]. Ying et al. (2018) confirm place cell degradation as plaque proteins build up due to the APP mutation and improper protein cutting [21].

These protein build-ups can rapidly lead to betaamyloid plaque formation, which decrease the number of neurons that code spatial periodicity in grid cells, such as lower-level cells receiving spatial sensory inputs[21]. Additionally, the J20 transgenic mouse model of AD, that similarly overexpresses human APP with two genetic mutations driven by a PDGF- β promotor, have abnormal levels of amyloid plaques and act as useful models for protein buildup in AD [22]. These J20 mice exhibit haphazard firing field locations in navigational tasks. Ying et al. (2018) also examined these mice in a path integrative task to use self-motion cues with

“Human research and intracranial recordings are revealing more about virtual navigation tasks and the relationship between human grid cells and theta oscillations."

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express high levels of APP and PSEN1, do not show learning deficits compared across groups [27]. Thus, current studies suggest that protein accumulation modeled in AD could cause earlier deficits in the inputs to grid cells, which could cause decreases in grid cell plasticity in forming an adaptable cognitive map in changing environments. Therefore, individuals with similar pathology may encounter difficulty in orienting themselves to the holistic environment due to early input, egocentric cue integration dysfunction. Further tests into the feedforward path from lower-level spatial inputs such as place and HD cells to the MEC and path integration signals important in phase locking of theta oscillations will aid in corroborating previous research, define the progression of the disease and clarify disease onset in this region.

Figure 3: Giocomo et al. (2014) display examples of grid cell activity in electrophysiology depending on different grid spacing and field size. Such uniform symmetry and spacing is not seen in pathologies related to this circuitry.

Adapted from the paper ‘Computational Models of Grid Cells’. Neuron. 10.1016/j. neuron.2011.07.023 Source: Wikimedia Commons

“Although stress negatively impacts hippocampal plasticity possibly through these proinflammatory proteins, such events can promote caudate nucleus—strongly associated with habit learning—activation in an inverse relationship."

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allocentric visual cues. Theta rhythmicity also reflects problems with the spatial cognition in the CA1 region of the hippocampus in J20 mice [23]. Theta frequency, consistent with gamma frequency findings, oscillations exhibited decreased power and irregular rhythmic behavior [23]. These mice displayed impaired path integration, especially in allocentric cuedeprived environments. Thus, grid cells in the MEC may incorporate egocentric noise that causes this dysfunction in navigational abilities.

Possible future work on compensating this system with stronger cue input signals from lower-level sensory signaling to the MEC may prove productive. Figure 2 shows how grid cells in the MEC respond neurotypically to an environment during awake behavior in navigation.

Though Tg2576 and J20 are useful models of disease in relation to revealing cognitive function especially in AD patients and normal aging individuals with higher levels of protein buildup, there are confounding issues in solely studying Aβ protein accumulation.

APOE knock-in mice (APOE 3 and APOE 4) allow for a perspective independent of protein accumulation and focus on genetic correlates and cardiovascular conclusions of AD [24].

Navigational exploration latency times are significant enough to disparage young mice with the APOE knock-in genes from the young control mice. This suggests earlier possible developmental concerns in relation to spatial memory that precede grid cell deterioration [25, 26]. Adversely, 5xFAD transgenic mice, which

Anxiety and emotional stressing arousal. The ubiquitous effects of stress on the hippocampus suggest that cytokines may alter hippocampal functions during neuropsychiatric pathologies [28]. Due to the plethora of receptors found in the hippocampus, proinflammatory protein interleukin, a cytokine, reveals the relationship between the caudate nucleus and the hippocampus in navigational tasks. Other interleukins, including IL-6 and TNF-α, increase in AD patients as the disease progresses. Although stress negatively impacts hippocampal plasticity possibly through these proinflammatory proteins, such events can promote caudate nucleus—strongly associated with habit learning—activation in an inverse relationship. Goodman et al. (2018) investigate the influence of stressing arousal on spatial and habitual (stimulus-response) memory in humans [28]. The virtual eight-arm radial maze task adapts from rodent spatial memory tasks.

In this task, participants use allocentric spatial cues in the radial maze to navigate on certain trials while they rely on egocentric navigation in a stimulus-response variation of the maze in the absence of allocentric spatial cues in other trials. Some trials evoke a painless electrical shock to induce stressing arousal. During spatial memory version of the task, subjects err more. In contrast, the participants exhibited no difference between shock and no shock trials in the stimulus-response version of the maze. In support of the inverse relationship between the hippocampus and the caudate, these findings show how memory deficits may function in psychopathologies like AD. Negative influences on hippocampal plasticity may diminish grid cells remapping abilities across environments and for such neurons to lose phase locking in theta oscillations.

DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S PAT I A L C O G N I T I O N Progression of normal aging in spatial cognition. Deficits in spatial navigation are not limited to pathologies of disease, but rather can also be described in normal aging.

Normal aging, defined by the natural process of cognitive decline without certain biology or symptoms of diseases, acts also as a model of delineating spatial cognitive process decline outside the scope of pathology. Through fMRI and a virtual reality navigation task, grid celllike activity in the whole-brain analysis of the entorhinal cortex displayed decreased stability of grid orientations suggesting problems in subjects orienting to novel environments in a virtual navigation task absent to self-motion cues. Additionally, the magnitude of grid-cell activation levels was significantly correlated to age-related deficits in path integration tasks between young and aged groups [31]. Stangl et al. (2018) show that the magnitude of gridcell representations is much higher in young individuals than older adults in fMRI data from the bilateral entorhinal cortex. Additionally, models testing for symmetrical significance (5-fold/7-fold) display that these magnitudes did not diverge from zero significantly. This suggests that grid cell signals are less tonically active, but not significantly different in cell type [31]. In aged individuals, an internal noise bias may the cause of issues in path integration seen in path integration error scores in both the visual navigation task and another self-motion and visual cue navigation task [30].

It is possible that aged individuals do not orient themselves to the environment correctly due to egocentric deficits in self-position computing during movement due to grid cell problems and decreased plasticity in this region.

Together, human grid cell representations could act as a biomarker for disease in dementia and otherneurodegenerativedisorders.Furthermore, the effects of theta in the role of naturally aging individuals in this path integration task need to be investigated in the future [31].

COGNITIVE TESTING PROXY Progress towards developing a method for early diagnosis of cognitive decline in AD, frontotemporal dementia, and vascular dementia, increasingly befits clinical settings with more accuracy. Coutrot et al. (2018) revisit the utility of mobile apps as a predictive tool for real-world navigation [32]. The app, Sea Hero Quest, includes multiple tasks such as wayfinding where participants are expected to study a map then navigate to multiple sequential goals. A group of 30 participants also engage in a real-world navigational task with the same goals. Individuals’ performances are consistent across tasks and show the significance of agerelated and gender-related factors on spatial FALL 2019

memory capabilities. Their current research maps the paths of players on the app by age and depicts the change in exploratory navigational behavior for those with normal aging. Moreover, researchers study subjects with genetic correlates of disease with human imaging techniques to reveal potential markers from individuals predisposed with the amyloid precursor protein (APP) gene, a cause of familial Alzheimer’s disease [31]. These studies with virtual reality correlate to cognitive decline and with further research will elucidate human populations that may aid in understanding the intersection of spatial cognition and disease.

Although Coutrot et al. (2018) did not explore cellular activity such as grid cells and theta waves, their work gives precedence to the use of virtual reality in cognitive decline testing. Current research uses mental rotation tasks to explain Unified Parkinson’s Disease Rating Scale (UPDRS) scores for early onset, spontaneous Parkinson’s disease [32]. Through the Mental Rotation Test Version A (MRT-A), along with other memory tests, MRT-A was significantly impaired between early-onset Parkinson’s disease patients and aged-matched individuals. The visual-spatial problems associated with cognitive decline can become a way to test for early PD prognosis coupled with other neuropsychological testing aids to reveal pathology stages. Such visuotemporal must be further tested in relation to eye movement correlated grid cell responses to localize deficits [33]. In conclusion, spatial memory and orienting tasks can be tools for diagnosis due to early deficits in this system.

“Through the entorhinal cortical regions, we capture a window into the anatomy and coding behind complex behaviors such as navigation and locomotion through path integration algorithms."

CONCLUSION Even though the intersection of spatial cognition and human disease research is still at its adolescent stages, researchers continue to ask questions surrounding the spatial cognitive system, with a focus on the orienting aspects of how cognitive mapping and oscillatory population potentials highlight human disease.

Through the entorhinal cortical regions, we capture a window into the anatomy and coding behind complex behaviors such as navigation and locomotion through path integration algorithms. Possible grid cell dysfunction could be a result of egocentric cue inputs not sufficiently achieving their targets in accord with silenced population signaling. This property of human pathology poses as a tool for cognitive testing capabilities in the clinical setting for earlier and more enriched timelines for diseases to better allocate drugs and treatment.

Applying learned methods from grid cell and theta oscillation research onto other cognitive processes to integrate human disease research is indispensable for the understanding 32

of the lower-level and higher-level cellular and population responses in the hierarchy of spatial memory. Such as framework may only then begin to illustrate the holistic view of spatial memory in humans. D CONTACT PELIN OZEL AT PELIN.OZEL.19@DARTMOUTH.EDU References 1. Rowland, D.C., Roudi, Y., Moser, M., & Moser, E.I. (2016). Ten Years of Grid Cells. Annual review of neuroscience, 39, 19-40.

2. O’Keefe J, Nadel L. (1978). The Hippocampus as a Cognitive Map. Oxford, UK: Clarendon.

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5. Tsanov, M., Chah, E., Vann, S.D., Reilly, R.B., Erichsen, J.T., Aggleton, J.P., O'Mara, S.M. (2011). Theta-Modulated Head Direction Cells in the Rat Anterior Thalamus. Journal of Neuroscience, 31 (26) 9489-9502; doi: 10.1523/jneurosci.0353-11.2011 6. Sheline, Y.I., Morris, J.C., Snyder, A.Z., Price, J.L., Yan, Z., D'Angelo, G., Liu, C., Dixit, S., Benzinger, T., Fagan, A., Goate, A., Mintun, M.A. (2010). APOE4 Allele Disrupts Resting State fMRI Connectivity in the Absence of Amyloid Plaques or Decreased CSF Aβ42. Journal of Neuroscience, 30 (50) 17035-17040; doi:10.1523/ jneurosci.3987-10.2010 7. Canto CB, Witter MP. 2012. Cellular properties of principal neurons in the rat entorhinal cortex. II. The medial entorhinal cortex.

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22. Mucke L, Masliah E, Yu GQ, Mallory M, Rockenstein EM, Tatsuno G, Hu K, Kholodenko D, Johnson-Wood K, McConlogue L. Highlevel neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation. J Neurosci. 2000 Jun 1;20(11):4050-8. PubMed. 23. Mondragón-Rodríguez, S., Gu, N., Manseau, F., & Williams, S. (2018). Alzheimer's Transgenic Model Is Characterized by Very Early Brain Network Alterations and β-CTF Fragment Accumulation: Reversal by β-Secretase Inhibition. Frontiers in cellular neuroscience, 12, 121. doi:10.3389/fncel.2018.00121 24. Getz GS, Reardon CA. ApoE knockout and knockin mice: the history of their contribution to the understanding of atherogenesis.

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DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

ALS

Emerging Treatments for Amyotrophic Lateral Sclerosis

BY GRACE HERRON '19

ABSTRACT Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons. There are two drugs currently approved to treat the disease, though both are insufficient. Consequently, there are a number of emerging therapies aiming to better treat patients with ALS. Treatments are targeted to various pathologies of the disease, including protein aggregations, dysfunctional synapses, and glutamate excitotoxicity. There are more novel treatments in development as well, such as gene therapies, repurposed drugs, and even brain-machine interface. The drug discovery process is supported by rapid screening assays and innovative drug delivery technologies. The future of ALS therapies is promising. However, a better understanding of the underlying mechanism is necessary before significant breakthroughs in treatment of this devastating disease can be made disease.

INTRODUCTION Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease of high unmet need. It is characterized by the progressive loss of upper motor neurons in the motor cortex, brainstem, and spinal cord, as seen in Figure 1 [1]. The main mechanism of motor neuron degeneration is unknown [1]. People diagnosed with ALS face a variety of devasting symptoms associated with upper motor dysfunction. As the disease progresses, they lose limb and respiratory function, as well as the ability to chew, swallow FALL 2019

and speak [1]. This disease therefore places an incredible burden on patients and their caregivers. Ultimately, patients die within two to five years of diagnosis [1]. Approximately 500,000 people in the United States will develop ALS in their lifetime [1]. Of those cases, around 10% are familial, meaning they have a known genetic cause [2]. The most prevalent genetic causes are mutations in the C9ORF72, SOD1 and TDP-43 genes [2]. The remaining 90% of cases occur sporadically and have no identifiable genetic correlate.

Currently, there are two approved treatments for ALS: Riluzole and Edaravone [3].

However, these treatments are inadequate. They mainly provide comfort to patients by slowing deterioration and preventing complications.

Unfortunately, they cannot reverse the damage or provide a cure for the disease [3].

There is therefore an urgent need for new and improved treatments. There are a variety of pathologies associated with ALS and new emerging therapies are targeting these different pathological features. This review provides an overview of the latest preclinical innovations in ALS, recent advances in drug discovery, as well as suggestions for future directions.

Figure 1: MRI scan shows atrophy of the motor cortex in an ALS patient, resulting from the degeneration of motor neurons.

Source: Wikimedia Commons

“Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease of high unmet need. It is characterized by the progressive loss of upper motor neurons in the motor cortex, brainstem, and spinal cord."

EMERGING THERAPIES Targeting protein aggregation. A common pathological feature seen in both sporadic and familial ALS is the accumulation of proteins in motor neurons [1]. It is believed that these accumulated proteins lead to 34

Figure 2: TDP-43 Histology in ALS. Spinal motor neurons with ALS display nuclear clearing of TDP-43 (E) whereas control neurons retain normal nuclear TDP-43 (F). In ALS motor cortex neurons, TDP-43 forms dense aggregates (G) that are not seen in control motor cortex neurons which have normal nuclear TDP-43 (H).

Source: Printed with permission from: Saberi S, Stauffer JE, Schulte DJ, Ravits J. Neuropathology of Amyotrophic Lateral Sclerosis and Its Variants. Neurol Clin.

2015;33(4):855-76.

“Silencing the expression of this [TGRFII] receptor in rodents was shown to increase the clearance of toxic protein aggregates associated with ALS."

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motor neuron death [1]. A major class of new therapies is attempting to reduce these protein accumulations through various mechanisms of action. One common target is TAR DNA Binding Protein 43 (TDP-43). TDP-43 is a DNA and RNA binding protein that functions as a regulator of gene transcription [4]. Dysfunctional or mis-localized TDP-43, as pictured in Figure 2, is found to accumulate in both types of ALS, making it a standout target for intervention.

Researchers at AC Immune in Switzerland are using monoclonal antibodies (mAb) to target dysfunctional TDP-43 [3]. They have created a new technology called SupraAntigen that can produce and screen large numbers of potential antibodies for dysfunctional TDP-43 [4]. When they identify the most specific antibody, it can be administered to patients to prompt an immune response to destroy dysfunctional TDP-43. This approach has the potential to be disease-modifying. SupraAntigen technology is currently used specifically for TDP-43, but it could be applied to other proteins commonly found to aggregate in the motor neurons of ALS patients.

Another class of TDP-43 therapies target aberrantly localized TDP-43 rather than dysfunctional TDP-43. The mechanism of action is therefore slightly different. Rather than targeting TDP-43 directly using antibodies, these small molecules intervene one step earlier in the pathway. They target the multiprotein complexes (MPCs) responsible for the proper localization of TDP-43 [5]. When multiprotein complexes are not working, TPD-43 is not localized correctly, which ultimately leads to TDP-43 accumulation and motor neuron death. Small molecule drugs can inhibit these dysfunction multiprotein complexes, which returns TDP-43 to its proper location in the nucleus and prevents its aggregation [5]. This novel approach more proactively treats protein accumulation by preventing TDP-43 from building up in the first place. It reflects a deeper understanding of the causal factors of protein accumulation.

Other approaches more generically target protein accumulation. They are not specific to just one protein like TDP-43, but any accumulated protein. This line of therapies harnesses the cell’s own ability to clear proteins via autophagy. Autophagy is the cellular process that directs misfolded proteins, toxic aggregates, and injured organelles to lysosomes where they are destroyed [6]. Inducing autophagy in motor neurons can help clear out accumulated proteins. Researchers at the Gladstone Institute in Southern California have identified a number of molecules that can cross the blood brain barrier and induce autophagy [6]. When they treated motor neurons in vitro with a select autophagy inducer, the motor neurons survived longer [6]. If this drug is capable of producing the same survival benefit in vivo, then autophagy could represent a major opportunity for ALS treatment. Because autophagy is not specific to one particular protein, it could be used to treat a broader patient population.

There are additional ways to activate neurons’ autophagic processes. BiAgil is a novel treatment that also aims to increase autophagy in motor neurons. BiAgil works by silencing the expression of Transforming Growth Factor-Beta receptor II (TGFRII) [7].

ALS patients commonly have elevated levels of TGRFII in their blood plasma, cerebrospinal fluid, and spinal cord which inhibits autophagy [7]. Silencing the expression of this receptor in rodents was shown to increase the clearance of toxic protein aggregates associated with ALS [7]. BiAgil and small-molecule autophagy inducers both increase autophagy, but through different mechanisms. Autophagy inducers directly promote autophagy, while BiAgil inhibits autophagy suppression. As research in this field continues, it will be important to consider possible off-target effects of autophagy inducers. These molecules non-specifically induce autophagic processes throughout the body, which could result in damage to unaffected cells. It will be critical to identify ways to induce autophagy specifically in affected neurons.

Promoting synaptic remodeling. At the synaptic level, ALS is characterized by loss of innervation at neuromuscular junctions.

Neurons appear to “pull back” from muscle cells as they degenerate, leaving muscle cells that lack innervation [8]. It may be possible to modify these synapses in ALS patients with drugs that promote synaptic remodeling.

This is accomplished by inducing the innate regenerative ability of axons, such as “adaptive sprouting.”Adaptive sprouting is a phenomenon seen in ALS patients where axons will “sprout” new processes to innervate neighboring muscle cells that lost their innervation [8].

DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

ALS Researchers at Genentech in San Francisco, California are attempting to artificially induce adaptive sprouting by inhibiting Beta-Amyloid Cleaving Enzyme (BACE1). BACE1 typically represses axonal sprouting [8]. When mice were administered a drug to inhibit BACE1, there was increased sprouting and neuromuscular junction innervation [8]. It would be interesting to see how these effects would transfer to human patients. It is possible that inducing axonal sprouting in humans could have unintended side effects by creating random or aberrant connections. As the field of synaptic remodeling advances, there should be increasing attention toward improving the specificity of these regenerative processes.

Reducing Glutamate Excitotoxicity.

Patients with ALS often have disrupted glutamate signaling [1]. It is believed that glutamate excitotoxicity may contribute to the pathology of the disease. One way to alter glutamate signaling is to target mutated glutamate receptors. Both ionotropic and metabotropic glutamatereceptors areimplicated in ALS pathology. Mutations in ionotropic AMPA receptors are commonly seen in the disease. In fact, the majority of sporadic ALS patients have dysfunctional AMPA receptors [9]. Since sporadic cases account for 90% of total ALS cases, targeting AMPA receptors would be applicable for most patients.These dysfunctional AMPA receptors commonly have an unedited GluA2 subunit, which makes the ion channel more permeable to calcium [9]. Scientists at the University of Tokyo identified a drug candidate named FN1040 that targets these dysfunctional, calcium-permeable AMPA receptors. FN1040 is an RNA molecule that specifically binds to mutated AMPA receptors [9]. When this drug was administered to mice, it demonstrated a robust neuroprotective effect against glutamate excitotoxicity [9]. One concern with inhibiting glutamate signaling is that it can have a sedative effect. However, FN1040 had “no sedative side effect� in mice [9]. It is still possible that sedative effects could emerge if FN1040 advances to a human clinical trial. Due to the potential for side effects with glutamate therapies, there should be particular caution about sedation if these drugs are administered to humans.

It is also possible to target metabotropic glutamate receptors. CTEP is a drug candidate that is a negative allosteric modulator of the mGlu5 metabotropic glutamate receptor (Figure 3). When ALS mice were administered CTEP, they experienced delayed disease onset, increased survival, and improved motor abilities [10]. Even if treatment was started after the onset of symptoms, it still prolonged life [10]. These findings are particularly promising; interfering FALL 2019

with metabotropic signaling not only prevents disease onset, but it improves symptoms of existing disease. Similar to FN1040, the benefits of CTEP are likely attributable to its protective effects against glutamate excitotoxicity. Mice that were administered CTEP had normalized levels of glutamate as well as decreased degeneration in motoneurons and glial [10].

The effects of FN1040 and CTEP on ionotropic and metabotropic receptors, respectively, are currently both limited to in vivo studies in rodents. It will be interesting to see how their efficacy translates to humans and if side effects emerge from disrupted glutamate transmission.

RNA-targeted gene therapies. The aforementioned therapies were mainly directed at pathologies seen in the sporadic form of the disease. There are emerging therapies to address the familial form of the disease as well. Because familial ALS has known genetic correlates, it is more amenable to precise, gene-targeted approaches. RNA-targeted therapies offer a potential solution. These drugs intervene at the level of mRNA. They are designed to bind to mutant mRNA transcripts before they can be translated into mutant proteins. If the mutant proteins are never translated, they cannot exert a toxic effect. Antisense oligonucleotides (ASOs) are an RNA-targeted therapy (Figure 4). ASOs are short strands of approximately 20 nucleotides that bind mutant mRNA and mark it for degradation [11]. ASOs have demonstrated good pharmacodynamic profiles in the central nervous system (CNS). Intrathecal injections of ASOs in rodents led to robust activity in all major CNS cells types [11]. ALS involves many cells in the CNS including microglia, astrocytes, and motor neurons, so it is critical to have activity in all of these cells [2, 10]. ASOs have potential to treat a variety of ALS mutations.

By slightly altering the base pair sequence, they become targeted to a different gene [11]. This presents a promising approach for the 10% of ALS patients with known genetic mutations.

