Neuroscience Communications April 2012
Using nanoprobes to image tumors PAGE 47
Peptide used to label nerves in surgery PAGE 13
Review of the progress of neuroimaging PAGE 7, 27 & 58
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Q&A Aslan: Hi Dr. Ju, why don’t you start by telling us what it is you are doing and what it is you hope to accomplish. Dr. Ju: When I was an undergraduate in neuroscience, I didn’t really enjoy learning the material very much. It was really dry and boring. I liked the experimentation and really enjoyed being able to design my own experiments later in graduate school but always thought back upon how I would change things if I ever had the opportunity. When I started teaching full time I was always trying to use what I had learned about neuroscience in the classroom and hoping to make a difference in how students learned. There’s been a lot of trial and error but I feel like I’m finding ways to unite technology with teaching a bit more effectively and hopefully this comes across to students. A: Those of us that have taken your courses know that in the past few years you have underwent changes in your mood due to lifestyle changes you implemented as a sort of quasi-experiment, can you tell us a bit about your experience and what it is you learned? DJ: This “self” experimentation came about because of the large number of students I would see (healthy, young individuals) who would develop major depressive disorder. One of my colleagues Dr. McIntyre gave a lecture about how metabolic disorders may underlie depression and as a result I wanted to see if changes in sleep cycles and eating habits could actually induce depression. I underwent sleep deprivation and changes in diet for only 3 weeks during February and at the end of the “experiment” didn’t really notice any changes other than a slight increase in body weight. Much later that year I started to notice that I stopped enjoying certain activities (anhedonia) and lacked normal sleep cycles (psychomotor effects) and eventually lost the ability to concentrate for long periods of time. I was really shocked by this as it started to affect my abilities to teach. I hoped that from this experience students could learn a little bit more about the practical aspects of neuroscience (it’s not all book relatad) and that these changes sometimes have long lasting effects. I also wanted to note that after 2 years I still bear some of the effects of this experiment - my concentration has improved but hasn’t fully recovered and I still have some issues with sleep. I’ve learned also that this type of experiment helps me to really understand the student perspective more and that mental health issues are something we should all talk about. I’ve had numerous students talk to me about their own struggles with depression as a direct result of opening up about my 10 months of depression which I’ve felt very personally about. A: What sort of changes have you made to your courses and teaching style to accommodate for this stress, and have you noted any appreciable results? DJ: I’m hoping that students have the opportunity to learn about neuroscience in both a traditional manner as well as by enhancing learning beyond the classroom - something that I refer to as the extended classroom. As a result I’ve tried to make learning a bit more relevant to students and to try and provide as many unique opportunities as possible (i.e. internships, mentorship, and publications/ projects such as this one). It’s still very early to say but I think that students learn better when things are run at their own pace and they retain the information longer in a non-stressful environment. I’m hoping that the results of students learning at their own pace (i.e. how many times have students looked something up on wikipedia or online because they couldn’t understand something or were just curious) will pay huge dividends later. I’m very hopeful that all of these unique opportunities can help students in some way and that many of them will share their experiences with me. A: What would you say are some obstacles that prevent professors from making a course that does not compromise the mental health of its students? DJ: I think professors simply forget about their own experiences as students. I have the opportunity to speak to many different professors and the ones that I’ve always been most impressed by are the ones that remember what it was like as a student, try to keep up with how students learn, and want to try and make learning an experience rather than a series of tests. By this I don’t mean to suggest that you have to have needless activities that don’t really add to the course, but to think of things that will be memorable AND valuable to students after graduation.
Acknowledgements The class of HMB420, 2012, would like to express our greatest appreciation to Dr. Bill Ju for creating a completely unorthodox but helpful and considerate learning environment and wish that more professors would adopt his degree of sympathy and understanding towards their respective students. The class of 2012 and the entire faculty and staff of the Neuroscience Human Biology Program at the University of Toronto wish to express their immense gratitude to Mr. Aslan Efendizade (also an author) and Ms. Laura Herrera for the commitment of their time and talents to this publication. The amazing work that they have done in formatting and planning this entire publication speaks to their professionalism and talents. Sincere thanks from Dr. Bill Ju on behalf of everyone in this publication.