Another class of RNA-targeted therapies are short-hairpin RNAs (shRNA). The molecular structure of these molecules interferes with mRNA and silences gene expression [12].

Currently, shRNAs are being used to target

“CTEP is a drug candidate that is a negative allosteric modulator of the mGlu5 metabotropic glutamate receptor."

Figure 3: Skeletal Structure of CTEP. CTEP is a potential ALS drug candidate that acts as an allosteric inhibitor of the mGlu5 metabotropic glutamate receptor.

Source: Wikimedia Commons

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Figure 4: Mechanism of Action of Antisense Oligonucleotides (ASOs).

ASO drugs bind directly to their target mRNA, thereby preventing translation into a protein.

Source: Wikimedia Commons

“In preclinical trials, Adapalenefilled nanoparticles induced retinoid signaling pathways in ALS mice, increased their life span, and improved their motor performance."

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mutations in superoxide dismutase 1 (SOD1) that cause ALS. SOD1 mutations account for around 25% of all familial ALS cases [1]. In a preclinical trial, a single dose of a SOD1-targeting shRNA resulted in the longest survival extension ever reported in a SOD1 mouse model [12]. This drug, named AVXS-301, is currently under IND submission to advance to human trials. The preclinical results are exciting, but there seems to be a major drawback to AVXS-301. In the study, the drug was administered immediately after birth. It is therefore unclear if the drug would have the same effect in adult patients that it did in pre-symptomatic, newborn mice.

Additionally, there is not a genetic screening for SOD1 performed at birth, so it is not possible to identify affected individuals as infants. This is a relevant issue for all gene therapies. AVXS-301 and other similar drugs will not be as effective unless they are accompanied by new genetic screening panels to promptly identify patients.

Repurposing Existing Therapeutics.

ALS shares many pathological features with other diseases. It may therefore be possible to use existing drugs off-label to treat ALS. There are benefits to repurposing already-available therapies. For one, it eliminates the research and development costs associated with creating a new drug. Existing drugs also have wellestablished safety and pharmacological profiles because they are FDA-approved. This mitigates the risk associated with clinical development.

There are multiple approved drugs being studied in ALS, including Adapalene, Apilimod, Acamprosate, and Baclofen. Adapalene is a topical therapy for acne that affects retinoic acid signaling [13]. Retinoic acid signaling is known to have a neuroprotective effect, so there was interest in using Adapalene to treat ALS [13]. However, Adapalene is poorly-water soluble, which makes it challenging to target to

the central nervous system [13]. To address this problem, scientists at the Barrows Neurological Institute in Phoenix, Arizona developed lactic acid nanoparticles that deliver Adapalene to the central nervous system [13]. In preclinical trials, Adapalene-filled nanoparticles induced retinoid signaling pathways in ALS mice, increased their life span, and improved their motor performance [13]. This acne medication could therefore be a potential treatment for ALS. Cancer drugs are also being tested for their efficacy in ALS. Apilimod is a kinase inhibitor that is currently approved for the treatment of B-Cell non-Hodgkin lymphoma [14]. This drug may be therapeutic for ALS patients that have the C9ORF72 mutation. This mutation is the most prevalent genetic cause of ALS, accounting for 40-50% of familial cases [1]. The mutation is associated with dysfunctional endosomes and lysosomes that leads to the accumulation of proteins and RNA [1]. In preclinical studies, Apilimod was able to rescue the effects of the C9ORF72 mutation and improve endosome and lysosome function [14]. Given that the study was in vitro, it remains to be seen if these effects translate in vivo or correlate with clinical outcomes such as prolonged life or improved motor performance.

It may also be possible to combine more than one existing drug into a new combinational therapy. PXT864 is a combination of two repurposed drugs: Acamprosate and Baclofen [3]. Acamprosate is a medication for alcoholism and Baclofen is a muscle relaxant [3]. Their structures are displayed in Figure 5a and b. When administered together, they acted synergistically to protect neuromuscular junctions from glutamate excitotoxicity [3].

Excitingly, PXT864 was better at protecting neuromuscular junctions than Riluzole, which is the current treatment for ALS [3]. The success of repurposed drugs in preclinical DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

ALS trials, especially combination therapies, is an exciting development for ALS drug discovery.

Further investigation is needed to identify novel combinations that may have synergistic effects.

This approach has the potential to minimize development costs and accelerate the drug discovery process.

Brain-machine interface. It may be possible to treat ALS non-pharmacologically using brain-machine interface (BMI) [15]. BMI is increasingly being considered as a therapy to restore motor control and function in patients with severely disabling diseases like ALS [15].

This approach does not cure the disease, but it could dramatically improve the quality of life.

There has been skepticism over whether BMI would work in ALS patients. BMIs need to be able to reliably decode brain signals. However, ALS patients have degenerated motor cortices.

It was therefore uncertain if electrical signals could still be decoded from these degenerated brain regions [16].

BrainGate, a company developing BMI technology for immobile patients, aimed to address this problem. They created intracortical brain computer interfaces and implanted them in the precentral gyrus of ALS patients [16]. Their main goal was to determine if the computer could still “decode” signals from ALS patients’ brains. In their study, they were able to reliably decode four different commonly used hand movements: power, pinch, key grasp, and forearm supination [16]. Importantly, the patient’s level of impairment from ALS did not affect decoding accuracy [16]. This suggests that motor neuron degeneration does not prevent reliable decoding of brain signals. These findings, although limited to hand movements, could lead to technologies that dramatically increase the independence of ALS patients.

The technology of brain-machine interfaces is advancing itself. New devices use flexible electrocorticography (ECog) electrode arrays and optogenetics to reduce the invasiveness [17].

Although not curative, BMI presents a nearterm opportunity to improve patients’ quality of life. Now that it is know that grasp type can be reliably decoded in ALS patients, future studies should investigate other movements that can decoded. In particular, they should focus on identifying movements that would restore functional independence, such as those involved in eating, drinking, brushing teeth, and other daily self-care tasks.

that accelerate the timeline of drug discovery.

New ALS-specific assays enable researchers to rapidly screen compounds and identify which ones could work best. Other innovations are aimed at improving drug delivery. These novel technologies better deliver drugs to their targets in the central nervous system. Together, these fields of research play a key role in advancing the search for an effective ALS treatment.

Assays and high output screening.

There is a need for ALS assays that can accurately and rapidly identify the most promising drug candidates. Charles River Laboratories has developed a disease-related assay in motor neurons. These motor neurons are derived from human induced pluripotent stem cells (hiPSC) of ALS patients [18]. In conjunction with their assay, they also developed a protocol to rapidly differentiate these motor neurons [18]. The ability to quickly produce motor neurons expedites the timeline of their assay.

It also makes the assay amenable to high output screening [18]. These features will allow researchers using the assay to screen different drug candidates at a rapid pace in motor neuron derived from actual ALS patients.

Charles River Laboratories is not alone in this area of innovation. BrainXell is another company developing ALS patient-derived motor neurons for screening. Their goal was to design an assay that would be sensitive and applicable to a wide variety of ALS drug classes. More specifically, their assay screens a drugs’ ability to restore normal levels of neurofilament light chain (NF-L) expression [19]. NF-L is a potential biomarker for ALS. It is significantly elevated in the cerebrospinal fluid of ALS patients compared to controls [20]. NF-L could soon be used as a clinical biomarker, making it an ideal marker to screen preclinical candidates. With their assay, BrainXell was able to screen over 6,000 potential therapies, 80 of which restored NF-L levels and were deemed “hits” [19]. These 80 compounds were advanced to secondary screening, where two in particular restored normal levels of NF-L

“The technology of brain-machine interfaces is advancing itself. New devices use flexible electrocorticography (ECog) electrode arrays and optogenetics to reduce the invasiveness."

Figure 5:Skeletal Structures of Acamprosate (top) and Baclofen (bottom). One potential treatment for ALS repurposes the alcoholism medication Acamprosate (5a) and the muscle relaxant Baclofen (5b) into a combination therapy.

Source: Wikimedia Commons

ADVANCING DRUG THERAPY The emergence of potential therapies for ALS has been facilitated by innovations to support drug discovery. There have been breakthroughs FALL 2019

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“Drug delivery to the central nervous system is uniquely challenging because drugs must cross the blood brain barrier."

Figure 6: Structure of a Liposome. Liposomes are spherical lipid bilayers that enclose an aqueous interior.

The aqueous interior enables the delivery of hydrophilic drugs across the blood brain barrier.

Source: Wikimedia Commons

with no apparent side effects [19]. The ability to quickly narrow down potential drugs will allow researchers to invest time and resources into the therapies most likely to be successful.

Ultimately, this should decrease drug discovery timelines and prevent failure of drugs in later stages of development.

Novel Delivery Methods. ALS pathology occurs in the central nervous system. Drug delivery to the central nervous system is uniquely challenging because drugs must cross the blood brain barrier. To overcome this challenge, new delivery methods are being designed for ALS drugs. Delivering drugs via liposomes may be one possibility. Liposomes are spherical droplets with a lipid bilayer; their structure (pictured in Figure 6) enables them to cross the blood brain barrier and enter the central nervous system [20]. Liposome delivery offers benefits over systemic drug delivery. For one, it reduces the amount of drug that needs to be administered. When neurological drugs are delivered systemically, they are diluted in the peripheral tissues [21]. More drug therefore needs to be delivered to exert the desired effect in the central nervous system. Delivering drugs in liposomes minimizes dilution, allowing smaller doses to have the desired effect. Smaller doses also have the added benefit of reduced toxicity [21]. In addition to smaller doses, liposomes offer more targeted delivery. They can be designed to target specific cell types within the central nervous system [21]. Overall, they enable a smaller amount of drug to be delivered exactly where it needs to go.

Liposomes are currently being used to deliver potential ALS drugs, such as H-Ferritin.

H-Ferritin is an iron sequestration protein that reduces oxidative stress [21]. As with many

neurodegenerative diseases, oxidative stress is commonly seen in ALS and can result from iron dysregulation [21]. H-Ferritin has ferroxidase activity that aids in the oxidation of iron to limit its toxicity [21]. When H-Ferritin was encapsulated in liposomes and delivered to ALS mice, it delayed disease onset and prolonged survival [21]. Additional findings of the study were perhaps more interesting. Researchers also tested the effects of delivering empty liposomes to ALS mouse models. Curiously, they found that empty liposomes also significantly delayed disease onset and extended life [21]. These results suggest that liposomes themselves may have therapeutic benefits rather than H-Ferritin.

Future studies must aim to better understand the mechanism of action of liposomes. If liposomes were indeed therapeutic, it is possible that empty liposomes could be an ALS treatment.

In addition to liposomes, viruses can also deliver ALS drugs to the central nervous system.

Viral delivery is particularly appropriate for gene therapies. Adeno-associated viruses (AAV) can be used to administer RNA-based drugs, like the short-hairpin RNAs (shRNA) previously discussed [22]. When these viruses are injected into the spinal cord below the pia mater (subpial injection), they distribute throughout the entire spine [22]. Once distributed, they induce the expression of the whatever genetic material they contain. When these genes are expressed locally, they can induce their desired effect. This viral delivery technique has been tested in shRNAs that silence the SOD1 gene. When SOD1-shRNA was delivered via an adeno-associated virus, it was found to be safe and effective in preserving motor function and offering neural protection [22]. The next step in advancing viral delivery will be determining ways to deliver viruses systemically so that spinal injections are not needed.

FUTURE DIRECTIONS The future of ALS treatment is promising given the number of emerging therapies.

However, all the therapies outline in this review are still in preclinical development. It remains to be seen if they will demonstrate the same positive results in humans that they did in animal models.

Only then can these candidates be considered for FDA approval. The FDA approval rate for drugs that enter clinical development is around 16% [23]. Unfortunately, this number is much lower for ALS drugs. In the past 50 years, more than 50 potential therapies for ALS have been tested in randomized controlled clinical trials [23]. Of those tested, only two were approved.

There are many possible explanations for the low rate of ALS drug approval. It could be attributed to the complexity of the disease.

There is a poor understanding about the main 39

DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

ALS mechanism of motor neuron degeneration which is a major barrier to drug development [23].

The high failure rate could also be attributed to poor clinical trial design. There is not a standard ALS biomarker to measure in clinical trials.

Consequently, most clinical trials test subjective clinical effects rather than objective biomarkers [23]. To remedy these two problems, there needs to be more research into both the mechanisms of ALS as well as potential biomarkers for the disease.

Towards a Better Understanding of ALS. The high failure rate of ALS drugs in clinical trials should be a red flag to scientists. Solving this problem will require a shift in how scientists approach ALS research. To start, there should be a greater emphasis on basic science over applied science. Basic science drives applied science; it provides the foundation knowledge that can then be translated to solve real world problems [24]. Without basic science, applied science has no basis. For ALS, basic science should focus on the life and death of motor neurons. This knowledge should lead researchers towards a more definitive understanding of the disease mechanism and ultimately better therapies.

While transitioning to basic science, scientists should also pay increasing attention to failed studies. Null results are not as exciting as positive outcomes, but they are equally as valuable. For example, a recent study on the novel compound CLR01, a protein assembly inhibitor, failed to demonstrate positive results in a preclinical trial [25]. This drug targets aggregated SOD1 protein, which was believed to cause motor neuron degeneration [25]. CLR01 significantly decreased levels of misfolded and aggregated SOD1 protein, but it had no effect on survival, disease onset, life span, or motor function [25]. These finding suggest that SOD1 aggregation may not be the direct cause of motor neuron death [25]. Even though this study did not produce a positive result, it provided valuable information about the pathology of SOD1-related ALS. The results of this and other null trials can advance the understanding of ALS and should not be overlooked.

Identifying Clinically Relevant Biomarkers. There is a pressing need to identify relevant biomarkers for ALS. Given the diverse pathologies seen in the disease, it has been difficult to identify a single biomarker that can be used in a clinical setting. The lack of a biomarker is likely contributing to the high failure rate of ALS drugs in clinical trials. It is possible that current clinical measures are not sensitive to the effects of ALS drugs [23]. This would mean that clinical trials cannot detect the benefit of a drug even if there were one.

FALL 2019

Improving the success rate in clinical trials will therefore require a valid biomarker.

Researchers at the National Institute of Mental Health and Neuroscience in Bangalore, India have investigated neurofilament light chain (NF-L) as a potential biomarker. This is the same molecule being used by BrainXell in their motor neuron assay previous discussed.

NF-L was found to be significantly elevated in the cerebrospinal fluid (CSF) of ALS patients compared to normal subjects [20]. In continuing their search for a biomarker, these researchers identified an even better candidate: Chitotriosidase-1 (Chit-1). Chit-1 could be a biomarker for sporadic ALS [20]. Considering that 90% of ALS patients have the sporadic form, this would apply to most patients. In their study, they found that Chit-1 is upregulated at least 15fold in the cerebrospinal fluid of ALS patients.

Although exciting, this biomarker is still limited in its clinical relevance. An ideal biomarker would be detectable in the blood rather than the cerebrospinal fluid so that spinal taps are not needed to collect samples. A biomarker that can be detected in the blood would be incredibly beneficial for future ALS clinical trials.

“Before advancing any further, there needs to be a better foundational understanding of the mechanisms of the disease."

CONCLUSION ALS is a disease of high unmet need.

Fortunately, there are a number of emerging therapies to better treat it. Scientists across the world are working to find effective treatments that target different pathological features of the disease. Simultaneously, there are new innovations aimed at expediting and improving the drug discovery and delivery process. The future of ALS treatment will require critical changes. Before advancing any further, there needs to be a better foundational understanding of the mechanisms of the disease. Until the exact causes of motor neuron death are determined, it is unlikely there will be a cure. In the nearterm, there are many promising preclinical candidates. The continued dedication of researcher to understanding and treating this disease will hopefully result in an effective therapy for patients in need. D CONTACT GRACE HERRON AT GRACE.C.HERRON.19@DARTMOUTH.EDU References 1. Peters, O. M., Ghasemi, M., & Brown, R. H. (2015). Emerging mechanisms of molecular pathology in ALS. Journal of Clinical Investigation, 125(6), 2548-2548. doi:10.1172/jci82693 2. Dennys, C.N., Likhite, S. B., Huffenberger, A., et al. (2018, November). Correlation between cellular markers, astrocyte toxicity and disease progression in ALS patients. Paper Presented at Society for Neuroscience 2018, San Diego, CA. 40

Program No. 355.05.

3. Boussicault, L., Laffaire, J., Rinaudo, P., et al. (2018, November). A combination of acamprosate and baclofen (PXT864) as a potential new therapy for amyotrophic lacteral sclerosis. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.05.

4. Afroz, T., Seredenina, T., Darmency, V., et al. (2018, November). Discovery and development of diagnostics and therapeutics for TDP-43 proteinopathies. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 188.02.

5. Selvarajah, S., Sahu, S., Moreira, A., et al. (2018, November).

Drug-like small molecules targeting catalytic multi-protein complexes that correct mislocalization of TDP-43 in ALS-FTD. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.12.

6. Javaherian, A., Castello, N., Chan, M., et al. (2018, November). Development of novel small molecule autophagy inducers for treatment of ALS. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.14.

7. Kuespert, S., Heydn, R., Peters, S., et al. (2018, November).

Targeting autopaghy in ALS by BiAgil - A specific TGFĂ&#x;RII LNA-ASO. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 209.02.

8. Marshall, K. L., Tallon, C., Kennedy, M. E., et al. (2018.

November). Pharmacological inhibition of BACE1 enhances peripheral nerve regeneration in ALS disease model. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.26.

9. Akamatsu, M., Yamashita, T., Teramoto, S., et al. (2018.

November). Target therapy for ALS with RNA aptamers -rescue of ALS phenotype resulting from loss of motor neurons with TDP-43 pathology in ALS model mice. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.04.

10. Usai, C., Milanese, M., Bonifacino, T., et al. (2018, November). In vivo pharmacological blockade of mGlu5 receptors by the negative allosteric modulator CTEP ameliorates the disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.27.

11. Jafar-Nejad, P., Powers, B., Soriano, A., et al. (2018, November). Pharmacodynamics of antisense oligonucleotides in the CNS of rodents and primates following central administration. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.09.

12. Thomsen, G. M., Likhite, S. B., Corcoran, S., et al. (2018, November). Intrathecal AAV9-SOD1-shRNA administration for amyotrophic lateral sclerosis. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.16.

13. Medina, D. X., Chung, E. P., Teague, C., et al. (2018, November). Retinoid activating nanoparticles increase lifespan and reduces neurodegeneration in the SOD1G93A mouse model of ALS. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.19.

14. Staats, K. A., Seah, C., Wang, Y., et al. (2018, November).

Apilimod rescues C9orf72 repeat expansion-induced phenotypes in vivo. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.21.

15. Lebedev, M. A., & Nicolelis, M. A. (2006). Brain–machine interfaces: Past, present and future. Trends in Neurosciences, 29(9), 536-546. doi:10.1016/j.tins.2006.07.004 16. Huang, K. T., Brandman, D. M., Saabi, J., et al. (2018, November). Multiple grasp types can be reliably decoded from the precentral gyrus of people with ALS with progressive levels of motor impairment. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 403.08.

17. Yoshida, F. and Hirata, M. (2018, November). Potential of bi-directional brain machine interface using neural recording and optogenetic neuromodulation. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.06.

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18. Jain, S., Bsibsi, M., Janus, M., et al. (2018, November).

Disease relevant in vitroassays for amytrophic lateral sclerosis in motor neurons derived from control and patient iPSCs. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 052.06.

19. Hendrickson, M., Kouznetsova, J., Zheng, W., et al.

(2018, November). ALS drug discovery via high-throughput phenotypic screening using iPSC-derived human motor neurons. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No 208.20.

20. K, V., Varghese, A. M., Shruthi, S., et al. (2018, November).

Enhanced CSF NF-L levels and deranged neurofilaments: Conjoined players of motor neuron degeneration in sporadic ALS. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 052.17.

21. Synder, A. M., Madhankumar, A. B., Neely, E. B., et al.

(2018, November). Liposome-mediated uptake of H-ferritin improves outcomes in the SOD1G93A mouse model of amyotrophic lateral sclerosis. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.02.

22. Bravo Hernandez, M., Takadoro, T., Platoshyn, O., et al.

(2018, November). A potent treatment effect after spinal subpial adeno-associated virus (AAV9) shRNA-SOD1 delivery in adults ALS SOD1G37R mice. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.10.

23. Mitsumoto, H., Brooks, B. R., & Silani, V. (2014). Clinical trials in amyotrophic lateral sclerosis: Why so many negative trials and how can trials be improved? The Lancet Neurology, 13(11), 1127-1138. doi:10.1016/s1474-4422(14)70129-2 24. Basic vs. Applied Research. (n.d.). Retrieved November 18, 2018, from http://www2.lbl.gov/Education/ELSI/researchmain.html 25. Malik, R., Meng, H., Fontanilla, C., et al. (2018, November).

The molecular tweezer, CLR01, inhibits SOD1 aggregation in vitro and in the G93A-SOD1 mouse model of ALS. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 208.25.

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A D O L E S C E N C E A N D D E V E LO P M E N T

Early-Life Stress Stress and and Adolescent Adolescent Early-Life Depression: Mechanisms Mechanisms and and Models Models Depression:

BY ANDREW BOGHOSSIAN '19

ABSTRACT Depression affects millions of individuals worldwide and has been linked to early life stress and trauma. Despite overwhelming evidence that supports the link between earlylife stress (ELS) and depression, very little is understood about the specific underlying neural mechanisms - especially in adolescents.

Studies have examined the effects of ELS on both the reward system, which centers around the basolateral amygdala and nucleus accumbens, and other cortical areas, including the norepinephrinergic locus coeruleus and serotoninergic dorsal raphe nuclei. Recent research has consequently led to the theory that depression may be the result of an imbalance of excitation and inhibition due to altered maturation rates in these key brain regions.

Future research on the link between ELS and depression may have important implications both in the treatment of depressive disorders and our understanding of the developing brain.

INTRODUCTION In recent years, mood disorders have come to the forefront of both academic research and popular discussion due to increased mental health awareness and education. Major depressive disorder, colloquially known as depression, is one of the most common mood disorders, and disabilities, affecting upwards of 200 million people worldwide [1]. Depression is characterized by a number of symptoms FALL 2019

including: constant sadness, anxiety, anhedonia, irritability, impaired decision-making, decreased motivation, and thoughts of suicide in extreme cases [2].

One of the primary risk factors for depression and other mood disorders is developmental stress or trauma, known as early-life stress (ELS) [3]. Unfortunately, ELS is relatively common and comes in many forms including childhood abuse, low socioeconomic status, and social isolation, all of which act as long-term stressors on the developing brain [36]. Such examples of ELS during adolescence have been strongly linked to the development of depression [3]. Many studies have confirmed the relationship between ELS and depression, leading to the development of paradigms utilizing ELS to induce and study depression in animal models [7]. However, the specific mechanisms through which ELS alters brain development are still poorly understood.

Many ongoing research theories posit that ELS leads to changes in a variety of neural systems including those involved in reward and emotional processing [3]. Research on ELS and its role in depression is also pertinent in our understanding of the adolescent brain, given that the consequences of ELS often lie dormant until adolescence, when they emerge as depression and mood disorders [8]. This review aims to address the specific neural mechanisms by which adolescent ELS may induce depression later in life through the modulation of reward pathways and other cortical regions.

Figure 1: An image of fluorescently labeled perineuronal nets (in red) surrounding neurons (in blue) in mouse cortex.

Source: Wikimedia Commons

“Many ongoing research theories posit that ELS leads to changes in a variety of neural systems including those involved in reward and emotional processing."

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Figure 2: An outline of the reward pathway of the brain. Of particular note in this review are the PFC, amygdala, habenula (LH), and nucleus accumbens (NAc).

Source: Wikimedia Commons

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BRAIN-WIDE CHANGES DUE TO EARLY-LIFE STRESS A majority of studies examining the impact of ELS on brain development have come from whole-brain fMRI studies [2]. These studies have found that ELS can lead to a broad variety of changes in brain structure. In particular, ELS has been associated with a smaller hippocampus, a limbic structure often associated with memory and implicated in the study of PTSD [2, 5]. Indeed, stress has long been known to alter various aspects of memory [2]. It has also been found to alter the volume of areas involved in reward and emotional processing, evidenced by an increase in amygdala volume in institutionalized children, and reduced medial prefrontal cortex volume in adults that have been mistreated as children [2, 5, 9].

Typically, imaging-based studies rely on extreme forms of ELS, such as childhood abuse, as they are the easiest to identify in a population; however, more recently studies have attempted to look at the effects of socioeconomic status, a proxy for stress, on the same brain areas. One study found that higher household income is correlated with larger cortical surface area in preadolescence [4]. Though it is difficult to attribute these cortical differences to stress specifically, stress is a likely candidate for the differences between brain development trajectories in privileged versus disadvantaged upbringings. Similar effects on cortical development were found to be correlated with childhood trauma, suggesting a similarity in the stress response evoked by trauma and low socioeconomic status [9]. Adolescents who indicated that they had difficult family lives growing up had decreased overall cortical thickness [9]. Interestingly, the middle occipital gyrus, a region associated with visual processing, was the only cortical area that was significantly thicker in adolescents with traumatic early-life experiences than controls [9]. This suggests that ELS alters aspects of perception, though little follow-up has been done on ELS and its effects on visual processing [9].

In an attempt to create a more nuanced metric for stress in the modern world, one study used neighborhood disadvantage (a combination of median-household income, education-level, and crime-rate among other factors) to examine the effects of stress on subcortical regions implicated in depression such as the thalamus, amygdala, and hippocampus [5]. Not only were children from disadvantaged neighborhoods more likely to develop depression in adolescence, they were also found to have a larger left amygdala and right thalamus than those from privileged neighborhoods [5]. Increased thalamus and amygdala volume are strongly correlated with depression, suggesting that these areas may mediate the development of depression associated with neighborhood disadvantage [5].

Despite these correlations, there exist many potentially confounding variables such as the variation in race of such study participants. Researchers examining the role of race in ELS found that African American adolescents were more likely to have witnessed or experienced violence, while European American adolescents were more likely to self-report as stressed [10]. These results seemingly upend the common association of trauma and stress, therefore requiring a new distinction between ELS and trauma. However, because of the use of self-reporting stress levels, the results may be more indicative of cultural differences between the two populations rather than actual stress response activation. Future experiments could use more objective measures to define stress, such as cortisol levels, to rule out this confounding factor. Nevertheless, ELS has been shown to alter brain volume and structure in many key cortical and subcortical areas related to depression. Although whole brain measures are useful for examining the gross effects of ELS on the brain and help confirm a link between ELS and depression, they elucidate very little about specific mechanisms of action.

ELS AND DEPRESSION: ANIMAL MODELS As previously mentioned, ELS is often used in animal models to induce depression.

Researcher primarily use a rodent-based limited bedding paradigm, which consists of providing insufficient materials with which mother rats can make their nests [8, 11]. By doing so the mother is put under stress and must leave the nest more frequently to acquire bedding [8, 11]. The mother is consequently hyper-vigilant and cortisol levels are heightened in both the mother and her pups DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

A D O L E S C E N C E A N D D E V E LO P M E N T [8, 11]. Limiting bedding has been shown to induce anxiety and depression in adolescent rats and can be measured through sucrose preference tests, forced swim tests, and elevated plus mazes [8]. Another common method of inducing depression in rats is through post-weaning social isolation [6, 7]. Rats housed alone after weaning consistently show symptoms of depression later in life [6, 7]. Despite the differences between these methods, both reliably induce depression in rats, though it is difficult to parse out social deficits from those caused by stress.

An emerging animal model of depression is social isolation in pigs [1]. Pigs are a useful animal model given their neuroanatomy and social behaviors, both of which resemble those of humans better than rodent analogs.

[1]. Pigs housed alone during development show increased helplessness, anhedonia, and dysregulation of the HPA axis, symptoms commonly associated with depression [1]. These symptoms can be treated with fluoxetine, a common antidepressant for humans, suggesting a similarity not only in behavior but also in mechanism [1]. Studies in pigs and rats have provided further insight into specific ways in which ELS may affect depression. The majority of these studies have focused on reward pathways in the brain.

THE EFFECTS OF ELS ON THE REWARD PATHWAY Symptoms of depression such as anhedonia and impaired decision-making suggest that depression is a disorder of reward pathways in the brain [12]. Dysfunction of the basolateral amygdala (BLA), nucleus accumbens (NAcc), prefrontal cortex (PFC), and habenula have all been implicated in depression [12]. These areas collectively form and influence the primary reward (mesolimbic) pathway in the brain, which drives motivated behavior through dopaminergic transmission [12, 13]. Normal functioning of this pathway relies on a delicate balance of excitation and inhibition of each of these areas (Figure 2).

The effects of ELS on inhibitory parvalbumin-expressing neurons in the BLA. The BLA is primarily associated with fear learning and expression [14]. Increased BLA volume has been associated with both stress and risk for depression [5, 14]. The primary inhibitory activity within the BLA comes from parvalbumin-expressing neurons (PV neurons), which inhibit excitatory non-PV neurons projecting from the BLA to other brain areas such as the VTA and NAcc [13]. ELS has been shown to up-regulate PV neurons in the BLA, shifting the activity balance toward net inhibition [8]. These FALL 2019

PV neurons were also found to be encapsulated in more, larger perineuronal nets (PNNs), which typically develop into adulthood [8, 15]. This effect was found to be especially prominent in females, suggesting an interaction between sex and stress [15]. PNNs thus enhance neuronal activity and stabilize neuronal connections, obstructing future plasticity and providing a possible mechanism for the impaired learning associated with depression [8, 11].

The combination of increased PV neurons and PNNs surrounding them heavily shifts the excitatory-inhibitory balance of the BLA. Interestingly, this shift was associated with an inability to express fear in juvenile and early adolescent rats, rather than the increased fear one might expect to characterize depression [8].

ThesedatamayexplainthedelaybetweenELSand depression later in life, suggesting a mechanism by which fear is suppressed by increased PV neuron activity during development. This delay could be adaptive, allowing the organism to undergo development without increased fear and other cognitive impairments [8, 11]. How impaired fear expression during development translates to adulthood depression is not wellunderstood, though it may be related to a failure to develop coping mechanisms [8].

“The combination of increased PV neurons and PNNs surrounding them heavily shifts the excitatory-inhibitory balance of the BLA."

ELS alters PFC in modulation of BLA activity. Further evidence for the BLA imbalance hypothesis of depression comes from examining projections from the cortex to inhibitory PV neurons in the BLA. GABAergic projections to the BLA inhibit the PV neurons, allowing the BLA to become hyperactive, resulting in fear and emotional expression [8, 15]. During development, the pre-limbic cortex, a major source of GABAergic BLA PV neuron inhibition, has not yet fully developed, resulting in decreased inhibition of the inhibitory PV neurons [8]. In conjunction with research on PV neurons in the BLA, this finding suggests that Figure 3: A diagram of connections within the amygdala. BLA PV neurons are represented by the yellow circle.

Source: Wikimedia Commons

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“Although direct injections of ChABC in adolescent humans may not be a viable therapeutic treatment, studying the effects of ChABC could be useful for developing drugs to treat depression in adolescents and potentially prevent its development."

Figure 4: A fluorescent image of the habenula of a mouse stained blue (lateral) and green (medial). The habenula has been implicated in ELS and depression.

Source: Wikimedia Commons

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ELS may lead to the early maturation of the BLA, which, in combination with still developing cortical input, prevents fear expression in juveniles. This potentially explains both the lack of symptoms of depression in pre-adolescents and the emergence of those symptoms in adolescence, a time when the cortex begins to further develop, re-establishing balance between the two areas.

Somewhat contradictory evidence, however, has been found in female rats.

Female rats subjected to ELS showed impaired reinforcement learning in adolescence [11].

These females showed blunted modulation of behavior to reward cues and increased modulation to negative cues when reward values shifted, suggesting a negative bias in their learning, indicative of depression [11].

During fear learning, females exposed to ELS also showed increased pupil diameter, a symptom of hyper-arousal often associated with anxiety and depression [11]. Through fluorescent microscopy researchers found that these female mice had larger PNNs in their dorsomedial PFC (dmPFC), an area known to project to and inhibit PV neurons in the BLA [11]. Furthermore, PV activity in these mice was synchronized to a greater extent [11].

Following injection of Ch-ABC (an enzyme that digests PNNs) into the dmPFC, reinforcement learning improved, confirming the role of PNNs in observed deficits [11]. If PNNs in the dmPFC are developed early as well, this complicates our understanding of suppressed depression as a result of an imbalance the BLA and PFC activity.

The role of the dmPFC implicates more complex interactions between the two areas.

For example, PNNs in the dmPFC may not increase inhibitory activity, but instead, stabilize

connections that increased synchronization and consequently increases inhibition.

Nevertheless,moreresearchmustbeconducted to fully understand this complex BLA-PFC interaction. Although direct injections of ChABC in adolescent humans may not be a viable therapeutic treatment, studying the effects of Ch-ABC could be useful for developing drugs to treat depression in adolescents and potentially prevent its development [11].

ELS and the NAcc: the effects of corticotropin-releasing hormone. The NAcc is thought to play a vital role in learning and habit formation through its place in the mesolimbic pathway [12, 13]. With dopaminergic input from the VTA, the NAcc is important for motivation and reward [12].

Some animal models of depression show that stress and depression activate VTA projections to the NAcc; this is somewhat paradoxical, as these pathways are thought to signal reward.

However, it has been proposed that depression may function as a sensitization to reward [12].

There is also evidence that subsets of the NAcc and VTA encode different aspects of reward and are not uniformly excitatory or inhibitory [12]. On this basis, many researchers turn to the NAcc as a possible center for anhedonia in depression.

Corticotropin-releasing hormone (CRH) expressing neurons in the BLA project to the shell of the NAcc and alter its neuronal activity [16]. Mice exposed to ELS in the form of fragmented, unpredictable maternal care showed increased expression of CRH in the shell of the NAcc [16]. Notably, CRH is also released as part of the stress response by the hypothalamic-pituitary-adrenal axis.

These mice also exhibited severe anhedonia, suggesting a relationship between CRH expression in the NAcc and pathological reward processing [16].

Though this study found that ELS was associated with the up-regulation of CRHexpressing neurons projecting from the BLA to the NAcc, little was done to confirm the role of CRH-expressing neurons in anhedonia.

Future studies should record the effects manipulations of this CRH pathway (such as infusing CRH into the NAcc or blocking its activity) on anhedonia and other symptoms of depression. These studies would not only confirm ELS-dependent changes within the NAcc and their role in anhedonia, but also strengthen the relationship between stress and depression due to the involvement of CRH, a known stress hormone. CRH is also implicated in the actions of the locus coeruleus, another brain region associated with depression discussed later in this paper.

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A D O L E S C E N C E A N D D E V E LO P M E N T The habenula and negative outcomes: a depression pathway? The habenula is thought to encode negative valenced outcomes via primarily inhibitory inputs to the VTA and NAcc [13]. In studies of adolescents, those with major depressive disorder were found to have higher habenula connectivity than normal adolescents based on whole-brain imaging [17]. In particular, anhedonia was positively correlated with habenula-VTA connectivity scores [17]. Interestingly, amount of anxiety was negatively correlated with the connectivity score between the habenula and the NAcc [17].

Increasing negative value input to the VTA, a reward center, logically would decrease reward; this study thus provides evidence supporting the reward hypothesis of anhedonia.

In mice with depression elicited by chronic, mild stress (CMS), the habenula was found to be hyperactive [18]. Importantly, the hyperactive neurons in habenula did not project to the dorsal raphe nuclei, another brain area associated with depression [18]. The habenula receives input from the entopeduncular nucleus; thus the entopeduncular nucleus to habenula to VTA pathway may be important for depression [18]. Future studies should examine the nature of these connections and the specific role the entopeduncular nucleus may play with regard to activity of the habenula.

In conclusion, if the habenula does encode negative outcomes, there is a significant likelihood that it is involved in the neural mechanisms underlying depression. Through modulation of the NAcc and VTA, the habenula could be responsible for anhedonia and other symptoms of depression.

ELS and reward: putting the parts together. ELS leads to many different changes in reward pathways by (1) increasing inhibition of the BLA via PV neurons, decreasing fear expression, (2) altering cortical inputs to the BLA, further dysregulating fear expression, (3) increasing CRH expression in the NAcc, and (4) increasing habenula activity [8, 11, 16, 17, 18]. The direct implications of these alterations are not fully understood, though each provides convincing evidence of how ELS may result in depression, explaining symptoms such as anhedonia, impaired decision-making, lack of motivation, and the delayed emergence of depression.

THE LOCUS COERULEUS Another brain area associated with depression is the locus coeruleus (LC). The LC is the major source of the neurotransmitter norepinephrine (NE) and is thought to mediate attention and vigilance [19]. Notably, depression FALL 2019

is often thought to involve a depletion of NE, suggesting its involvement in depression [20].

Interestingly, studies have also associated depression and anxiety with hyperactivity of the LC [20, 21]. The LC is activated by CRH, as previously discussed, which is induced by stress [20, 21]. This down-regulates the number of opioid receptors there, which mediate noradrenergic activity, causing LC hyperactivity [20].

Additionally, subjecting adult and adolescent mice to a single stressful episode is sufficient to induce electrophysiological changes in the LC via CRH release [21]. These changes include increased spontaneous firing and excitability of LC NE neurons [21]. The effects of CRH in adolescents lasted up to a week after the stressor, while the effects in adults did not, suggesting an increased susceptibility to stress in adolescence [21].

These studies show that stress directly sensitizes the LC and causes hyper-vigilance for extended periods of time, which depend on the individual’s stage of developmental. Thus the LC may play an important role in ELS-induced depression and anxiety. These data also suggest that typical depression treatments may actually worsen LC-based symptoms, exacerbating overactivity. Based on its role in vigilance, the LC seems to be a more likely candidate for anxiety than depression; nevertheless, these two disorders often have similar symptoms and have both been shown to be linked with ELS.

Figure 5: A diagram of serotonin (5HT) pathways.

Serotonin is a widespread neuromodulator released from the raphe nuclei.

Source: Wikimedia Commons

THE DORSAL RAPHE NUCLEI AND SEROTONIN The neurotransmitter most commonly associated with depression is serotonin (5HT) 46

"Through modulation of the NAcc and VTA, the habenula could be responsible for anhedonia and other symptoms of depression."

is a diffuse monoamine released by the dorsal raphe nuclei and implicated in mood and sleep [7]. Like norepinephrine, depression is often associated with low 5HT levels to the extent that every major class of antidepressant increases 5HT transmission [6]. Because the dorsal raphe is the primary source of 5HT in the brain, its dysfunction has been implicated in depression.

In male rats showing anxiety and depression after exposure to post-weaning social isolation, dorsal raphe activity was lower than that of controls [7]. In female rats, the opposite was found; dorsal raphe neurons were hyper-excitable when compared to controls.

As expected, the differences in 5HT expression between male and female rats translated into opposite behavioral effects [6]. For example, socially isolated females showed significantly more exploratory behavior than normal females, while socially isolated males showed less than normal males [6]. Such sex-specific effects of stress and isolation are somewhat common, though poorly understood [6, 7, 15].

This phenomenon can potentially be explained by the activity of SK channels on dorsal raphe 5HT neurons [7]. SK3 channels limit the activity of 5HT neurons, and are upregulated in isolated males, and blockade of SK3 channels improved symptoms of depression [6, 7]. Females did not show the same effects, suggesting that SK channels are differentially regulated in the female brain [6, 7]. Within the dorsal raphe, there is a delicate balance between excitation and inhibition that, when disturbed in either direction, can result in depression.

CONCLUSION There is substantial evidence linking ELS to depression and its symptoms, though little is known about the particular underlying neural mechanisms and substrates that alter brain function. Likely candidates include dopamine in the reward system, NE for vigilance in the locus coeruleus, and 5HT mood-alteration in the dorsal raphe nuclei [7, 12, 19]. ELS has been shown to alter the excitatory-inhibitory balance within these systems, many times inducing early maturation [7]. The notion of ELS leading to rapid maturation is intriguing, especially given the association between stressful upbringings and “growing up fast”. In conjunction these systems orchestrate a variety of large-scale behaviors, altering the activity of many complementary systems through modulation of motivation, attention, and emotion. These functions fit well with the symptoms of depression, which has far reaching effects on behavior. Only through further study of the interaction between ELS and these neural systems can a better concept of the developing brain can be achieved. D 47

CONTACT ANDREW BOGHOSSIAN AT ANDREW.S.BOGHOSSIAN.19@DARTMOUTH.EDU References 1. Menneson, S., Menicot, S., Fau, A., et al. (2018, November). Validation of a psychosocial chronic stress model in pigs. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 110.08.

2. Centers for Disease Control and Prevention (2018, April 23). Mental Health Conditions: De-pression and Anxiety. Retrieved from https:// www.cdc.gov/tobacco/campaign/tips/diseases/depression-anxiety.html.

3. Heim, C. and Binder, E. B. (2012). Current research trends in early life stress and depression: review of human studies on sensitive periods, gene–environment interactions, and epigenetics. Experimental Neurology, 233, 102-111.

4. Gonzalez, M. R., Urban, K. A., Hertring, M. M. et al. (2018, November).

Associations be-tween socioeconomic factors and brain structure in preadolescence. Paper Presented at Socie-ty for Neuroscience 2018, San Diego, CA. Program No. 281.04.

5. Bell, K., Harnett, N. G., Mrug, S., et al. (2018, November). The influenceofneighborhooddisadvantageduringadolescenceonvolume of the adult amygdala, hippocampus, and thala-mus. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.05.

6. Oliver, D. K., Inston, K., Sivakumaran, S., et al. (2018, November).

Sex-specific behavioral consequences of chronic social isolation. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 110.11.

7. Inston, K., Oliver, D. K., Sivakumaran, S., et al. (2018, November). Sexspecific effects of so-cial isolation on dorsal raphe serotonin neurons and behavior. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 110.09.

8. Bath, K. G. (2018, November). Early life stress has asymmetric effects on cortical and subcor-tical development. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 007.06.

9. McQuinn, S. (2018, November). The effects of early childhood trauma and gender on brain structure. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.03.

10. Davis, E., Goodman, A. M., Orem, T.R., et al. (2018, November).

Race, violence exposure, and the psychosocial stress response. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.02.

11. Johnson, C. (2018, November). Early life stress alters neural processing of reward and pun-ishment. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 007.07.

12. Nestler, E. J. and Carlezon, W. A. (2006). The mesolimbic dopamine reward circuit in depres-sion. Biological Psychiatry, 59, 1151-1159.

13. Volkow, N. D. and Morales, M. (2015, August). The brain on drugs: from reward to addic-tion. Cell, 162, 712-725.

14. Hölzel, B. K., Carmody, J., Evans, K. C., et al. (2010). Stress reduction correlates with struc-tural changes in the amygdala. SCAN, 5, 11-17.

15. Lim, K. Y., Santiago, A. N., Opendak, M., et al. (2018, November).

Early life abuse alters GABAergic synaptic contacts in the basolateral amygdala of juvenile rats in a sexually dimor-phic manner. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 154.17.

16. Chen, Y., Itoga, C. A., Short, A. K., \et al. (2018, November). Aberrant CRH expression in the nucleus accumbens of adolescent mice after early-life adversity: A mechanism of anhedonia? Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 315.05.

17. Ely, B. A., Xu, J. G., and Gabbay, V. (2018, November). Habenula connectivity in adolescent mental illness. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 320.11.

18. Cerniauskas, I., Winterer, J., et al. (2018, November). Architecture of habenu-la circuitry underlies a distinct stress-induced depression phenotype. Presented at Socie-ty for Neuroscience 2018, San Diego, CA. Program No. 146.25.

19. Bast, N., Poustka, L., and Freitag, C. M. (2018). The locus coeruleus– norepinephrine system as pacemaker of attention – a developmental mechanism of derailed attentional function in au-tism spectrum disorder. European Journal of Neuroscience, 47, 115-125.

20. Lovegrove, T., Borodovitsyna, O., and Chandler, D.J. (2018, November). Behavioral suscep-tibility to acute stress is associated with decreased opioid receptor expression in the locus co-eruleus. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 067.16.

21. Borodovitsyna, O. and Chandler, D.J. (2018, November). Age and stress-dependent changes in locus coeruleus physiology and anxietylike behavior. Paper Presented at Society for Neuro-science 2018, San Diego, CA. Program No. 067.17.

DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S T R O K E R E C OV E R Y

A Body of Work in Progress: the State of Stroke Recovery

BY HUY DANG '19

ABSTRACT

INTRODUCTION

Stroke is a leading cause of death and longterm debilitation worldwide. Current treatments target a limited window of recovery, during which physical and mental rehabilitation tasks are used to facilitate the process of neuroplasticity.

Neuroplasticity has been shown to rewire damaged brain areas and repair cognitive and motor deficits; however, this process has a high rate of failure and is extremely taxing on the patient and inefficient. Ongoing investigations into the structure of the neurovascular unit, a complex consisting of neurons, endothelial cells, pericytes, astrocytes, microglia, and the extracellular matrix, are elucidating each component’s critical contributions to recovery of the post-stroke brain and underlying mechanisms of action. Through neuroprotective and neuroregenerative processes, trophic factors upregulate survival mechanisms but are hindered by inflammation, excitotoxicity, and necrotic factors. This review attempts to synthesize disparate information across the field and suggest a path forward to understanding the intricate plan of neural plasticity as well as to develop novel therapeutic strategies, such as stem cell therapy and hypothermia-induction, to optimize stroke recovery outcomes.

Stroke is currently the fifth leading cause of death in the United States, claiming nearly 130,000 lives per year and accounting for one out of every twenty deaths [1]. In addition, stroke is the leading cause of adult-onset longterm disability, and its increasing incidence in younger populations is adding to its emergence as one of the fastest growing expenses in medical management [2].

The outcomes of stroke are variegated and depend on variables such as the nature, location, and severity of the initial infarct – as well as rapidity of treatment. Long-term motor symptoms can range from weakness or spasticity to complete limb paralysis, while cognitive impairments can include aphasia, hemispatial neglect, and behavioral deficits.

Most patients have lifelong difficulties with daily activities such as using the toilet, dressing, bathing, eating, and motility. Only ~25% of patients return to their prior levels of everyday activity and cognitive functioning [3].

Stroke is broadly defined as damage to the brain resulting from interruption of its blood supply; there are consequently several etiologies.

Hemorrhagic stroke (fig. 2a) occurs when a blood vessel ruptures within the brain, causing bleeding inside the skull, a rigid structure

FALL 2019

Figure 1: Hemorrhagic stroke.

Hemorrhagic strokes are one of the leading cause of disability amongst the elderly.

Source: Wikimedia Commons

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plasticity.

CURRENT STATE OF STROKE TREATMENTS, SHORT-TERM AND LONG-TERM

Figure 2: Different etiologies of stroke. (A) Hemorrhagic stroke, left. Hemorrhagic stroke is caused by bleeding in the brain that causes pressure to build up inside the skull, leading to tissue damage.

(B) Ischemic stroke, right.

Ischemic stroke is caused by a blockage of blood vessels that perfuse the brain due to a lodged blood clot.

Source: Wikimedia Commons

“There is increasing research suggesting that scientists can take advantage of and manipulate natural biological mechanisms of recovery to help patients suffering from stroke, neurodegenerative disorders, and traumatic brain injury regain functionality."

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that is unable to expand to accommodate the accumulation of blood. This can arise from trauma, bleeding disorders, cerebral aneurysms, or malformations of cerebral vasculature.

Increased intracranial pressure compresses brain tissue, damaging neurons and leading to a decline in motor and/or behavioral function. The more common ischemic stroke (fig. 2b) is caused by the formation of a thrombus, a blood clot that blocks blood flow, or a traveling embolus, a blood clot that is dislodged from its original location and becomes trapped in a distant artery.

In ischemic strokes caused by both thrombi and emboli, arteries which supply the brain with oxygen are blocked, causing hypoxia-induced cell death in brain tissue downstream of the site of occlusion [4]. Ischemic strokes are more common in the geriatric population and are often due to life-time accumulations of plaque in the blood vessels, which lead to the formation of clots.

Prodigious amounts of time and funding have been poured into stroke research due to the syndrome’s debilitating consequences, increasing incidence, and monetary burden on the medical insurance system. Treatments, including novel applications of technology and brain-machine interfaces, are currently being developedtocircumventnervoussystemdamage in order to bring agency and independence back to patients. However, there is increasing research suggesting that scientists can take advantage of and manipulate natural biological mechanisms of recovery to help patients suffering from stroke, neurodegenerative disorders, and traumatic brain injury regain functionality. However, the mechanisms underlying biological recovery have been largelyunderstudied, andendogenous processes are often hindered by other bodily processes (such as the immune response), making them difficult to harness as therapeutic tools. This review will focus on the biological mechanisms of stroke recovery, including pharmacological and non-pharmacological approaches that encourage endogenous regeneration. We will start by summarizing the current state of stroke treatment and proceed to explore up-to-date discoveries of the endogenous mechanisms that underlie functional recovery and facilitate brain

Acute interventions: “time is brain”.

Immediately after a stroke, there is a small timeperiod during which much of the underperfused tissue can be saved. As time goes on, however, more irreversible tissue damage occurs and radiates outward, causing increasingly severe neurological deficits and diminished chance of survival and recovery. Thus, acute interventions involve immediate restoration of blood flow to the brain. In the case of ischemic stroke, thrombolysis via intravenous injection of tissue plasminogen activator (tPA), a “clot-buster,” clears the occlusion and allows reperfusion. Aspirin is often taken afterwards, which significantly reduces the risk of further disability or death by decreasing chance of reinfarction [5]. Specific surgical interventions can also physically break up or remove the blockage, opening the blood vessel. However, these interventions are meant to prevent rather than reverse further damage.

Rehabilitation and long-term management. Brain plasticity, including both reorganization and compensatory processes, is the base for neurological recovery and the substrate by which physical therapy leads to motor function restoration [6]. However, this process is time-intensive and inefficient.

Constraint-induced movement therapy (CIMT) is one method of physical therapy shown to be effective in encouraging the brain to rewire its damaged circuitry. CIMT increases functionality in the lagging appendage by restraining the less-affected appendage and training the hemiplegic side. Constraintinduced language therapy (CILT) uses a similar concept and restrains communication strictly to verbal means (no writing or gesturing) for patients experiencing aphasia or speaking difficulties [7]. These therapies can potentially restore motor and certain cognitive functions, though they are extremely time-intensive and stressful to the patient. Recent research looks to bridge the gap between acute intervention and physical rehabilitation by taking advantage of endogenous biological mechanisms of neural recovery to facilitate rehabilitation.

FUNCTIONAL RECOVERY FOLLOWING BRAIN DAMAGE 88In the event of a stroke, tissue damage is most severe at the focal point of the infarct, a small localized area of dead tissue downstream of the occluded vessel. This area, called the DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S T R O K E R E C OV E R Y ischemic core, is irreparably damaged and is electrically inactive; the cells in this area are beyond repair and quickly necrotize. Around the core is a region called the ischemic penumbra (or peri-infarct region), where damage is less severe (fig. 3). These cells can carry out limited oxidative metabolism and have diminished electrical activity; however, they are still salvageable with intervention [8].

The neuronal death in the core and damage in the underperfused penumbra both contribute to the loss of function seen after stroke, but the infarct tissue affects more than the immediate area due to the projections to and from remote brain areas, which are also severed after injury.

This loss or change in function of projection areas is termed diaschisis and results from tissue hypometabolism, neurovascular uncoupling, and widespread aberrant neurotransmission.

It appears there are already mechanisms in place to aid brains recovery, including increased proliferation of neural stem cells and lowered levels of axonal regeneration. However, these mechanisms are often overshadowed by other endogenous mechanisms, which often maladaptively lead to regeneration failure [9, 10]. Functional recovery is a process encompassing three phases: 1) reversal of diaschisis and activation of cell repair, 2) functional cellular plasticity allows changes in the properties of existing neuronal pathways, and 3) neuroanatomical plasticity leads to the formation of new connections [11]. Steps two and three are achievable through normal learning and can be further enhanced by creating a supportive milieu after injury. Classic studies of stroke have focused on neuronal recovery; however, introduction of the neurovascular unit (NVU) has replaced the neuron-centric view of research on ischemic stroke. This approach places equal emphasis on the roles of neurons, endothelial cells, pericytes, astrocytes, microglia, the extracellular matrix, and their complex interactions. Under the purview of the NVU, our understanding of the endogenous plasticity process expands to a whole brain view of recovery. These processes can be readily studied in rodent and monkey experimental models with specificity to type of stroke and the type of brain matter involved (gray matter stroke vs. white matter stroke), yielding useful information.

The surviving penumbra and early post-stroke processes. Regaining neurological function after stroke is a dynamic and multifactorial process, highly dependent on the severity of ischemia. Cellular stress caused by repetitive depolarizations occurring for several hours after onset of ischemia hampers recovery of penumbral tissue. Resultant edema and inflammation from the injury also contributes FALL 2019

Figure 3: The stroke core and penumbra after cerebrovascular ischemia.

The green area represents healthy tissue that has not been damaged. The yellow area (2) is the stroke penumbra of damaged tissue that still has the possibility of recovery given timely intervention. The red zone (3) is the stroke core where the most damage occurs. Tissue in this area is dead and cannot be recovered.

to the difficulty of recovery. Surviving neurons that are damaged by catabolic processes can rapidly repair themselves and resume metabolic function; however, such neurons may still exhibit aberrant neurotransmission, partly due to dendritic spine dysfunction [13].

The penumbra shows potential for survival post-stroke, and neurons in this area display the ability to rejuvenate and regain functionality. In fact, a recent study has shown that the penumbra is critical for successful and rapid post-stroke recovery [14]. Wahl et al. induced photothrombic stroke of the forelimb motor cortex in mice and allowed a period of recovery, during which the mice improved their motor function (tested by two grasping tasks).

Optical mapping indicated a shift in location of the neural center for forelimb function to an area typically representing the hindlimb. A virus that reversibly inactivates local neurons was injected into the peri-infarct area, where the forelimb function was originally mapped.

Wahl et al. noticed a decline in the extent to which motor performance was regained, indicative of the functional relevance of the penumbra in recovery and re-establishment of motor engrams. Thus, the penumbra not only represents an area with salvageable tissue, but also may play a critical role in network plasticity and relearning post-stroke. Further investigation of this phenomenon could support the idea that damaged tissue is necessary for neural plasticity.

Angiogenesis: the role of vasculature in neuroprotection and neurorestoration.

Angiogenesis, the formation of new capillaries from existing vessels, is upregulated after ischemic stroke, and has beneficial effects such as the restoration of blood flow to hypoxic tissue, neurorestorative effects including NSC recruitment, sustained cell survival, upregulation of repair processes, and cleanup of necrotic brain tissue. Angiogenesis has even been proven to strengthen the blood brain barrier via the proliferation of new endothelial cells that prevent incoming contaminants from crossing into the brain. Numerous links have

Source: Wikimedia Commons

“The penumbra shows potential for survival post-stroke, and neurons in this area display the ability to rejuvenate and regain functionality."

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“Pericytes are a relatively unstudied cell type in the brain, often found wrapped around the endothelial cells that line capillaries and venules throughout the body."

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been made between the blood concentration of vascular growth factors and neural plasticity following stroke; several of these growth factors are involved in guiding the migration of newborn neurons [15]. Caveolin-1 (Cav-1), expressed in endothelial cells, connective tissue, and adipocytes, are best known for their role in creating invaginations of the plasma membrane for endocytosis, transcytosis, and exocytosis, although many functions of these growth factors are unknown. Blochet et al. found that Cav-1 knockout (KO) exhibited more severe post-stroke outcomes, evidenced by larger lesion volumes (97% increase), lower behavioral scores decreased angiogenesis, and modified astrogliosis (changes in astrocyte morphology), leading to new interest in Cav-1 as a modulator of ischemic recovery [16].

Pericytes are a relatively unstudied cell type in the brain, often found wrapped around the endothelial cells that line capillaries and venules throughout the body. Their classical role is to regulate blood flow and, in the brain, sustain the blood brain barrier [17]. Pericyte proliferation – especially in the ischemic core and away from vasculature – has been found to be upregulated following stroke [18]. It has thus been proposed that pericytes may contribute to glial scarring, the result of a mechanism that protects damaged tissue and begins the healing process.

NSC migration after stroke and the contribution of angiogenic factors. NSC migration to the area of the OB occurs even in the absence of the OB, suggesting that their migration is not target-mediated [19]. Thus, the underlying neural mechanism guiding NSC divergence from their migratory path during injury has been largely unknown.

Recently, however, new evidence has pointed to the possibility of post-stroke progenitor cell migration depending on migratory cues from the remodeling vasculature around the injury site [20]. Ischemic injury seems to initiate a limited endogenous repair program, generating new vasculature and stimulating neuron and glia genesis. Ongoing research has identified novel neurovascular signaling systems that appear to regulate neural repair after stroke, enhancing the brain’s limited capacity to regenerate after insult. Via whole-genome sequencing, Abduljawad et al. found novel candidate ligands that were upregulated in vascular endothelial tissue immediately following stroke. Similarly, the corresponding receptors of these ligands were found to be upregulated in NSCs seven days after stroke, expanding the pool of previously discovered signal ligands involved in neural progenitor recruitment [21]. The chemokine stromal-derived factor 1 (SDF1) and angiopoietin-1 (Ang1) are examples of such

ligands and are induced in peri-infarct blood vessels. They were found to serve as trophic signals for migrating neuroblasts localized to the ischemic area.

Fujioka et al. take this one step further and proposes that blood vessels increase the migratory efficiency of neuroblasts by providing the vascular structures along which neuroblasts can travel towards their destination. ß-1 integrin, a transmembrane receptor for extracellular matrix proteins, was found to have significant effects on the chain formation and blood vessel-guided migration of neuroblasts.

In vitro experiments showed that ß-1 integrin is involved in adhesion of neuroblasts to laminin coated dish surfaces (a proxy for the basil lamina, a protein network foundation for most cells and organs), providing a mechanism for efficient somal translocation during migration.

Injection of laminin-containing self-assembling gel into the post-stroke mouse brain promoted chain formation and migration towards the ischemic tissue [22]. This data suggests a mechanistic role of laminin signaling via ß-1 integrin that provides guidance for neuronal migration along vascular scaffolds, increasing the efficiency of supplying NSCs to the ischemic tissue. Extending these findings, Williamson et al. induced photothrombic stroke in mice and found that vascular patterning was altered by ablation of the compensatory NSCs that traveled to the penumbra, suggesting a bidirectional interaction between newborn SVZ-derived progenitors and the remodeling vasculature underlying post-stroke plasticity [23].

Flanagan et al. conducted a similar study combining, where an optimized 3D biomaterial scaffold made of fibrin, hyaluronic acid, and laminin reminiscent of the mechanical characteristics of CNS neurovascular niches was used to test the tight coupling between angiogenesis and neurogenesis. Exposure of the scaffold to human NSCs enhanced vessel formation, substantiating the beneficial effects of human neural cells on vessel formation. In stroke-induced rats, transplantation of neural stem cells along with the scaffold into the periinfarct area showed greater neuron survival and recruitment in the penumbral tissue when compared to rats that were only treated with transplanted NSCs. The increased number of surviving neurons translated to improved functional recovery of motor skills, which were tested by performance on rotarod tests [24]. Through both in vitro and implantation experiments, Flanagan et al. substantiated the bidirectional communication between NSCs and endothelial cells, which seem to promote the growth of the other. If verified mechanistically by other studies, refinement of these coupled signals could prove to be a powerful tool in DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S T R O K E R E C OV E R Y Figure 4: The anatomy of the neurogenic subventricular zone in the adult rodent and human brain. (A) Sagittal view of a rodent brain showing the sites of neurogenesis in the subventricular zone/olfactory bulb (SVZ/OB) system. (B) Schematic drawing of the composition and cytoarchitecture of the adult rodent SVZ. (C) Coronal view of the adult human brain showing the basal ganglia and lateral ventricles. (D) Schematic drawing depicting the cellular composition and cytoarchitecture of the adult human SVZ, consisting of four layers: Layer I – ependymal cell layer (green), Layer II – hypocellular gap, Layer III – astrocytic ribbon, containing astrocytes and migrating neuroblasts, Layer IV – transitional zone, containing oligodendrocytes and separating the SVZ from the striatum rich in neurons. RMS: rostral migratory stream; DG: dentate gyrus; LV: lateral ventricle.

Source: Wikimedia Commons

directing neuronal migration to peri-infarct/ damaged brain regions as well as promoting vascularization of underperfused brain tissue.

NSC survival depends on neurotrophic factors. Upon migration to their destination, stroke-responsive neural precursors localize near burgeoning vasculature – a neurovascular niche in which angiogenesis causally upregulates neurogenesis [25, 26]. It has been proposed that these newborn neurons contribute to functional recovery following stroke and that efforts to promote proliferation and differentiation can have beneficial effects. NSCs differentiate into both mature striatal neurons and astrocytes in the striatal penumbra, but few survive over a long time and eventually most disappear from the area [27].

During normal migration, NSCs reach their destination and integrate themselves into the OB where they differentiate to mature neurons; however, many are eliminated to prevent redundancies and false synaptic connections. Their survival is dependent on the presence of neurotrophic growth factors such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF), granulocyte colony stimulating factor (G-CSF), and VEGF (a previously mentioned endothelial vascular growth factor), all of which decrease apoptosis and oxidation, as well as suppress autophagy by stimulating expression of anti-apoptotic Bcl-2 proteins. Additionally, EPO has been found to express anti-apoptotic effects in addition to its role in angiogenesis [28, 29]. Absence of factors like the apoptosis promoting members of the FALL 2019

Bcl-2 family (BAX, BAK, PUMA, and BIM), which normally support the apoptotic cascade by stimulating the p53 tumor suppressor gene, decreases cell death and increases survival.

Interestingly, conflicting results have been found regarding the regulatory function of p53, an important cell cycle and apoptosis regulatory factor. Although its presence upregulates cell death, its complete absence (knockout) has been found to paradoxically cause apoptotic brain lesions making its role ambiguous and a topic of future research [28]. In places like the OB, neuroblasts and newly differentiated cells must compete for growth and survival factors; thus, only 30 to 70 percent of migrating cells survive when attempting to form synaptic connections and integrate themselves into existing neurocircuitry. In a normally functioning system, many cellular factors that moderate cell death and survival fine tune and optimize the brain’s circuitry and functionality. During tissue injury, however, activated glial cells proliferate and secrete anti-apoptotic molecules like nerve-growth factor (NGF), glial-cell derived neurotrophic factor (GDNF), and NT-3, supporting neuron survival and recovery [30].

Activated astrocytes contribute to recovery by decreasing the amount of excitotoxic glutamate released by damaged and dead cells [31].The role neurotrophins play in increasing neurogenesis and cell survival make them a promising area of research in stroke treatment. Even so, our understanding the interactions of these growth factors and endogenous recovery processes is dubious at worst and germinal at best.

“NSCs differentiate into both mature striatal neurons and astrocytes in the striatal penumbra, but few survive over a long time and eventually most disappear from the area."

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Figure 5: Pi3K/AKT pathway.

The Pi3K/AKT pathway promotes neuron survival, maintenance, cellular growth, and proliferation of neural stem. Several growth factors can stimulate this pathway and cause cellular proliferation.

Source: Wikimedia Commons

“Many signaling pathways have also been implicated in neural stem cell proliferation, migration, differentiation, and maintenance."

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Recent studies on penumbral recovery paint an even more complex picture in which different isoforms and precursors of neurotrophic factors can impose opposing effects. For example, proBDNF has neurodegenerative effects while the mature BDNF has neuroprotective effects [30].

This complication may explain why different studies, which do not differentiate between forms of BDNF, show contradicting results with regards to the effects of BDNF on recovery.

Furthermore, neurotrophic efficacy is highly dependent on the availability of receptors on target cells, the presence of coreceptors and cofactors, and genetic variation. Furthermore, neurotropic efficacy may be mediated by either neuroprotective or injury promoting factors, resulting from complex biochemical and molecular cascades triggered by stroke and traumatic brain injury. Utilization of techniques to parse these components will ultimately contribute to the creation of a supportive neurogenic environment that allows optimal cell growth and survival. Despite these complications, a promising starting point in establishing a role for neurotrophins in stroke recovery comes from a recent study showing concurrent rise in serum pro-BDNF and cognitive function following rehabilitation therapy [32].

At the very least, pro-BDNF levels can serve as a biomarker for cognitive function and depressive mood in stroke patients; at best, measuring proBDNF levels could serve as a preliminary step in establishing the neuroprotective effects of neurotrophins after stroke.

Growth-promoting pathways. Many signaling pathways have also been implicated in neural stem cell proliferation, migration,

differentiation, and maintenance. Enrichment of the Pi3K/AKT pathway (fig. 5), important for neuronal survival, maintenance, cellular growth, and proliferation of NSCs, leads to a robust increase in post-stroke neurogenesis though its role in NSC differentiation has been a subject of debate. BDNF, FGF, transforming growth factor ß (TGF-B), insulin-like growth factor (IGFB), and SDF-1α all activate the Pi3K pathway and have been shown to enhance brain ischemia-induced NSC proliferation and prolong survival [28, 33]. Upregulation of Pi3K via SDF-1α binding its receptor has been shown to enhance NSC migration towards the site of SDF-1α production, suggesting a critical role of the Pi3K pathway in mediating NSC migration in addition to its effects in promoting proliferation and maturation of neurons.

Notch signaling has a fundamental role in many cellular processes, and context dictates downstream effects including cellular proliferation, differentiation, or cellprogrammed death [34]. The effects of Notch signaling are critical but only play a small role in the multifactorial process of NSC maturation in the post-stroke brain; furthermore, the pleiotropic nature of Notch dictates that only the correct developmental context will elicit desired effects. For example, Li et al. illustrated that the blockage of Notch signaling by DAPT can rescue cerebral hypoperfusion, reduce penumbral apoptosis, and reduce infarct size, leading to improved neurological and cognitive function [35]. One possible explanation for the unexpected results of this experiment could be an interaction between Notch and signals from other pathways, illustrating the importance of keeping these complex interactions in mind when investigating cerebroplastic mechanisms or developing therapeutics. Oftentimes, small changes can lead to larger divergent effects.