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CONTENTS April 2012 | Volume 1 | Issu No 1
RESE ARCH 2 Q&A - Acknowlegements 4 NEUROIMAGING rt-fMIR: Current and future applications in neuroscience Phoebe Bao 7 NEUROIMAGING Eavesdroping on the human auditory cortex Cynthia Chan 11 NEUROIMAGINg Harnessing the power of electrical activity in the brain Farah Din 13 NEUROSURGERY Fluorescence labelling and imaging in the operating room Aslan Efendizade 16 NEUROPHYSIOLOGY Spinal Cord Injury: Current breakthroughs and future promises in therapy Daniel Etarsky 19 NEUROPHARMACOLOGY Your attention, please: On methylphenidate helping us transcend our boundaries Athena Hau 22 PERSONALITY DISORDER The behavioural disorder of the future: Internet addiction disorder Nicola Hyslop
24 NEUROPHYSIOLOGY Treating stroke with music Falisha Karpati 27 NEUROIMAGING Reconstruction of visual experiences and the future of mind-reading Priscilla Kwa 30 NUTRITION A review of neuroprotective properties of caloric restriction Ceilidh MacPhail 33 STRUCTURAL IMAGING Visualizing concussions: DTI & H-MRS in diagnosis & return-to-play Monica Maher 37 NEUROIMAGING Putting the fun back into functional near-IR spectroscopy Alex Mihaescu
47 NANOTECHNOLOGY The evolution of nanotechnology Dushyaan Sri Renganathan 51 MOLECULAR NEUROSCIENCE Somatic L1 retrotransposition in the human brain: Generating somatic diversity between neurons Jessica Suddaby 55 OPTOGENETICS Optogenetics and its recent applications Vivek Verma 58 NEUROIMAGING Neuromarketin: The search for the brain’s “buy button” Joshua Villafuerte 63 MOLECULAR BIOOGY Gene editing nucleases Alan Xi
40 MOOD DISORDER MRI-guided accelerated repetitive TMS over two days achieves results comparable to full traditional treatment Lily Qiu 43 NEUROPATHOLOGY Role of inflammation in the development of depression Alicia Rodrigues
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Neuroimaging Real time functional magnetic resonance imaging (rt-fMRI): Current and future applications in neuroscience Phoebe Bao Human Biology Department, New College, University of Toronto, Toronto, ON Real time functional magnetic resonance imaging (rtfMRI) is an emerging neuroimaging technique that utilizes on-line fMRI data processing to provide brain activation data. Real time fMRI enables instantaneous monitoring of brain states, which has widespread applications in brain machine interface control and biofeedback protocols. This review focused on research by Hampson and colleagues (Real-time fMRI biofeedback targeting the orbitofrontal cortex for contamination anxiety, 2012). Using a neurofeedback protocol, subjects with contamination related anxiety were taught to explicitly control their orbitofrontal cortex (OFC). Individuals with successful control of OFC activation levels showed lower anxiety ratings at the end of the study, whereas individuals that received sham neurofeedback showed no changes in anxiety ratings. These findings suggest that rt-fMRI enabled neurofeedback has clinical applications in the treatment of mental illnesses linked to dysfunctional activations in a specific region of interest. Limitations of rt-fMRI and its future applications are also discussed. I. BACKGROUND Functional magnetic resonance imaging (fMRI) is a developing neuroimaging technique that can be used to monitor neural activations. Similar to fMRI, real time fMRI (rt-fMRI) is a non-invasive technique that relies on the blood oxygen level dependent (BOLD) response, with the added ability to deliver instantaneous fMRI data. During its initial development in 19951, rt-fMRI was seen as a breakthrough in neuroimaging research because it allows for live monitoring of data quality and delivery of interactive paradigms to observe changing mental states. Currently, this technique’s widespread applications include biofeedback protocols, on-line monitoring of brain states and control of brain machine interfaces. Real time fMRI requires extensive computation to instantaneously process fMRI data. One method involves computing an incremental general linear fit model to each time point in the fMRI data collection1,2. In the general linear fit model approach, at subsequent time points, the model fit is updated, expected fMRI signal is subtracted from the fit and the remaining signal is categorized as noise or variance in the data. More recent studies utilizing rtfMRI have incorporated advanced data-processing steps critical to post-scan processing of MRI data. These include: 3-D head motion correction, spatial normalization to stereotaxic space and rt-fMRI specific statistical analysis (for review see Weiskopf et al. 2007 3). Real time fMRI’s unique property of concurrent data processing allows for the immediate monitoring of mental states and the corresponding neural correlates. Brain states and the corresponding neural correlates tend to remain consistent across scan sessions and this consistency allows for classification of neural