Like Notch signaling, Wnt signaling is a conserved pathway involved in neural progenitor proliferation and lineage specification during development, though its mechanism of action post stroke has not been well elucidated. Wei et al. used Wnt-3a intranasal treatment on strokeinduced mice, and Wnt signaling seems to reduce infarct size. The intranasal treatment also upregulated production of BDNF, increasing proliferation and migration of neuroblasts from the SVZ [35]. Through promotion of BDNF, Wnt-3a signaling seems to act in both a neuroprotective role and as a regenerative factor for the treatment of ischemic stroke. The Sonic hedgehog (Shh) signaling pathway seems to have similar effects as Wnt signaling by mediating enhanced neurogenesis and cortical remodeling; however, this pathway has also been understudied in the context of ischemic stroke and its unique properties and underlying DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S T R O K E R E C OV E R Y mechanism in the peri-infarct tissue are still being uncovered [36].

Further elucidation of these signaling pathways may prove beneficial to understanding the complex orchestration of mechanisms underlying plasticity. Neurotrophic factors influence the various signaling pathways in yet undiscovered ways, and these pathways in turn modulate different aspects of the growth factor production and efficacy. Taking one step back, this bidirectional influence has a functional effect on the cells that receive and process the signals, increasing or decreasing movement towards or away from ischemic regions. Both cell genesis and angiogenesis are tightly coupled, and their interplay will be difficult but important to understand in the development of effective therapeutic options to treat the diversity of stroke cases.

PHARMACOLOGICAL AGENTS AND EXPERIMENTAL THERAPEUTICS Our efforts to unveil the underlying mechanisms of neurovascular regeneration has pushed the limits of discovery and enabled scientists to develop therapeutic techniques that combat ischemic stroke and neurodegenerative damage. Much of this research is in line with the goal of finetuning endogenous repair programs, which can themselves facilitate functional recovery. Creation of a growth promoting environment can be induced with pharmacological Induced hypothermia prevents reperfusion injury. Recent clinical trials have shown that delayed thrombectomy/ thrombolytic treatment can be beneficial for a subset of stroke patients. While re-establishment of blood flow to the ischemic area ensures sustained cell survival and repair processes, reperfusion injury is a well-documented cause of brain deterioration following ischemic insult. Rapid restoration of circulation causes inflammation and oxidative damage via induction of oxidative stress rather than (or concurrent with) restoration of normal function.

Thus, slow reperfusion treatments may alleviate the damage caused by rapid reperfusion. Zhao et al. showed that pharmacologically induced hypothermia reduced post-stroke inflammation, was protective of the blood brain barrier, and reduced functional impairment. After 3 days post reperfusion treatment, infarct volume and neuronal cell death was significantly reduced [37].

Immunotherapy promotes intrinsic recovery. Neurite outgrowth inhibitor Nogo-A has negative regulatory effects on angiogenesis during development through its interaction with sphingosine-1-phosphate FALL 2019

receptor-2 (S1PR2), which restricts both neural and vascular growth. Interestingly, Nogo-A or S1PR2 are present at high levels in the injury zone following stroke, detrimentally inhibiting growth of new vasculature and neuronal migration. After stroke, increased inflammation coupled with high levels of Nogo-A decreasing vascularization and neuron growth results in necrotic lesions. Genetic deletion of either Nogo-A or S1PR2 results in increased levels of vascular regeneration in the peri-infarct zone and improved functional performance on motor function metrics [38]. Rust et al. introduced antiVEGF antibodies in Nogo-A-deleted animals, which decreased angiogenesis and nullified the effects of Nogo-A knockout. The ability to improve perfusion after ischemic injuries by targeting Nogo-A with immunotherapy illustrates the viability of immuno-targeting as a method of inducing a proliferative environment.

Nih et al. extend this line of thinking by injecting (1) antibodies targeting angiogenesis inhibiting genes and (2) therapeutic VEGF, which increases angiogenesis. Doing so results in decreased inflammation and significantly increases vascularized networks of regenerated functional neuron networks [39]. This work lays the groundwork for the future implementation of dual immuno-modulator and angiogenic factors to repair damaged tissue.

Stem cell treatments. Although there are several mechanisms at play in the process of brain regeneration, treatment options and normal brain repair tend to fail in promoting recovery. This mechanism of failure is largely unknown but has been proposed to be due to inflammatory immune responses, edema, and a loss of trophic support mediating the failure of endogenous repair processes. Tobin et al. administered mesenchymal stem cells (MSCs), which are known to secrete trophic factors as well as dampen immune system activation, into the ischemic regions of stroke induced rats. Subjects administered the MSCs and activated forms of MSCs had rapid and sustained improvement in both sensory and motor function [40]. One week post stroke, the MSC-treated mice showed decreased levels of microglia activation normally sustained 3 weeks after stroke, indicating that functional recovery resulted primarily from decreased inflammation.

Microglia in MSC treated mice showed decrease in levels of pro-inflammatory cytokines like IL-6 and TNF-Îą and an increase in pro-regenerative IL-10 and IL-4 up to 7 days in culture. Taken together, this constellation of findings suggests that MSC treatment has regenerative effects via decreasing inflammatory response as well as increasing oligodendrogenesis, allowing for the myelination of novel axons.

“Our efforts to unveil the underlying mechanisms of neurovascular regeneration has pushed the limits of discovery and enabled scientists to develop therapeutic techniques that combat ischemic stroke and neurodegenerative damage."

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FUTURE DIRECTIONS FOR RESEARCH

“Interactions between the different systems of neurogenesis, angiogenesis, pericytes, and the contributions of other factors involved in recovery are only starting to be parsed. "

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Neural regeneration is a complex and extremely rich web of intracellular and intercellular communication involving a wide variety of cell types and an even greater number of signals. Each signal has several different functions, leading to cascades of cellular events that together orchestrate angiogenesis, neural and glial proliferation and recruitment, pro- and anti-inflammatory responses, cellular differentiation, apoptotic and survival signals, and hundreds of other processes that are subject of ongoing investigation in the regeneration of the brain post-injury. Though our current therapeutic strategies are limited to rehabilitation during a critical window of recovery, an exuberant body of work is emerging using endogenous targeting and strategic manipulation of the intrinsic repair mechanisms to create an environment of proplasticity, enhancing our own body’s internal regenerative properties. Many preliminary trials are looking at therapeutic angiogenesis, stem cell transplants, and immunological strategies to decrease inflammation and the destructive processes that occur after stroke.

Though none of these studies have progressed to human trials, prospects for the development of effective treatments for stroke victims look very promising with evidence of expedient recovery in murine and primate models.

However, our knowledge of the intrinsic recovery processes of the brain is still in its infancy, and our understanding of NSC proliferation, survival, and differentiation are still limited, so our ability to extrapolate these findings to neurodegenerative and neurotraumatic models is also limited.

Interactions between the different systems of neurogenesis, angiogenesis, pericytes, and the contributions of other factors involved in recovery are only starting to be parsed.

Studying the factors that are released and that affect this bidirectional system will naturally progress towards more detailed understanding of the underlying signaling pathways that affect these changes. Important also will be the elaboration and confirmation of the roles that pericytes, oligodendrocytes, microglia, and astrocytes play in post-stroke recovery as integral parts of the neurovascular unit.

Their growth-permissive mechanisms could prove to be integral in permitting recovery by neurovascular regeneration.

Pursuance of the various treatments that have already shown to be efficient in animal models may lead to a therapeutic strategy in humans ahead of complete understanding of the regenerative mechanistic picture, which

will likely take a long period of time. Fine tuning MSC therapy, hIPSC-GEP therapy, and glia to neuron transformation is a good starting point in the development of treatments for brain damage and will likely even serve as a model for other bodily recovery systems. These therapeutic developments in and of themselves will contribute information to complete the picture of brain plasticity. Though there is much work to be done, a treatment for stroke and neurodegenerative/traumatic recovery is close at hand and our understanding of the brain and its processes are ever advancing. D CONTACT HUY DANG AT HUY.Q.DANG.19@DARTMOUTH.EDU References 1.Impact of Stroke (Stroke statistics). (2018). Retrieved from http://www.strokeassociation.org/STROKEORG/AboutStroke/ Impact-of-Stroke-Stroke-statistics_UCM_310728_Article. jsp#.W-30BqeZO8p 2. Sultan, S., & Elkind, M. (2013). The Growing Problem of Stroke among Young Adults. Current Cardiology Reports, 15(12). doi: 10.1007/s11886-013-0421-z 3.Musuka, T. D., Wilton, S. B., Traboulsi, M., & Hill, M. D. (2015). Diagnosis and management of acute ischemic stroke: speed is critical. CMAJ : Canadian Medical Association journal = journal de l'Association medicale canadienne, 187(12), 887-93.

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6. Hatem, S., Saussez, G., della Faille, M., Prist, V., Zhang, X., Dispa, D., & Bleyenheuft, Y. (2016). Rehabilitation of Motor Function after Stroke: A Multiple Systematic Review Focused on Techniques to Stimulate Upper Extremity Recovery. Frontiers In Human Neuroscience, 10. doi: 10.3389/ fnhum.2016.00442 7. Carter, A. R., Connor, L. T., & Dromerick, A. W. (2010).

Rehabilitation after stroke: current state of the science.

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Antioxidants & Redox Signaling, 14(10), 1889-1903. doi: 10.1089/ars.2010.3474 9. Taylor, S., Smith, C., Harris, B., Costine, B., & Duhaime, A. (2013). Maturation-dependent response of neurogenesis after traumatic brain injury in children. Journal Of Neurosurgery: Pediatrics, 545-554. doi: 10.3171/2013.8.peds13154 10. He, Z., & Jin, Y. (2016). Intrinsic Control of Axon Regeneration. Neuron, 90(3), 437-451. doi: 10.1016/j. neuron.2016.04.022 11. Wieloch, T., & Nikolich, K. (2006). Mechanisms of neural plasticity following brain injury. Current Opinion In Neurobiology, 16(3), 258-264. doi: 10.1016/j.conb.2006.05.011 12. Katsman, D., Zheng, J., Spinelli, K., & Carmichael, S. (2003). Tissue Microenvironments within Functional Cortical Subdivisions Adjacent to Focal Stroke. Journal Of Cerebral Blood Flow & Metabolism, 23(9), 997-1009. doi: 10.1097/01. wcb.0000084252.20114.be 13. Zhang, S. (2005). Rapid Reversible Changes in Dendritic Spine Structure In Vivo Gated by the Degree of Ischemia.

Journal of Neuroscience, 25(22), 5333-5338. doi: 10.1523/ jneurosci.1085-05.2005 14. Wahl, A.S., Achenbach, C.V., Schroeter, A., et al. (2018, November). Functional reorganization of the peri-infarct sensorimotor cortex to compensate for the fine motor skill impairment. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 385.03 15. Liu, J. (2015). Poststroke angiogenesis: blood, bloom, or brood?. Stroke, 46(5), e105-6.

16. Blochet, C. E., Buscemi, L., Clement, T., Badaut, J., Hirt, L. (2018, November). Caveolin-1 involvement in early tissue remodeling after stroke: effects on angiogenesis and astrogliosis. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 137.11 DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

S T R O K E R E C OV E R Y 17. Yang, S., Jin, H., Zhu, Y., Wan, Y., Opoku, E. N., Zhu, L., & Hu, B. (2017). Diverse Functions and Mechanisms of Pericytes in Ischemic Stroke. Current neuropharmacology, 15(6), 892905.

18. Pham, T., Carmichael, S. (2018, November). Pericyte contribution to scar after stroke. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 383.10 19.Kirschenbaum, B., Doetsch, F., Lois, C., & Alvarez-Buylla, A. (1999). Adult Subventricular Zone Neuronal Precursors Continue to Proliferate and Migrate in the Absence of the Olfactory Bulb. The Journal Of Neuroscience, 19(6), 2171-2180. doi: 10.1523/jneurosci.19-06-02171.1999 20.Koutsakis, C., & Kazanis, I. (2016). How Necessary is the Vasculature in the Life of Neural Stem and Progenitor Cells? Evidence from Evolution, Development and the Adult Nervous System. Frontiers in cellular neuroscience, 10, 35. doi:10.3389/fncel.2016.00035 21. Abduljawad, N., Brumm, A. J., Machnicki, M., Coppola, G., Carmichael, S. (2018, November). Vessel-derived signaling factors regulate neural progenitor cell responses to cortical stroke. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 054.29 22. Fujioka, T., Kaneko, N., Ajioka, I., et al. (2018, November).

Beta1 integrin signaling promotes neuronal migration along vascular scaffolds in the post-stroke brain. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 662.04 23. Williamson, M.R., Drew, M.R., Jones, T.A. (2018, November). Influence of subventricular zone-derived precursors on neurovascular remodeling after ischemic cortical lesions. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 136.03 24. Flanagan, L.A., Arulmoli, J., Phan, D.T.T., Hughes, C.C.W. (2018, November). A 3D scaffold and human cell neurovascular niche to test interactions between human neural and vascular cells in vitro and in stroke models. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 662.06 25. Ohab, J., Fleming, S., Blesch, A., & Carmichael, S. (2006).

A Neurovascular Niche for Neurogenesis after Stroke.

Journal Of Neuroscience, 26(50), 13007-13016. doi: 10.1523/ jneurosci.4323-06.2006 26. Lin, R., Cai, J., Kenyon, L., Iozzo, R., Rosenwasser, R., & Iacovitti, L. (2018). Systemic factors trigger vasculature cells to drive Notch signaling and neurogenesis in neural stem cells in the adult brain. STEM CELLS. doi: 10.1002/stem.2947 27. Wiltrout, C., Lang, B., Yan, Y., Dempsey, R., & Vemuganti, R. (2007). Repairing brain after stroke: A review on postischemic neurogenesis. Neurochemistry International, 50(7-8), 1028-1041. doi: 10.1016/j.neuint.2007.04.011 28. Lu, J., Manaenko, A., & Hu, Q. (2017). Targeting Adult Neurogenesis for Poststroke Therapy. Stem cells international, 2017, 5868632.

29. Chen, S. D., Wu, C. L., Hwang, W. C., & Yang, D. I. (2017).

More Insight into BDNF against Neurodegeneration: AntiApoptosis, Anti-Oxidation, and Suppression of Autophagy.

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Neurotrauma: The Crosstalk between Neurotrophins and Inflammation in the Acutely Injured Brain. International journal of molecular sciences, 18(5), 1082. doi:10.3390/ ijms18051082 31. Seidel, J. L., Escartin, C., Ayata, C., Bonvento, G., & Shuttleworth, C. W. (2015). Multifaceted roles for astrocytes in spreading depolarization: A target for limiting spreading depolarization in acute brain injury?. Glia, 64(1), 5-20.

32. *Chang, W., Lee, J., Lee, A., Kim, H., Kim, Y. H. (2018).

Relationship between serum levels of BDNF and cognitive function in stroke patients. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 054.11 33. Koh, S. H., & Lo, E. H. (2015). The Role of the PI3K Pathway in the Regeneration of the Damaged Brain by Neural Stem Cells after Cerebral Infarction. Journal of clinical neurology (Seoul, Korea), 11(4), 297-304.

34. Gonçalves, J., Schafer, S., & Gage, F. (2016). Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior. Cell, 167(4), 897-914. doi: 10.1016/j.cell.2016.10.021 35. Zhang, H. M., Liu, P., Jiang, C., Jin, X. Q., Liu, R. N., Li, S. Q., & Zhao, Y. (2018). Notch signaling inhibitor DAPT provides protection against acute craniocerebral injury. PloS one, 13(2), e0193037. doi:10.1371/journal.pone.0193037 36. Zhang L., Chopp M., Meier D. H., et al. Sonic hedgehog signaling pathway mediates cerebrolysin-improved neurological function after stroke. Stroke. 2013;44(7):1965– 1972. doi: 10.1161/STROKEAHA.111.000831 37. Zhao, Y.Y., Wei, Z.Z., Wei, L., Zhang, Y.B., Yu, S. (2018, November).Protectiveeffectsofpharmacologicalhypothermia against reperfusion injury after severe ischemic stroke in adult mice. Paper presented at Society for Neuroscience 2018, San FALL 2019

Diego, CA. Program No. 137.09 38. Rust, R., Gantner, C., Enzler, A., et al. (2018 November).

Nogo-A neutralization enhances VEGF mediated angiogenesis and functional recovery after stroke. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 054.04 39. Nih, L.R., Carmichael, S., Segura, T. (2018 November).

Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke. Paper presented at Society for Neuroscience 2018, San Diego, CA. Program No. 384.12 40. Tobin, M.K., Lopez, K., Pergande, M.R., et al. (2018 November). Treatment with activated mesenchymal stem cells increases long-term functional recovery following ischemic stroke via reduction of microglia activation and induction of oligodendrogenesis

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From Emotional Contagion to Altruism: Toward Animal Models of Empathy

BY MIA DRURY '20 Figure 1: Prarie voles are one of many animals capable of displaying altruistic behaviors.

Source: Wikimedia Commons

“The insula has been implicated in a wide range of processes in addition to empathy, including sensual touch and the enjoyment of music."

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ABSTRACT The role of empathy in a vast number of human conditions has been theorized about for many decades. Neural characterizations of empathy in humans have only been elucidated in the past decade. Animal behavioral and neurobiological models of empathy are further behind this trend and have only emerged in the past few years. Although new techniques are on the horizon, there is vast room for advancement in studies of basic empathic processes and modulators. This review will explore the conceptual and technological boundaries of empathic studies in both animals and humans.

An emphasis will be placed on how studies of empathy may apply to studies of altruistic behavior.

BEHAVIORAL AND NEURAL CHARACTERIZATIONS OF EMPATHY IN HUMANS Previous psychological and neuroscience research on empathy has recruited a wide variety of methods to study diverse working definitions of empathy. These definitions are empathy for pain, affective empathy, and cognitive empathy.

The neural correlates of empathy for pain and affective empathy will be explored here, as most evidence supports the conservation of these processes across species.

Empathy for pain can be considered a form of affective empathy and was the first aspect

of empathy to be explored. In 2004, Singer et al. determined that empathy for pain recruited cortical areas involved in affective pain processing, but not sensory pain processing.

These affective pain areas include the anterior insula, and anterior cingulate cortex (ACC) [1].

The insula has been implicated in a wide range of processes in addition to empathy, including sensual touch and the enjoyment of music [2]. Generally, the insula is hypothesized to play a vital role in integrating and attending to interoceptive cues, such as those related to heart rate, breathing, and muscle tension, consequently producing a subjective awareness of emotional feeling [2]. The ACC was also determined to be an important component of emotional representation networks [1]. Many experiments have built off of this foundational study to explore the close interaction between empathy and emotional representations of pain.

Humans also display empathy for other emotions. With affective empathy, an individual vicariously experiences the internal state of another individual, while knowing that the source of the emotion is the state of the other, rather than themselves [3]. A meta-analysis of 129 empathy studies recently concluded that empathy for both pain and other, nonpain related emotional states produced similar neural activation patterns [4]. Notable areas of overlap included the anterior insula, and the cingulate cortex [4]. Thus, it appears that human neural imaging studies have started to converge on core candidate regions for affective DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

A LT R U I S M A N D E M PAT H Y Table 1: Empathic processes can be conceptualized on multiple levels. At each level, emotional processing occurs to varying degrees, and may be interpreted in different ways to produce empathy or empathy related behavior. [1][4][30] Source: Mia Drury

empathy. Most human studies discussed later in this review also point to similar activation for empathy-inducing tasks.

Modulation of empathy by social context in humans. Recent studies have focused on the nuances of empathy in humans – particularly the influences of social context and personal history. Affective empathy has been studied the most extensively, and findings from such studies are relevant to the development of animal models; thus, affective empathy will be the focus of our discussion. Empathy for pain appears to be influenced by a variety of factors including prior similar experience with pain, familiarity and likability of the recipient, how individuals express their emotion or distress, and individual differences in empathic processes.

Seemingly contradictory studies have explored the degree to which familiarity with painful experiences modulates empathic responses to pain. On one hand, Yamamotova et al. demonstrated that empathetic individuals with previously painful experiences underestimated levels of observed pain in others [5]. On the other hand, EEG studies from Meconi et al. (2018) illustrated that humans who have shared similar experiences with pain will display increased empathy for each other [6]. In this study, researchers demonstrated that this type of empathy is precipitated by autobiographical memory activation, independent of any emotional judgement of the other’s pain.

The differences between these two studies may lie in the type of empathy being elicited.

Yamamotova et al. (2018) exclusively activated empathy in response to pain, while Meconi et al. required that subjects underwent a training period during which EEG patterns for painful memories were classified. Thus, the subjects would have recently recalled such memories, making them more readily available to support an empathic decision in a later task. The addition of autobiographical memory information may have provoked a more cognitive form of empathy, rather than an affective one.

Likability. How much one individual“likes” FALL 2019

another also modulates empathy [7]. A metric of likeability can be studied using a vicarious reward paradigm, where a demonstrator agent in the study becomes “likeable” by performing well on a task that rewards both the observer and the demonstrator. An agent becomes “unlikeable” if they perform poorly. When these two different agents are exposed to pain, observers will have more empathy for the agent that they “like”. fNIRs imaging revealed more activation in the premotor cortex, although the area of highest activation in these results occurs right where the anterior insula is located [8]. Likeability would be an interesting construct to analyze in a naturalistic setting, perhaps through techniques discussed later in this review.

Communicating internal state.

Empathic capacities in a demonstrator may be just as important as those in an observer.

Interestingly, empathy in one individual appears to modulate empathic responses of both the observer and demonstrator of pain. In a demonstrator, higher levels of empathy cause the observer to overestimate pain. If the observer has higher levels of empathy, they will also overestimate pain [7]. Rather than improve the accuracy of empathic understanding, it seems as though empathy serves to polarize how another person’s pain is interpreted, on the part of both the observer and demonstrator. Individuals with high affective empathy might be generally more emotive or socially communicative. Additional studies would be needed to determine how robust this effect is. Measuring the degree of emotional communication within an empathy paradigm will be important for determining how a demonstrator can affect empathic processes in an observer.

“Seemingly contradictory studies have explored the degree to which familiarity with painful experiences modulates empathic responses to pain. "

ANIMAL MODELS OF EMPATHY Animal models generally support the notion that emotional states can be shared between individuals. How and to what degree this information is communicated, instantiated in the brain, and influences behavior has captured the attention of researchers studying a 58

Figure 2: Empathic neural activation in humans, summed across multiple studies.

Both pain-related and nonpain related activation is represented. Overlap between studies occurs within the ACC and the anterior insula [4].

Source: Frontiers in Behavioral Neuroscience

Figure 3: Observational fear learning occurs in an observer when a demonstrator receives a shock [12].

Source: Frontiers in Behavioral Neuroscience

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wide variety of species. Methods of determining empathic capacity are also applicable to animal models of diseases and disorders where there is evidence of changed empathic function.

Thus far, animal neurobiological correlates of empathy have strongly aligned with human findings [8][9].

Emotional contagion as an empathic construct. Current animal models and methods appear to be pedagogically divided between those that study emotional contagion and those that study empathy. Emotional contagion is process where one individual perceives the internal state of another and exhibits an appropriate response [10]. Emotional contagion is thought to be a more highly conserved phenomenon than empathy and refers to a wide range of behaviors including mimicry, mirroring, and the perception of emotional displays [10].

In humans, infectious laughter, panic, and yawning may all be considered instances of emotional contagion. The primary question currently being addressed in non-human studies is whether all emotional contagion occurs due to mimicry of other individuals’ actions, or due

to a changed internal state that might closely relate to empathy. This question is particularly applicable to processes that produce strong subjective experiences in humans such as fear, pain, and stress. In animals, a change in internal state might point to the subjective experience of another’s emotions, rather than simply a behavioral response that reflects or replicates the emotional display of another [11]. While this question has not been completely answered, methods are being developed to access the neurobiology underlying emotional contagion.

With this information, there is potential to draw stronger conclusions regarding the communication of emotion between animals.

Thus, while emotional contagion is a broad construct, it may be a useful inroad to animal studies of empathy.

Observational fear learning as emotional contagion. Recent emotional contagion studies in animals focus on how fear responses are transferred between individuals and within groups, also known as observational fear learning. Observational fear learning occurs when animals learn to be fearful of a particular stimulus through social observation.

Human and primates learn about another’s distress through both verbal and facial signals, and this learning has been robustly investigated [1]. The neurobiology of fear and emotional contagion in non-primates has been primarily demonstrated in mice and rats (with the exception of a zebra fish study discussed in the “Candidate Neural Circuits” section) [26]. Jeon et al. (2010) demonstrated that mice will exhibit freezing behavior after observing a conspecific receiving foot shocks [9]. Jung et al. (2018) are similarly investigating the ability of observing mouse defensive behavior to trigger freezing behavior in the observer. In one experiment, a naturalistic “looming stimuli” is used to evoke DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

A LT R U I S M A N D E M PAT H Y a fear response in a “demonstrator” mouse.

The fear response is then transferred to an observer mouse, who exhibits freezing behavior upon viewing the demonstrator. Measuring the autonomic response of the observer mice revealed that the fear transference evokes a change in the observer’s internal state.

Furthermore, anatomical mapping indicates that this response is mediated by the ACC, supporting the results from the foot-shock paradigm described by Jeon et al. [9, 10]. In sum, observational fear learning is a form of emotional contagion, which may involve the activation of empathic neural circuits in nonprimate species.

Empathy from observing pain. Living with a conspecific in pain has been shown to elicit a greater empathic response in both human and rodent models. One study employed a paradigm, which paired two male mice, one of which had undergone painful reconstructive nerve surgery [13]. The study found that the cage-mate who had not undergone surgery displayed heightened pain responses on a writhing test, indicating that the experience of pain sensitivity and suffering was somehow transferred between closely interacting mice [13]. Researchers then chemically inhibited both the amygdala and the insula and were able to decrease the cage-mate’s hyperalgesia [13]. More recently, the same group measured neural activity in the cage-mates using FosB. Heightened responses were found in the basolateral nucleus of the amygdala, insular cortex, and ACC – the same areas activated in human empathic paradigms [4].

Operant conditioning has also been used to study empathy from observing pain. Contreras et al. (2018) trained rats to press one of two cued levers for food. Afterwards, in an empathy task, one of the levers would deliver a foot shock to a fully visible conspecific. Rats considered more empathetic learned to avoid the shockinducing lever. It was concluded that empathic responses are modulated by social context and increased empathic responses were correlated with increased anterior insula activity from electrophysiological studies.

A final paradigm for empathy from observing pain involves aiding a distressed conspecific. Previous studies have used a twochamber paradigm, where a rodent learns to press a lever to release a distressed conspecific into their own cage, thus gaining a cage-mate to interact with [14]. In these paradigms, it is unclear whether animals are motivated by social reward or the ability to relieve the distress of another. To eliminate the social reward aspect of the task, Cox et al. (2018) developed a threechamber apparatus where a distressed rat could be released into a visible (but separate) FALL 2019

Table 2: Vast space for analyzing empathic pairings [15] Source: Mia Drury

chamber from an observer rat pulling a chain [14]. Cox et al. found that rats quickly learned this task, indicating that empathic behaviors can be disentangled from social reward.

Additionally, the anterior insula displayed potentiated cFos activation in rats that complete this task, compared to control, home cage rats. Cox et al. are currently exploring an interesting application of this paradigm that relates to substance use disorder; preliminary result suggest that rats cannot perform this task during heroin withdrawal. Future studies might investigate whether deficits in empathic processing are a result of (or vulnerability to) underlying drug addiction and will likely focus on the insula as a region of comparison.

Pairings. Generally, many of the studies referenced can be improved upon by analyzing more diverse observer-demonstrator pairings (Table 2). Notably, female – female pairings or female-male pairings are often left out or neglected in these experiments. Such pairings are difficult due to the complexity of female hormone levels and interactions.

Future studies must attempt to make animal models a reliable tool for analyzing human behavior and neurobiology by taking sex into consideration. Differences or similarities between sexes could help target circuits where

“Living with a conspecific in pain has been shown to elicit a greater empathic response in both human and rodent models."

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“Whereas the insula is thought of as a communication center, the ACC is thought to play a role in gating and integrating top-down signals."

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hormones have differential action. Foundations for this type of analysis have begun in humans, using neuroimaging and autonomic response measurement techniques to determine whether women process emotional stimuli differently than men [16]. Neuroimaging has revealed an increase in frontal and amygdala activation in women during empathic responses; decreased parasympathetic nervous system activity was correlated with more empathic concern in women, although no such correlation was seen in men [15][16]. Future research measuring neural activation in rodents could support these findings, given the known similarities in brain regions activated by empathy in both humans and rodents. Additionally, more invasive techniques such as optogenetic and hormone manipulation could provide more intimate access to empathic neural circuitry.

Familiarity. Multiple studies have revealed that animals must share a social history with each other (such as having lived together) in order to demonstrate empathy for another.

Pairing mice with strangers tends to decrease or abolish evidence of empathy from observing pain [17][18]. Stress is thought to mediate this response, with situations involving a stranger evoking a greater stress response. Lidhar et al. (2018) investigated this phenomenon by administering corticosterone to both familiar and unfamiliar pairings of mice. Before corticosterone injections, unfamiliar pairings of mice elicited smaller empathic responses in comparison to mice that had a social history.

After corticosterone injections, however, both familiar and unfamiliar pairings of mice showed similar empathic responses [19]. Such findings corroborate human studies of empathy, where unfamiliarity induces less empathic activation.

One drawback of most animal studies in this area is that they exclusively use male-male pairings. Rogers et al. addressed the sex-paring part of this issue using the social affective preference paradigm previously described [20].

Interestingly, female rats were more likely to approach familiar stressed conspecifics, while males were not [21]. These preliminary findings bring up questions regarding how hormones alter the degree to which familiarity changes empathic responses.

Personal history. An individual’s prior experiences can also affect the empathic response. Sanders et al. (2013) demonstrated that mice having previously experienced a foot shock exhibited greater freezing behavior when observing another mouse receiving the same foot shock [39]. The reaction of the observer mouse suggests that it was able to recognize a familiar, painful situation in the observed mouse and react appropriately. Mice will not freeze as much if they experienced a different prior

stressor, indicating that observing others induced an internal state change drawing upon previous relevant learning and memory [39].

CANDIDATE NEURAL CIRCUITS UNDERLYING EMPATHY To advance our neurobiological understanding of empathy, it will be necessary to further elucidate the connectivity between candidate brain regions. Gu et al. (2013) argue that both the anterior insular cortex and anterior cingulate cortex (ACC) are necessary for emotional integration and awareness, although each has a distinct functionality [21]. The insula has long been thought to be a likely candidate for the brain region critical in integrating internal and external inputs in order to invoke a perceivable, internal state that matches the that of another (interoception) . The insula communicates with autonomic, limbic, sensory, and motor processing areas within the brain, supporting the idea that the insula acts as a center for cortical integration as a part of emotional processing [21].

Whereas the insula is thought of as a communication center, the ACC is thought to play a role in gating and integrating top-down signals [4]. Activity of the ACC in both rodents and humans has been associated with a wide range of empathy-inducing scenarios [4][22] [23]. Recent genetic research suggests that a specific population of neurons within the ACC may be responsible for fear contagion in mice.

Kim et al. (2018) found that selective deletion of the Nrxn3 gene in ACC somatostatin-expressing interneurons greatly increases observational fear acquisition in mice. Optogenetic manipulations, there is evidence that these neurons are an integral part of an inhibitory circuit in the ACC responsible for gating top-down inputs involved in observational fear learning [23].

If the anterior insula and ACC represent a minimal set of regions involved in empathy, how do other brain regions interact with these areas to generate a diversity of empathic responses?

In addition to generating emotional awareness, this empathic neural circuit would likely be used to calculate an emotional value and assign the given emotion to another individual, rather than the self. Such emotional calculations could either occur solely within the anterior insula or in a larger circuit involving higher level cognitive areas. Although animal studies support the idea that insula activity is necessary for generating empathic responses, it might not be sufficient.

Contreras et al. (2018) have demonstrated that the anterior insula can be activated solely by ultrasonic vocalizations of a conspecific in distress, despite the fact that animals will not respond to vocalizations alone [18]. This finding provides compelling evidence of some type of DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

A LT R U I S M A N D E M PAT H Y empathic-action inhibition that occurs in a brain region other than the anterior insula.

Determining the degree to which the anterior insula is solely responsible for generating emotional awareness could have important implications for the study of consciousness, in addition to empathy.

There is also evidence that empathic circuits and stress systems have a reciprocal relationship.

Increased activation of stress systems may inhibit empathic processing. Researchers hypothesize that effects of familiarity on empathic processing occur as a result of the stress induced by socially interacting with non-familiar individuals [24]. Social ostracism consequently impairs empathy and is correlated with increased basolateral amygdala functional connectivity [25]. In both interactions with unknown conspecifics, and situations of social ostracism, increased stress levels seem to be contributing to decreased empathic abilities. Paradoxically, decreased stress responses are also associated with psychopathy and deficits in empathic processing [20]. Thus, empathic circuits likely have a dynamic interaction with stress systems.

Due to this delicate relationship, disturbances in the stress response result in impaired empathic abilities. Manipulations of stress systems in animal models could help determine the nature of the stress-empathy interaction. For example, Lidhar et al. (2018) investigated the regulation of empathic activity by glucocorticoid receptors in pre-limbic areas. It was found that blocking glucocorticoid receptors in the prelimbic cortex increased empathy during interactions with unfamiliar mice, while activating receptors with corticosterone decreased empathy during interactions with familiar mice. These results suggest a top-down stress-related gating input to empathy circuits [19]. Future studies may seek to determine the specific neuronal connections between pre-limbic glucocorticoid-expressing neurons, and populations of neurons in the ACC and anterior insula.

A role for oxytocin. This review has demonstrated that social context plays a strong modulatory role in the control of empathic behaviors. Oxytocin, a neuropeptide and hormone, has been integral in the study of social neurobiology, and its role in empathy appears to be conserved across species. Given that social context is integrated into the empathic circuit, oxytocin may have a modulatory role in this circuit. A zebra fish study demonstrated that the administration of oxytocin enhances fear contagion within a school of fish, supporting oxytocin’s role in amplifying incoming social signals [26]. In rats, Contreras et al. (2018) demonstrated that the administration of oxytocin increases empathic responses, though only in response to familiar conspecifics.

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Similarly, upon seeing a familiar conspecific in pain, prairie voles display allogrooming “consolation” behaviors, and mice exhibit a similar type of investigative sniffing. These behaviors are thought to be elicited by empathic processes; knocking out oxytocin receptor impairs these empathic behaviors in both mice and prairie voles [27].

Although oxytocin has a demonstrated role in supporting social harmony, it also enhances antagonistic feelings towards non-group members – even in humans [28]. Oxytocin could serve to both amplify effects of familiarity on empathy and reduce empathetic consideration in response to strangers or outgroup members.

A role for oxytocin in humans antagonistic sentiment has become strikingly clear in the case of racial bias. Racial bias—even if unintentional—has been shown to decrease fMRI responses in the anterior insula and ACC [29]. Polymorphisms in the human oxytocin receptor gene result in differing degrees of empathic neural activation [29]. Future studies could determine whether the administration of oxytocin increases the effects of racial bias on empathic neural responses.

Figure 4: In addition to food-sharing, vampire bats engage in allogrooming, the act of cleaning, grooming, or maintaining the body of an unrelated individual.

Source: Wikimedia Commons

“Although oxytocin has a demonstrated role in supporting social harmony, it also enhances antagonistic feelings towards non-group members – even in humans."

EMPATHY PROVIDES INSIGHT TO ALTRUISTIC CAPACITIESY Altruism poses an interesting question for evolutionary biologists. Why would one individual help another at a potential cost to its own fitness? Although humans are often considered to be uniquely altruistic, evidence suggests that other species likely experience empathy and might therefore have the capacity for compassion and altruism.

Altruistic behavior is likely driven by multiple processes in addition to empathy, leading to two primary hypotheses for the proximate causes of altruism. The empathyaltruism hypothesis posits that empathy, “the capacity to have feelings that reflect the emotional dynamics of another’s situation,” drives altruistic behavior [citation].

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Figure 5: Mobile EEGs have been used in combination with other data sources, such as virtual reality (VR). As interest in naturalistic behavioral studies grows, VR technology will likely improve, potentially approaching the spatial imaging abilities of fMRI. Source: Michael Gaebler (2019)

Table 3: Altruism-generating scenarios for naturalistic experiments. Note: game theorists or philosophers could likely help develop the most informative virtual reality scenarios.

Source: Mia Drury

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Alternatively, seemingly altruistic actions could be driven by personal distress; altruistic behaviors would consequently work to alleviate one’s own pain by helping another [30]. In either of these cases, a variety of factors including cost to the individuals and social context (i.e. whether the distressed individual is a friend or an adversary) may still play a major modulatory role [31, 32]. Thus, studying the neurobiology of altruism likely involves the integration of social neuroscience, emotion, motivation, and reward.

Due to its complexity, the study of altruism may be greatly impacted by cutting-edge technology and techniques. The following sections will explore the interplay between developing technologies and potential future experiments in the context of altruistic behavior.

Vampire bats and altruism. Very few species seem to display purely altruistic behaviors, although several studies have revealed instances of purely altruistic behavior among vampire bats (Figure 4). For example, Carter et al.

(2013) discovered that male bats will share blood with non-kin in times of energy deprivation [33]. Blood-sharing behaviors are thought to be a form of reciprocal altruism. Individuals will share blood with others, even in the absence of direct benefit. Researchers hypothesize that these ostensibly altruistic bats may share blood to develop a broad network of social support.

Consequently, in times of unsuccessful feeding, vampire bats can rely on this network of social support to survive, thus gaining indirect fitness benefits. Studies of blood-sharing behaviors typically rely on the manipulation of social contexts and the measuring the quantity of food shared between individuals as a function of their social and genetic relationship [33].

Neurobiological analyses of blood-sharing behaviors have mirrored those used in rodent studies. For analyses, individuals must be killed immediately following altruistic interactions, preventing repeated studies of the same individual participating in future interactions.

Unfortunately, these future interactions are key to understanding reciprocal altruistic behavior.

However, Zhang et al. (2018) recently developed a neural assay, which could potentially allow for real time-brain imaging of freelyinteracting bats. With this methodology, the neural activity of vampire bats can be measured using wireless electrophysiology, which allows for unrestricted, natural movement. Researchers can also record the neural activity in two bats simultaneously, using this data to provide evidence for the convergence of neural activity between socially-interacting fruit bats [35].

Interestingly, the effects of manipulating social contexts, altering oxytocin levels, and lesioning cortical regions of candidate empathic circuits on altruistic behavior can thus be measured.

Human altruistic studies. Naturalistic behavioral experiments in humans are on the horizon of neuroscience research, with potential applications to studies of empathy and altruism.

Humans do not display stereotyped altruistic behaviors; consequently, the most informative studies will focus on unique instances of altruism in natural settings. In the near future, virtual reality will be combined with mobile neuro-imaging technology to simulate and analyze altruistic events (Figure 5). Virtual reality will potentially allow researchers to utilize both robust, emotionally salient stimuli and realistic cost variables – particularly in comparison to stimuli currently used in fMRI studies. Furthermore, altruistic decisionmaking behaviors could be analyzed in different contexts, both within and across subjects.

Table 3 provides a description of three possible scenarios, created to show the potential breadth of altruism-inducing situations that could be generated with virtual reality. In conclusion, DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

A LT R U I S M A N D E M PAT H Y virtual reality would allow researchers to generate situations that subjects would realistically find themselves in – perhaps based on their profession, income level, or personal history.

CONCLUSION Studies of empathy and altruism in humans may potentially shed light on how human society operates – especially on the individual level. Considerations of antisocial behaviors, thought to oppose empathy, may provide valuable insight into ethical issues.

Antisocial behaviors such as acts of extreme aggression play a major role in social contexts, and instances of violent behavior are often repeatedly perpetrated by individuals with psychopathy [37]. Research on neural empathic circuits and altruism may help us understand psychopathy and elucidate their underlying mechanics.

The study of empathic neural circuits and altruistic is a developing field and has only recently begun to take shape within the past decade. As this review has demonstrated, potential areas of study are numerous, and techniques will range from conventional experimental paradigms in animal models to naturalistic experiments in non-traditional animal models to innovative virtual reality technology. Studies of empathy are an in-road to greater acceptance, and investigations of altruistic behavior and may expose critical ethical inconsistencies in modern society. D CONTACT MIA DRURY AT MIA.J.DRURY.20@DARTMOUTH.EDU References 1. Singer, T., Seymour, B., O’Doherty, J., Kaube, H., Dolan, R. J., & Frith, C. D. (2004). Empathy for Pain Involves the Affective but not Sensory Components of Pain. Science, 303(5661), 1157. https://doi.org/10.1126/science.1093535 2. Critchley, H. D., Wiens, S., Rotshtein, P., Öhman, A., & Dolan, R. J. (2004).

Neural systems supporting interoceptive awareness. Nature Neuroscience, 7, 189.

3. Center for Empathy and Compassion UCSD. (2017) SEEC Research. Retrieved from: http://empathy.ucsd.edu/research/index.html.

4. Timmers, I., Park, A. L., Fischer, M. D., Kronman, C. A., Heathcote, L. C., Hernandez, J. M., & Simons, L. E. (2018). Is Empathy for Pain Unique in Its Neural Correlates? A Meta-Analysis of Neuroimaging Studies of Empathy. Frontiers in Behavioral Neuroscience, 12, 289. https://doi.org/10.3389/fnbeh.2018.00289 5. A. Yamamotova, P. Duratna, Z. Fellerova, A. Morozova; Charles Univ, 3rd Fac Med., Prague, Czech Republic. The role of empathy in own pain perception and pain estimation by others. Program No. 077.25. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

6. F.Meconi, I. Apperly, S. Hanslmayr. (2018). Empathy draws on autobiographical memories. EEG pattern classifier reveals memory reactivation in empathy for pain. Poster presented at the Society for Neuroscience Annual meeting, San Diego Convention Center.

7. M. Nakajima, S. Shimada. (2018). Enhancement of empathy for other’s pain by vicarious reward: A skin con-ductance response study. Poster presented at the Society for Neuroscience Annual Meeting, San Diego Conven-tion Center.

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Observational fear learning involves affective pain system and Ca v 1.2 Ca 2+ channels in ACC. Nature Neuroscience, 13(4), 482–488. https://doi.org/10.1038/ nn.2504 9. H. Jung, A. D. Huberman. (2018). Parsing the neural circuits for visuallyevoked emotional contagion. Poster presented at the Society for Neuroscience Annual Meeting, San Diego Convention Center.

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10. Dezecache, G., Jacob, P., & Grèzes, J. (2015). Emotional contagion: its scope and limits. Trends in Cognitive Sciences, 19(6), 297–299. https://doi.org/10.1016/j. tics.2015.03.011 11. Olsson, A., Nearing, K., Phelps, E. (2007) Learning fears by observing others: the neural systems of social fear transmission, Social Cognitive and Affective Neuroscience, 2(1) , 3–11, https://doi.org/10.1093/scan/nsm005 12. Panksepp, J. B., & Lahvis, G. P. (2011). Rodent empathy and affective neuroscience. Pioneering Research in Affective Neuroscience: Celebrating the Work of Dr. Jaak Panksepp, 35(9), 1864–1875. https://doi.org/10.1016/j. neubiorev.2011.05.013 13. D. B. Souza, R. L. Nunes-De-Souza, A. Canto-De-Souza. (2018). Empathy for pain: Heterogeneity activation of ACC, insula and amygdaloid complex in mice living with a conspecific in chronic pain. Poster presented at the Society for Neuroscience Annual Meeting, San Diego Convention Center.

14. S. Cox, C. M. Reiche. (2018). Rats will aid a distressed conspecific independent of social reward. Poster presented at the Society for Neuroscience Annual Meeting, San Diego Convention Center.

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Emotional contagion in mice: The role of familiarity. Behavioural Brain Research, 263, 16–21. https://doi.org/10.1016/j.bbr.2014.01.020 18. Contreras, M. (2018). Involvement of The Anterior Insular Cortex In Empathic Response In Rats. Poster pre-sented at the Society for Neuroscience Annual Meeting, San Diego Convention Center.

19. N. Lidhar, S. Sivaselvachandran, H. N. Turner, et al. (2018). Glucocorticoid receptor activity in the medial prefrontal cortex prevents emotional contagion in mice. Poster presented at the Society for Neuroscience Annu-al Meeting, San Diego Convention Center.

20. M. M. Rogers, A. Djerdjaj, K. B. Gribbons, J. P. Christianson. (2018). Insular cortex projections to the nu-cleus accumbens core modulate social affective behaviors. Poster presented at the Society for Neuroscience An-nual Meeting, San Diego Convention Center.

21. Gu, X. , Hof, P. R., Friston, K. J. and Fan, J. (2013), Anterior insular cortex and emotional awareness. J. Comp. Neurol., 521: 3371-3388. doi:10.1002/cne.23368 22. Shirtcliff EA, Vitacco MJ, Graf AR, Gostisha AJ, Merz JL, et al. (2009) Neurobiology of empathy and cal-lousness: implications for the development of antisocial behavior. Behavioral sciences & the law 27: 137–171.

23. A. Kim, S. Keum, J. Shin, J.-H. Kim, J. Park, H.-S. Shin. (2018). Nrxn3-dependent somatostatin-expressing neurons in the ACC control the degree of socially transmitted fear. Poster presented at the Society for Neuro-science Annual Meeting, San Diego Convention Center.

24. Gonzalez-Liencres, C., Juckel, G., Tas, C., Friebe, A., & Brüne, M. (2014).

Emotional contagion in mice: The role of familiarity. Behavioural Brain Research, 263, 16–21. https://doi.org/10.1016/j.bbr.2014.01.020 25. S. Jung, J.-H. Yoon, J. Chung, Y. Jeong. (2018). Decreased empathy and functional network alteration in a mice model of ostracism. Poster presented at the Society for Neuroscience Annual Meeting, San Diego Conven-tion Center.

26. D. Akinrinade, G. Levkowitz, R. F. Oliveira. (2018). Oxytocin regulation of social buffering and social con-tagion of fear in zebrafish. Poster presented at the Society for Neuroscience Annual Meeting, San Diego Con-vention Center.

27. S. Yada, K. Horie, S. Hidema, K. Nishimori. (2018). Analysis of empathic neural circuits regulated by oxyto-cin. Poster presented at the Society for Neuroscience Annual Meeting, San Diego Convention Center.

28. Luo, S., Li, B., Ma, Y., Zhang, W., Rao, Y., & Han, S. (2015). Oxytocin receptor gene and racial ingroup bias in empathy-related brain activity. NeuroImage, 110, 22–31. https://doi.org/10.1016/j.neuroimage.2015.01.042 29. Contreras-Huerta, L. et al. (2013) Racial Bias in Neural Empathic Respsonses to Pain. PLos One. https://doi.org/10.1371/journal.pone.0084001.

30. Feldman et al. (2015) Empathic Concern Drives Costly Altruism.

NeuroImage,105, 347–56, doi:10.1016/j.neuroimage.2014.10.043.

31. Hein, Grit, et al. (2010) Neural Responses to Ingroup and Outgroup Members’ Suffering Predict Individual Differences in Costly Helping. Neuron, 68(1), 149–60, doi:10.1016/j.neuron.2010.09.003.

32. Singer, Tania, et al. (2006) Empathic Neural Responses Are Modulated by the Perceived Fairness of Others. Nature. 439, 466.

33. Carter, G. G., & Wilkinson, G. S. (2013). Food sharing in vampire bats: reciprocal help predicts donations more than relatedness or harassment. Proceedings of the Royal Society B: Biological Sciences, 280(1753). https://doi.org/10.1098/ rspb.2012.2573 34. Oasalehm. Vampire Bat Allogrooming. Wikimedia Commons. 13 April 2017.

User:OgreBot/Uploads by new users/2017 April 13 19:30 35. W. Zhang, M. Yartsev. (2018). Correlated neural activity across the brains of socially interacting bats. Post-er presented at the Society for Neuroscience Annual Meeting, San Diego Convention Center.

36. M. Gaebler. Michael Gaebler (2019). Retrieved from http://www.michaelgaebler. com/?pageid=332 37. Harris, G. T., Rice, M. E., & Cormier, C. A. (1991). Psychopathy and violent recidivism. Law and Human Behavior, 15(6), 625–637. https://doi.org/10.1007/ BF01065856 38. Oasalehm. Vampire Bat Allogrooming. Wikimedia Commons. 13 April 2017.

User:OgreBot/Uploads by new users/2017 April 13 19:30 39. J. Sanders, M. Mayford, D. Jeste (2013). Empathic Fear Responses in Mice are Triggered by a Recognition of Shared Experience. PloS One, 8(9).

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Music and the Brain: A Call for a Therapeutic Future

BY CAROLINE PUSKAS '19 Figure 1: Research suggests that music may play an important role in the development of therepeutic techniques.

Source: Wikimedia Commons

“With a stronger understanding of music’s role and depiction in the brain, music could be better utilized in therapy to ameliorate symptoms of disorders that individuals face daily."

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ABSTRACT The representation of music in the brain differs greatly across studies, although the positive impact of music and its therapeutic quality is undisputed. Recent advances in neuroscience highlight popular hypotheses regarding the neurological mechanisms underlying the role of music in the brain, musicianship, speech and language, and even diseases and conditions including Alzheimer’s disease, fear extinction, high blood pressure, speech disorders, and PTSD. Due to music’s overarching influence in numerous disorders, the need for scientific inquiry into the neurochemical effects of music is more relevant than ever. With a stronger understanding of music’s role and depiction in the brain, music could be better utilized in therapy to ameliorate symptoms of disorders that individuals face daily.

INTRODUCTION Music makes people happy physiologically, as there is a direct causal role of dopamine in musical pleasure [1]. Thus far in 2018, the global music industry generated 51.5 billion U.S. dollars, with 19.6 billion U.S. dollar from solely the United States [2]. This desire for music and its production is rooted in the human brain, irrelevant of the location or culture of the population listening. Numerous studies are underway that investigate the effect of music and its representation in the brain, but for the most part are largely unrelated and span numerous disorders and diseases. The benefits of music are applicable to anyone and everyone. Therefore, the hunt to understand the neurological pathways and mechanisms behind

the neurological effects of music is an exciting and noninvasive opportunity to help alleviate symptoms of countless disorders through music therapy.

MUSIC AND EMOTION Although the correlation between music and emotion is uncontested, its neurological explanation is far from understood. In the study “Decoding Music Induced Emotions,” Amna Ghani addressed the neurological effect of music on emotional state. The emotional response to music in the brain was measured with both electroencephalography (EEG) recordings and 2D self-rating responses, expressing the reorganization of brain networks in correlation with changes in emotion [3]. The emotional components were continuously measured utilizing the circumplex model, which classifies emotions in two-dimensions; as a function of pleasure/displeasure (valence) and emotional intensity (arousal) [4].

With a goal of mapping out the largescale brain networks that relate to musicinduced emotions, the results of this experiment confirmed through subject-specific models and cross-frequency coupling analyses the dense connectivity of cortical regions. However, the link between these cortical regions with the rest of the brain remains unclear, thus limiting the cogency of the conclusions [3]. Further research is necessary to uncover the connections between the cortical regions and structures of the deepbrain in order to convincingly determine the large-scale brain networks resulting in musicinduced emotions.

Like Ghani, Shakil at the University of Toronto conducted a similar study. He extracted data on brain states using EEG in combination DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

MUSIC with mouse pointer data based on the musical perception of each participant [3,5]. He then mapped 2D manifolds of the EEG matrices for each subject using uniform manifold approximation and projection (UMAP). Each point of the 2D manifold represented the brain state of the subject at a particular point in time [5]. While Ghani examined the EEG findings in relation to the emotional response at particular locations within the brain, the results of Shakil’s study formulated more general clustering of EEG signals in relation to each other across thirty separate emotional pieces [3,5]. Again, this study demonstrated the relationship of music perception (mood) and corresponding brain states, but similarly was unable to link these findings further towards more specific brain states and their larger connections.

Going further, Jamal A. Williams of the Princeton Neuroscience Institute approached a similar question of the representation of music-induced emotions in the brain but pinpointed more specific higher-order cortical regions while focusing on the brain’s event segmentation [3,5,6]. In general, higher-order cortical regions correspond to the higher brain functions they enable, including cognition and behavior, both largely unique to humans [7]. With the hypothesis that “neural event transitions correspond to human-labeled song structure,” he used fMRI to record the brain activity of participants listening to 16 familiar classical and jazz songs. Next, Williams asked a separate group of subjects to mark when “meaningful” changes occurred in those songs [6]. The experiment detected activity in the higher-order brain regions mPFC and PMC when analyzing the model boundaries to human annotations.

A potential follow-up study could be conducted that uses the same set of individuals for both the fMRI recordings and human annotations rather than separate groups of subjects. As individuals do not perceive or emote in an identical manner, the analysis of overlapping fMRI recordings and human annotations would hold more value if conducted consistently across participants (Figure 3).

Williams’ acknowledgement and explanation of weaker pattern stability and differing findings from other labs in the field was a valuable addition to their poster. Being transparent about a study’s strengths and weaknesses and providing potential explanations is as a necessity for progressing research and discovery.

THE VIRTUAL BRAIN Furthermore, in “Decoding Music Induced Emotions,” Ghani proposed the next steps in further understanding music and the brain include the creation of virtual brains [3].

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Figure 2: Russell’s model of arousal and valence Source: Wikimedia Commons

His belief in the virtual brain’s ability to “capture the origin and information flow in emotion processing,” although innovative and forward-thinking, is premature. There remain tremendous gaps in the understanding of the functional human brain and its function and impact in relation to music. Thus, it is too early to embark on the electronic modeling of the brain until more of its live, physiological functions can be experimentally confirmed.

Perhaps this virtual brain is the next generation of neuronal research, although the current field of neuroscience has not quite reached an exhaustive level of understanding.

“There remain tremendous gaps in the understanding of the functional human brain and its function and impact in relation to music."

THE MOZART EFFECT “The Mozart Effect,” the argument that simply listening to Mozart’s music can improve the intelligence of an individual, abuses accepted knowledge of the relationship between music and the brain. However, it is such a pervasive myth that two posters presented at Neuroscience 2018 made the mistake of including the term “The Mozart Effect” in their poster description. L. Nuñez-Arcos presented “Improvements of cardiorespiratory parameters following music stimulation in children with autism” and solely tested the value of one song in particular, Mozart’s Sonata for two pianos in D major K448, in improving autistic children’s blood pressure [9]. Nuñez-Arcos played Mozart’s K448 sonata twice a week for a year to nine autistic child participants from five to twelve years old and measured their cardiac and respiratory frequencies along with blood pressure using an “intelligent wristband” [9]. He stated that although the heart rate of the participants would reduce to normal levels after musical stimulation only temporarily, their high blood pressure was reduced to lasting normal levels even when children were absent on vacation. Although no up-to-date research confirms the direct relationship between blood 66

Figure 3: Representation of cortical activation while listening to the brain according to the study conducted by Williams et al.

Source: Williams et al. (2018)

“This concept of music stimulating the parasympathetic nervous system introduces yet another neuroanatomical pathway affected by music, as previously mentioned experiments proposed the involvement of higher-order cortical regions in representing musicinduced emotions."

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pressure and mood, the persistent improvement in blood pressure experienced by autistic children in this experiment could in part result from an improvement in mood induced by music. Although his reasoning for mentioning the vacation was to suggest the lasting effects of improved blood pressure from musical stimulation, it is possible that the vacation itself improved the mood (and thus the blood pressure) of the participants [although this itself is debatable, as the change in routine during a vacation might be psychologically difficult for autistic children]. Perhaps Nuñez-Arcos should conduct additional research on participants with more strict and consistent time frames to support his current hypothesis, as reducing any potential sources of variation is vital.

Further, the discussion argued for the activation of the vagus nerve by Mozart’s K448 Sonata. This concept of music stimulating the parasympathetic nervous system introduces yet another neuroanatomical pathway affected by music, as previously mentioned experiments proposed the involvement of higher-order cortical regions in representing music-induced emotions. Lastly, this experiment emphasizes the great benefit of music stimulation as a means to avoid the use of additional medication with autistic children. The decreased costs and avoided side effects from additional medication make music therapy a safer and more accessible option.

Similarly, “The effect of classical music on the extinction of fear memory in rats,”conducted

by P.H.M. Oliveira, aimed “to analyze the influence of Mozart’s Sonata K.448 and Classical Music exposure on the extinction of fear memory in rats” [10]. The experiment involved 16 pregnant mice exposed to either Mozart, other Classical music, or ambient sound during the gestational period. Their male offspring later underwent a fear conditioning test, and Oliveira found the rats with musical exposure in utero were able to more efficiently extinguish aversive memory. Before this experiment can be viewed as credible in supporting “The Mozart Effect” and the power of classical music, it requires

more testing with other genres of music. The groupings in this experiment included “Mozart”, “Classical”, and “Ambient”. Why would Mozart, a classical composer, receive his own category separate from classical music? Although Mozart is considered one of the greatest composers in history, it is not as though his music is unlike other classical composers from his time. This experiment praises classical music and Mozart’s Sonata in particular without testing the effect of simply music in general. It is possible that any sound or music other than ambiance would improve fear extinction, but this study leaps to conclusions in support of “The Mozart Effect”.

Similar to Oliveira supporting the role of music in extinguishing fear memory, Hoffman argues that damage to the thalamo-amygdala pathway of the brain by a traumatic brain injury alters the sensory-emotional network processing of fear and sound [11]. Although no music was involved in this experiment, the auditory fear conditioning experiment found that white noise alone evoked defensive fear behavior in adult male rats 48 hours after Fluid Percussion Injury (FPI), while the rats that received pure tones did not display defensive behavior.

Hoffman expressed a potential explanation for this distinction in fear behavior as resulting from the range of frequency of white noise in comparison to the singular frequency of a pure tone [11]. As the brain and its relationship with music and sound is closely related to emotion, Hoffman’s findings on the emotional response of rats with traumatic brain injuries to sound shows the negative aspects of this correlation.

As Hoffman notes, the risk for post-traumatic stress disorder (PTSD) greatly increases after traumatic brain injury (TBI) [11]. This study further emphasizes the relationship between sound and emotion in the brain and highlights how sound/music can have detrimental effects under certain circumstances. These findings support the substantial effect of sound, and by extension music, on the brain particularly after traumatic brain injury (TBI).

Continuing the discussion of music’s effect on the brain, another area of inquiry DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

MUSIC is whether or not certain brain areas can dictate musicianship. T.L. Henechowicz at the University of Toronto studied the prevalence of Val66Met BDNF SNP polymorphism, a genetic mutation, in musicians and nonmusicians, as this polymorphism is associated with deficits in motor learning and plasticity [12]. He hypothesized that the population of musicians would have a reduced prevalence of the polymorphism, as musicianship includes high skill demands with performances and motor learning. He conducted genotypic and allelic frequency analysis, and tested for the Val66Met using a SNP genotyping assay for both musicians in professional training and members of the general population [12]. In the end, Henechowicz found no difference in the genotypic and allelic frequencies and no decreased prevalence of Val66Met BDNF polymorphism in musicians versus nonmusicians. For the professional musicians who had the polymorphism, Henechowicz argued that they must have exceptional musical skills to reach strong musicianship while also having the Val66Met BDNF polymorphism. Although, with the brain and all its complexities, weighing musicianship on a singular polymorphism appears to be a questionable hypothesis.

Similarly, there persists an ongoing battle of nature versus nurture with regard to musical talent. It remains disputed as to whether musicianship is mainly genetic or a talent that can be achieved through training and practice, or to what extent either factor dominates the other.

One opposition to Henechowicz’s experimental focus on the genetic basis of musicianship is the belief that there are in fact biological distinctions in auditory processing between musicians and non-musicians, but that these distinctions are a product of brain plasticity; created from musical practice and exposure rather than biologically inherited [13]. Henechowicz argues that genetics are mainly responsible for musicianship, while Kraus in the Annals of The New York Academy of Sciences argues that musical drive and repetition is the primary factor [12,13]. Kraus approaches musicianship with the mindset of top-down control over sensory processing, arguing for the shaping of the descending auditory system by interactions with the auditory environment, particularly during early childhood [13]. Numerous arguments for inherited musicianship, practice, and/or a combination of both remain disputed in the field of musical neuroscience.

MUSIC AND LANGUAGE Many theories exist concerning the locational response of the brain to music, with FALL 2019

a recurring question being the overlap between music, speech, and language. Norman-Haignere claims that distinct neural selectivities for music, speech, and song in human auditory cortex and confidently argues against prior studies claiming music’s ability to co-opt the mechanisms of speech and language [14]. His findings suggest that the fMRI creates blurred responses that falsely propose music’s overlap with speech and language [14].

Instead, Norman-Haignere measured cortical responses to various natural sounds using human electrocorticography (ECoG). With higher spatial and temporal resolution, he found clear selectivity for speech in some electrodes, and selectivity for music in other electrodes [14].

The results suggest that the anterior regions of the superior temporal gyrus create music and song-selective responses, while the middle STG expresses speech selectivity [14]. A fascinating result was the response to music with vocals, as it was not simply a summation of the music and speech selectivity in the electrodes. This finding suggests a lack of two distinct pathways at play in music and language, but it remains unclear what the neurological interaction of the two entail.

In a similar study, Sarah E.M. Faber at the University of Toronto sought to analyze the relationship between music and language. But instead of conducting a specific experiment she approached her poster as an opportunity to build a network model of musical improvisation [15]. The introduction and base of Faber’s model surrounded Cross and Morely’s theory on the evolution of music. In Communicative musicality: Exploring the basis of human companionship Cross and Morley argue that language emerged to fulfil “communicative, ostensive and propositional functions with immediate efficacy,” while music functions to manage social interactions and integrate experiential information over longer timescales [16]. With these differing original functions,

Figure 4: Representation of the auditory pathway.

Source: Wikimedia Commons

“It remains disputed as to whether musicianship is mainly genetic or a talent that can be achieved through training and practice, or to what extent either factor dominates the other."

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functions of music on the brain is invaluable in improving treatment for speech disorders.

Without a consistent hypothesis on the relationship between music and language, Faber shared her aspirations for collaborative findings with fellow musical neuroscientists. Her creation of a network model of musical improvisation was rooted in a drive to consolidate and synthesize the present information and research on musical neuroscience to make headway towards reaching a stronger understanding of the brain’s response to music. She candidly shared the hardships of working in speech therapy, a field without complete scientific proof of effectiveness. Music therapy has faced a loss of funding due to an insufficient understanding of the effect and benefits of music on the brain.

Thus, the time to collaborate in the field of music neuroscience is now.

CONCLUSION

Figure 5: A standard network model of musical improvisation by Sarah E.M. Source: Faber et al. (2018)

“Various theories assert differing opinions on the effects of music on the brain, although they all share a fundamental belief that no matter the location in the brain, music plays an impactful role and maintains a representation."

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Cross and Morley made an argument for the distinction of music and language in the brain. However, Faber shared other research

by Brown (2000) which proposed a shared auditory processing network with analogous syntactic processing between music and language before further divergence [17]. Thus, in this research approach Faber provided recent findings on the relationship of spontaneous music (improvisation) and language creation, specifying similarities and differences present in the networks.

Faber expressed the use of improvisation in music therapy with non-verbal individuals as a means for communication [15]. Without understanding the neurological mechanisms at play, music nonetheless represents an effective tool for alleviating symptoms of numerous states and disorders, including PTSD, fear extinction, Alzheimer’s, speech disorders, and more.

MUSIC THERAPY Of great significance, results show that singing can reduce the frequency of stuttering by over 90 percent [18]. Stuttering and stammering, disorders believed to manifest themselves psychologically through fears, can debilitate an individual’s ability to communicate [18]. But it is fairly well known that speech disabilities of these sort disappear when singing. Could this knowledge function as proof of an overlap of music and speech in a common auditorymotor mechanism? As the effect that musical speech therapy can have on speech disorders is incredibly significant, understanding the specific

Various theories assert differing opinions on the effects of music on the brain, although they all share a fundamental belief that no matter the location in the brain, music plays an impactful role and maintains a representation.

Although there is merit to the findings of a single experiment, the agreement of many different experiments on a certain conclusion is much more powerful. The labs of Ghani, Shakil, and Williams could have saved a great deal of time, energy, and money if there had been more open discussions to collaborate and build off of each other’s ideas and results. The role of music is not simply for listening enjoyment. Music functions as a tool doctors and scientists could harness to help people with countless diseases and disorders. But before treatment can occur, the first step is collaboration. D CONTACT CAROLINE PUSKAS AT CAROLINE.H.PUSKAS.19@DARTMOUTH.EDU References 1. Ferreri, L., & Mas-Herrero, E. (2019). Dopamine modulates the reward experiences elicited by music. Proceedings of the National Academy of Sciences of the United States of America.

2. U.S. music- statistics & facts. (n.d.). Retrieved November 17, 2018, from Statista website: https://www.statista.com/ topics/1639/music/ 3. A. Ghani1, J. Zhang, A. McIntosh, K.-R. Müller, P. Ritter.

Decoding music induced emotions. Program No. 340.20. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

4. Posner, J., Russell, J. A., & Peterson, B. S. (2005). The circumplex model of affect: an integrative approach to affective neuroscience, cognitive development, and psychopathology. Development and psychopathology, 17(3), 715-34.

5. S. Shakil, S. Faber, A. R. McCulloch, T. M. Brown, K. Shen, A. R. McIntosh. Relationship of EEG and music features through low dimensional manifolds. Program No. 340.19. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

6. J. A. Williams1, C. Baldassano, J. Chen, U. Hasson, K. Norman. Exploring event structure in music perception.

DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

MUSIC Program No. 335.06. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

7. Tranel D., Cooper G., Rodnitzky R.L. (2003). Higher Brain Functions. Neuroscience in Medicine. Humana Press, Totowa, NJ 8. A. M. Belfi, A. Reschke-Hernandez, E. Guzman-Velez, D. Tranel. Music and emotion in Alzheimer's disease. Program No. 157.04. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

9. L. Nuñez-Arcos, I. Fernandez-Lechuga, P. Carrillo, R. Toledo, M. Hernandez, J. Manzo. Improvement of cardiorespiratory parameters following music stimulation in children with autism. Program No. 368.06. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018.

Online.

10. P. H. Oliveira1, D. H. Pietrobon, L. L. S. Lemos, A. C. D. DE Andrade, et al. Effect of classical music on the extinction of fear memory in rats. Program No. 327.16. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

11. A. N. Hoffman, E. Hsieh, Z. T. Pennington, S. Watson, D. A. Hovda, et al. Projection specific mechanisms of auditory sensitivity that contribute to enhanced fear after traumatic brain injury. Program No. 413.14. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018.

Online.

12. T. L. Henechowicz1, J. L. Chen, L. G. Cohen, M. H. Thaut.

Prevalence of BDNF polymorphism in musicians: Evidence for compensatory motor learning strategies in music. Program No. 402.14. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

13. Krause, N., & Strait, D. L. (2015). Emergence of biological markers of musicianship with school-based music instruction.

The Neurosciences and Music, 1337 (1), 163-169. Retrieved from Annals of The New York Academy of Sciences database.

14. S. V. Norman-Haignere, J. J. Feather, *P. Brunner, A. Ritaccio, J. H. McDermott, G. Schalk, N. G. Kanwisher. Distinct neural selectivities for music, speech, and song in human auditory cortex. Program No. 191.04. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

15. S. E. Faber, A. R. I. McIntosh. Toward a standard network model of musical improvisation. Program No. 340.08. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.

16. Cross, I., & Morley, I. (2009). Communicative musicality: Exploring the basis of human companionship. Oxford University Press.

17. Brown S. (2000). “The ‘musilanguage’ model of music evolution,” in The Origins of Music, eds Wallin N.L., Brown B., Merker S., editors. (Cambridge, MA: MIT Press; ), 271-300.

18. Clements-Cortés, A. (2012). Can music be used to help a person who stutters? The Canadian Music Educator, 53(4), 45-48.Retrievedfromhttps://search-proquest-com.dartmouth. idm.oclc.org/docview/1022996038?accountid=10422

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The Influence of Stressors and Adverse Life Experiences on Brain Development

BY CHLOË CONACHER Figure 1: Diagram of MRI brain scans throughout human development from 1 week to 10 years.

Source: National Institutes of Health, distributed under a Creative Commons License.

ABSTRACT There are many factors that influence brain development, including stressors and adverse life experiences. Despite extensive research that demonstrates the mental, physical, and social effects of stress, as well as a wide acceptance that stress is detrimental to human health, exactly how stress impacts brain development is not well understood. Recent studies have focused on examining the brain areas that are particularly vulnerable and adaptable throughout child and adolescent development. This review focuses on integrating research about various adverse factors that could potentially influence brain development.

INTRODUCTION “Studies have found that there are differences in brain development between individuals with stress exposure, childhood trauma, and those who have not had to face similar adversities."

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Dr. Vincent Felitti and Dr. Robert Anda started their ground-breaking investigation on the effects of Adverse Childhood Experiences (ACEs) in the 1990s. This study examined ten different criteria of adverse childhood experiences, including emotional abuse, physical abuse, and sexual abuse, to determine what long-term effects occur as a result of these experiences [1]. Felitti and Anda found that a higher ACE score directly correlated with increased negative mental, physical, and social well-being later in life. For example, individuals with ACE scores of four in comparison to individuals with ACE scores of zero are “twelve times more likely to have attempted suicide, seven times more likely to be an alcoholic, and ten times more likely to have injected street drugs” [1] Since then, there have been

many variations of Felitti and Anda’s study utilizing similar ACE score criteria; as a result, awareness about the detrimental effects of stress has greatly increased. However, the scientific understanding about how brain development is influenced by stress, and therefore, experiments examining how brain development influences negative life outcomes is fairly limited. This review will focus on new research that explores a variety of stressors and how these stressors influence brain development. Various studies have focused on general stress exposure and childhood trauma as well as specific factors such as race, socioeconomic status, neighborhood quality, and substance-use. Additionally, there is research outlining potential positive factors that may influence brain development.

STRESS EXPOSURE AND CHILDHOOD TRAUMA Studies have found that there are differences in brain development between individuals with stress exposure and childhood trauma and those who have not had to face similar adversities; however, the specific differences have not been further explored [2]. Many studies have addressed cortical thickness variation in relation to stress.

Further research shows that individuals who have not endured any childhood trauma have a higher cortical thickness in the inferior frontal gyrus and the occipital lobe. This difference in cortical thickness is especially clear in the visual association area [2]. Additionally, females and males have significant differences in the cortical thickness of their fusiform gyrus which plays a role in facial recognition and social cues. These DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

B R A I N D E V E LO P M E N T Figure 2: The number of ACEs for an individual directly influences the risk of negative health and well-being outcomes.

Source: US. Department of Health and Human Services, Center for Disease Control and Prevention, distributed under a Creative Commons license.

findings give insight into the possibility that there is a relationship between sex and trauma, specifically in the fusiform gyrus [2].

In addition to visual association, facial recognition, and social cues, trauma has been found to impact different memory processes.

Individuals who have experienced more stressful life events show significantly more hippocampal activation during contextual memory tasks [3]. One study concluded that activation in the hippocampus increases during the Context Separation Completion task, but there is no confirmed relationship between stressful life events and accuracy on this test [3].

This contradicts previous research that found memory impairment in individuals with early life stress and trauma [3]. This may be true because the increased activation in the hippocampus is compensating for memory impairment.

However, this finding could also just be a result of the small sample size; the experiment only examined 18 healthy participants [3].

Those results based on increased hippocampus activation also contrasts other findings that show that individuals with major depressive disorder have decreased volume in their hippocampi [4]. Major depressive disorder has been linked to childhood adversity [4]. If childhood adversity increases hippocampus activity, then one might infer that there would be increased hippocampal volume for individuals with major depressive disorder, rather than decreased. It is important to note that the increased hippocampal activity is shown specifically in contextual memory activities, therefore this inference may be discounted by decreased activation in other types of memory activities and different hippocampal functions.

Further research found that hippocampal volume is negatively correlated with Childhood Trauma Questionnaire scores, supporting the finding that adverse experiences negatively impact hippocampus development [4]. More research needs to be done regarding the role of volume in comparison to activation of the hippocampus as FALL 2019

a result of childhood adversity. Furthermore, for the amygdala, there is a negative relationship between the basal and accessory basal nuclei of the amygdala with emotional abuse [4].

These studies broadly contribute to the understanding of stress exposure and childhood trauma. These findings include a correlation between increased cumulative stress exposure and increased hippocampus activation during contextual memory, a positive relationship between early life stress and cortical thickness, and in vivo evidence for a negative relationship between childhood adversity with hippocampus and amygdala volume [2, 3, 4]. While these studies provide a key perspective on the effects of the accumulation of stressors on brain development, they do not delve into the changes of specific factors. Researching the effects of specific factors is necessary for working towards a holistic understanding of adverse experiences and the impact of adverse experiences on brain development.

“Researching the effects of specific factors is necessary for working towards a holistic understanding of adverse experiences and the impact of adverse experiences on brain development."

RACE AND SOCIOECONOMIC FACTORS Race and socioeconomic factors both directly contribute to early life stress and adversities that individuals face throughout their lives. African Americans are regularly exposed to more violence, have lower family incomes, and live in more disadvantaged neighborhoods than European Americans [5, 6]. Although African Americans experience greater violence exposure, European Americans self-report higher stress ratings [5]. In addition to experiencing greater violence exposure, the self-reported stress ratings decrease while stress exposure increases for African Americans [5].

This negative relationship is not reflected in European Americans, but European Americans do show greater activation in the bilateral dorsal lateral prefrontal cortex, bilateral parahippocampal gyrus, dorsal medial prefrontal cortex, and the posterior cingulate cortex in response to stress stimuli in comparison to 72

Figure 3: Diagram of a brain with the hippocampus highlighted in red.

independently predict larger surface area of the left superior frontal, right superior frontal, left pars orbitalis, right pars orbitalis, and total cortical surface area [7]. In contrast to higher parental income and education, the number of environmental adversities one faces, such as neighborhood quality, is negatively associated with cortical surface area and cognition [7].

One of the environmental adversities that impacts cortical surface area and cognition is neighborhood quality.

Source: Photograph by Washington Irving, distributed under a Creative Commons license

NEIGHBORHOOD FACTORS

“The many effects of race on the brain show that a variety of factors can influence brain development and function and that life stressors are often tied to other factors, such as race and socioeconomic status."

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African Americans [5]. Additionally, African Americans show greater amygdala activation in comparison to European Americans. These findings suggest that racial differences in stress activation in young adulthood is likely caused by racial differences in adverse life experiences [5].

The amygdala plays an integral role in emotional processing, and racial differences directly impact amygdala functions. The positive relationship between adverse life experiences from racial differences with amygdala activation slightly contradicts the negative relationship between stress exposure and amygdala activation [4, 5]. Similar to contradicting evidence about hippocampus development, it is possible that amygdala activation is compensating for other developmental differences.

In addition to racial differences contributing to increased adverse life experiences, racial differences contribute to changes in neural threat responses [6]. African Americans show decreased activation in the prefrontal cortex, hippocampus, and amygdala while completing a Pavlovian fear conditioning procedure [6].

These are all areas of the brain that impact emotional response, revealing that the adverse experiences that African Americans endure minimizes emotional response to fear stimuli.

African Americans are also less likely to expect predictable threats and exhibit lower skin conductance in response to threats in comparison to European Americans [6].

The many effects of race on the brain show that a variety of factors can influence brain development and function and that life stressors are often tied to other factors, such as race and socioeconomic status. Higher socioeconomic status is associated with multiple differences in brain structure. For example, higher parental income and higher parental education both

Neighborhood quality is extremely varied across different regions, strongly influencing home lives of children. Children who grow up in disadvantaged neighborhoods have a higher likelihood of dealing with mental illness, and many of these mental illnesses, such as anxiety and depression, have been linked to the amygdala, hippocampus, and thalamus [8].

Variance in neighborhood quality has been found to impact these brain areas. The volume of the bilateral hypothalamus varies for children who experience neighborhood disadvantage, which potentially influences information processing and sensory skills [8]. Additionally, the volume of the right amygdala varies for children who experience neighborhood disadvantage, which potentially influences emotional expression and regulation [8]. Feng, et al. found contradictory evidence to Bell, et al. regarding amygdala development [8, 10].

The contradictory evidence shows that there is no relationship between amygdala volume and neighborhood quality in the positive and negative valence systems [10]. However, it is important to note that participants in Feng, et al.’s study all qualified as having mood/anxiety disorders [10]. This reveals that neighborhood disadvantage is not inherently tied to mental illness and that developmental variance correlated with mood and anxiety disorders does not align with developmental variance correlated with neighborhood disadvantage.

It is vital to conduct further research that specifically focusses on the amygdala under different conditions in order to understand the relationship between neighborhood quality and brain development.

In addition to these contradictory findings, there is minimal evidence that shows that neighborhood quality impacts reward systems.

A study looking at reward response for participants with mood and anxiety disorders found that neighborhood differences are not responsible for a variety of differences in reward response and brain activation, such as the nucleus accumbens, during reward tasks [9].

This aligns with findings that show that there is no effect of neighborhood quality on nucleus DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

B R A I N D E V E LO P M E N T accumbensvolume[10].Althoughneighborhood disadvantage may be associated with mental illness, neighborhood disadvantage is not associated with reward response differences in individuals with mental illness [8,9]. In addition to the reward system, there is no evidence that supports a relationship between neighborhood quality and volume differences in the dorsal lateral prefrontal cortex in the cognitive system [10]. There is, however, a relationship between left insula volume and neighborhood affluence.

This evidence is preliminary, but there is a current understanding that neighborhood disadvantage is related to variable development in brain areas that are responsible for emotional expression and regulation, environmental and sensory processing, and arousal response.

SUBSTANCE-USE FACTORS Adolescent substance-use has life-long impacts on behavioral functions, such as susceptibility to addiction, but it is unclear whether there are life-long brain changes that occur due to substance-use [11]. Some preliminary research shows that there is an association with alcohol use before age 11 and decreased cortical volume in the dorsal medial prefrontal cortex and the dorsal lateral prefrontal cortex [12]. One influencing factor for this finding could be socioeconomic status because “differences in SES predict differences in brain structure during a time when youth are at-risk of substance use� [7]. Greater number of days spent drinking at age 19 and average number of drinks per day both have positive relationships with cortical volume and thickness, particularly in the ventral medial prefrontal cortex, dorsal lateral prefrontal cortex, and insula [12].

There is also a positive relationship between binge drinking and cortical thickness in the ventral medial prefrontal cortex, ventral lateral prefrontal cortex, and the insula [12].

The finding that alcohol-use is associated with increased hippocampal and amygdala volumes challenges the findings that show a correlation between increased adverse childhood experiences and decreased amygdala and hippocampal volume [4, 12]. There is extensive evidence for adverse life experiences as predictors for increased susceptibility to substance-use, therefore the differences in these findings are counterintuitive [13]. A potential explanation for this contradiction is that alcohol-use during development could inhibit synaptic pruning in the hippocampus and amygdala, while other adverse life experiences do not. There is little evidence for development alterations as an effect of tobaccouse and cannabis-use [12]. Additionally, there is little evidence that maternal substance-use of alcohol alters brain development in adolescents FALL 2019

who are not diagnosed with Fetal Alcohol Syndrome [14]. Substance-use is common among teenagers, making it a prominent issue in society. More research needs to be conducted to further explore other substances, specific developmental periods that are particularly vulnerable to substance-use alterations, and other factors involving substance-use that may impact development.

POTENTIAL POSITIVE FACTORS While this review has highlighted many negative factors that may be influencing brain development, there are also many potential positive factors, such as parental-involvement, physical health, and sleep. Parenting is instrumental in the development of children and there have been many studies that highlight the positive effects of warm and supportive parenting on emotional well-being and lifeoutcomes [15]. Parents who are rated as less involved by their adolescent offspring have less insula activation when the parents and offspring are completing an emotional regulation activity using error processing [15]. The bilateral decrease in insula activation may play a role in parental inability to perceive pain, and therefore, empathize with the child. This suggests that empathetic responses are essential to parentchild relationships and positive parenting behavior. In order to understand this association in greater depth, it is necessary to further look at activation in the adolescent offspring brains.

Increased activation in the medial prefrontal cortex in adolescent brains when processing

“Parenting is instrumental in the development of children and there have been many studies that highlight the positive effects of warm and supportive parenting on emotional well-being and life-outcomes."

Figure 3: Diagram of a brain with the amygdala highlighted in red.

Source: Memory Loss Online, distributed under a Creative Commons license.

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Figure 3: Diagram of a brain with the prefrontal cortex highlighted in blue.

Source: Photograph by Erik Lundstrom, distributed under a Creative Commons license.

“Physical activity and sleep are both essential for overall health, but there is minimal understanding of how these factors influence brain development."

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parental error during an emotional regulation activity is associated with higher levels of parental depression [16]. Increased activation of the medial prefrontal cortex in parents when processing child error contributes to decreased anxiety and depression [16]. These findings further highlight the impact of parental response and parent-child relationships on brain development and overall well-being.

Negative parental factors can be detrimental in comparison to positive parental factors that can alleviate the effects of other adverse life experiences. Emotional regulation, which is highly influential throughout development, is formed within the parent-child relationship.

Physical activity and sleep are both essential for overall health, but there is minimal understanding of how these factors influence brain development. Some research suggests that physical activity increases cognitive ability, showing that physical activity has diffuse effects across the brain in early adolescents [17].

Increased fitness, regularity of exercise, lower body mass index, and lower resting heart rate are associated with increased white-matter and increased neurite-density [17]. Although this research is preliminary, it supports previous research that shows the positive effects of physical activity on cognitive ability. The various effects across the brain reveal possibilities for increased plasticity and physical activity as a potential mitigating factor of the negative effects and adverse experiences.

Sleep is another potential mitigating factor.

The brain during childhood and adolescent development shifts substantially and sleep may play a role in the substantial shifts. Sleep is one factor that The Adolescent Brain Cognitive Development (ABCD) study examines and follows longitudinally. The first progression of this study found that African American children are getting significantly less sleep than their Caucasian American and Asian American peers [18]. These differences in sleep time directly influence the inter-network and intra-network of functional connectivity [18]. Shorter total

sleep time in 9- to 10-year-olds is associated with weaker functional connectivity and these associations are specific to particular cortical networks [18]. The continuation of ABCD will create a more in-depth understanding of the relationship between sleep and functional connectivity, providing further insight into what the trajectory of functional connectivity looks like and how variations in brain developments influence brain function. As the study progresses, it is also important to look at factors other than race. ABCD can provide information about physiological stressors, such as physical activity, and emotional stressors, such as family conflict, and these factors could provide essential insight into how various factors impact sleep in addition to how sleep impacts brain development [7].

CONCLUSIONS AND FUTURE DIRECTIONS The research presented at the Society of Neuroscience Annual Conference 2018 contributes significantly to advancing our overall understanding of factors that influence brain development, specifically from stressors and adverse experiences. Cortical thickness is a common area of study when examining potential developmental variations. Some key findings involving cortical thickness are that childhood trauma increases cortical thickness in the inferior frontal gyrus and the occipital lobe and increased alcohol consumption increases cortical thickness in the prefrontal cortex and insula [2, 12]. Examining activation of certain parts of the brain is another approach to studying brain development. Some key findings include the association between increased stressful life events and increased hippocampus activation during contextual memory tasks, activation differences in bilateral dorsal lateral prefrontal cortex, bilateral parahippocampal gyrus, dorsal medial prefrontal cortex, and the posterior cingulate cortex, and amygdala activation in response to stress stimuli for African Americans and European Americans, and the association between decreased insula activation and decreased parent involvement [3, 5, 15]. Another common approach is looking at the volume of particular brain areas. Some key findings regarding volume include hippocampal volume being negatively correlated with childhood trauma, variance in bilateral hypothalamus and right amygdala volume in children who experience neighborhood disadvantage, and a positive relationship between adolescent alcohol- use and hippocampal and amygdala volume [4, 8, 12]. These methods have facilitated significant expansion of our knowledge about stress and brain development.

DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE

B R A I N D E V E LO P M E N T It is important to study a variety of factors in conjunction with each other. For example, exploration of racial differences in development should be done in conjunction with research examining the role that mental health plays in racial differences. Investigating the relationships between a variety of experiences, such as early childhood, early adolescence, and late adolescence, will cultivate a comprehensive understanding of the factors affecting brain development. In addition to expanding and specifying the types of factors and studying the interactions of these factors, it is also vital that the methods and analysis of brain development when looking at the effects of stress factors are improved. One possible way to do this is to examine differences between grey and white matter volume, rather than just volume. Further investigations may include volume in concurrence with structures and activation on a smaller scale. Additionally, it is important to link brain development findings to associated behavioral outcomes. It may be difficult and time consuming to work towards these steps, but the detrimental effects that disadvantaged individuals experience due to undeserved adversities must be understood. By understanding the multifaceted mechanisms that contribute to these detrimental effects, organizations will be able to develop effective intervention and prevention programs. There is currently a major disconnect between research and social interventions and prevention programs. By increasing overall knowledge about how stress affects brain development, research can better inform successful programs.

Therefore, in addition to expanding our overall understanding of the brain, this research can be used as an influential tool for change in addressing societal disparities. D

experiences contribute to racial differences in the neural response to threat. Paper Presented at Society of Neuroscience 2018, San Diego, CA. Program No. 078.30.

7. Gonzalez, M.R., et al. (November, 2018). Associations between socioeconomic factors and brain structure in preadolescence. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.04.

8. Bell, K., et al. (November, 2018). The influence of neighborhood disadvantage during adolescence on volume of the adult amygdala, hippocampus, and thalamus. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.05.

9. Forthman, K.L. et al. (November, 2018). Evidence indicating no effects of neighborhood affluence on brain functions and behaviors of positive/negative valence systems among mood/ anxiety disorders. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 158.07.

10. Feng, C., et al. (November, 2018). Neighborhood affluence accounts for inter-individual variations in the left insula volume among mood/anxiety disorders. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 158.08.

11. Hammond, C. J., Mayes, L. C., & Potenza, M. N. (2014).

Neurobiology of adolescent substance use and addictive behaviors: treatment implications. Adolescent medicine: state of the art reviews, 25(1), 15-32.

12. Purcell, J. B., et al. (November, 2018). Alterations in gray matter volume of the prefrontal cortex, hippocampus, and amygdala persist into adulthood following alcohol, tobacco, and cannabis use during adolescence. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.08.

13. LeTendre ML, Reed MB. The Effect of Adverse Childhood Experience on Clinical Diagnosis of a Substance Use Disorder: Results of a Nationally Representative Study. Subst Use Misuse. 2017;52(6):689-697.

14. Sharp, T., et al. (November, 2018). The association between maternal alcohol consumption in pregnancy and offspring brain morphology: A population-based MRI study. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 499.22.

15. Ratliff, E. L., et al. (November, 2018). Parental involvement predicts posterior Ă­nsula hemodynamic activation in response to child's costly error. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.09.

16. Cosgrove, K. T., et al. (November, 2018). The relationship between brain activation during parent-child fMRI hyperscanning and symptoms of anxiety and depression.

Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.10.

17. Salvan, P., et al. (November, 2018). Physical health and active lifestyle in 12-year-old children is linked with brain measures. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.17.

18. Goldstone, A., et al. (November, 2018). Typical total sleep time is associated with intra- and inter- resting-state network functional connectivity in 9-10 year olds. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.18.

CONTACT CHLOE CONACHER AT CHLOE.S.CONACHER.19@DARTMOUTH.EDU References 1. Stevens, J. E. (2015). The Adverse Childhood Experiences Study - the largest, most important public health study you never heard of - began in an obesity clinic.

2. McQuinn, S. (November, 2018). The effects of early childhood trauma and gender on brain structure. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.03.

3. John, R.A., et al. (November, 2018). The relationship between cumulative stress exposure and hippocampal activation during contextual memory. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 227.09.

4. Aghamohammadi Sereshki, A., et al. (November, 2018).

Effects of depression and childhood adversity on the volumes of the amygdala subnuclei and hippocampal subfields. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 631.04.

5. Davis, E., et al. (November, 2018). Race, violence exposure, and the psychosocial stress response. Paper Presented at Society for Neuroscience 2018, San Diego, CA. Program No. 281.02.

6. Harnett, N.G., et al. (November, 2018). Negative life FALL 2019

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DARTMOUTH UNDERGRADUATE JOURNAL OF SCIENCE