activations to predict the corresponding brain states4. This data classification training approach has been applied to lie detection in individuals with a high predictive accuracy5. Real timefMRI’s immediate data analysis process also has clinical applications. Real time fMRI has been used to delineate the primary motor cortices in brain tumor patients to guide surgical incisions6. Traditional fMRI post-scan data processing requires long periods of time, which can delay time-sensitive neurosurgeries. Thus, rtfMRI may be relevant to legal settings in the future (lie detection application) and provide improvement in surgical care. The classification of brain states through rt-fMRI technology also enables brain computer interface applications to operate more smoothly. Yoo and colleagues7 used an rt-fMRI enabled brain-computer interface to allow subjects to navigate in a maze. Individuals were trained to associate four distinct thought processes (i.e. mental calculation) with four directional cursor movements (i.e. up, down, left and right) on the computer screen. In brain-machine interfaces, rt-fMRI classified brain states can correspond to specific motor movements, which can guide the subject’s mental control of the machine because immediate and continuous visual feedback of the machine’s movement is provided8. Through biofeedback protocols, rt-fMRI also enables the explicit control of specific brain structures, which can have therapeutic applications. In a biofeedback protocol, rt-fMRI data is converted to a scale that conveys to the participant the degree with which they are activating a specific brain region. Research shows that continuous feedback on the level of activation enable subjects to develop explicitly control over activation of the insular cortex9, amgydala10 and auditory cortex11. Explicit control of brain regions can individuals to reduce their subjective experience of pain (DeCharms, 2005) and craving tendencies in chronic smokers 12. Thus rt-fMRI enabled biofeedback protocols have potential clinical applications. II. PROPOSED RESEARCH This review will focus on the biofeedback aspect of rt-fMRI applications. Hampson and collegues13 used a neurofeedback protocol to train subjects to explicitly control their orbitofrontal cortex (OFC), a region implicated in contamination-related anxiety. The objective of this study is to examine whether explicit regional brain control translates into explicit control of psychological outcomes, in this case, control over contamination related anxiety. Methodology Hampson and colleagues recruited healthy subjects with high scores in the Obsessions and Washing Compulsions Subscale for this study. Participants were divided into two age and gender
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matched groups to receive neurofeedback and sham biofeedback. The protocol was divided into five main stages. The first stage involved exposing subjects to contamination related images (i.e. images of rotting fruit, needles and garbage etc.) and neutral images during an fMRI scan to assess contamination related OFC activation in each subject. The second stage required subjects to meet with a clinical psychologist to develop an individualized cognitive strategy to control contamination anxiety for each subject. Strategies include reappraising the risk of contamination and embracing a meditative mindfulness. This subset of strategies was used to help participants to reduce to reduce activity in the OFC. To enable subjects to increase OFC activation, they were instructed to imagine being touched with contamination related objects. These cognitive strategies were proposed as an initial starting point for subjects to gain explicit control over their OFC activation levels; however, all subjects were encouraged to develop their own strategies during the neurofeedback session. In the third stage, each subject’s baseline OFC control abilities were assessed with fMRI. Subjects were shown contamination related images (Fig. 1a, 1b) and instructed to increase or decrease their OFC activation level. Each stimuli included a red, blue, or white arrow next to the contamination image to instruct the participant to increase, decrease, or rest OFC activity, respectively.
sham and experimental conditions, participants were informed of a 6-8 second delay between neural activation and neurofeedback. In the final stage of the study, subjects participated in an out-of-scanner assessment session where they were shown a new subset of contamination associated images. Subjects were asked to subjectively report their anxiety level on a scale of 1 to 5. Results Hampson and colleagues found that anxiety levels decreased following neurofeedback intervention for subjects that gained successful control over their OFC activation level (Fig. 2). However, in subjects that did not effectively gain control over OFC activation, anxiety ratings were not significantly reduced following the intervention. Subjects that received sham feedback did not have significant changes in their anxiety ratings.
Figure 2 Subjective anxiety ratings was significantly lower for one participant receiving neurofeedback intervention.
Discussion and Significance The findings of this study provide evidence that rt-fMRI enabled neurofeedback can teach individuals to gain explicit control over the OFC. With increased explicit control of the OFC through employment of cognitive strategies, contamination related anxiety levels decreased. These results suggest that explicit control Figure 1 Stimuli and neurofeedback employed by Hampson and colleagues (2012). a) Contamination related image with instruction to of specific brain regions is not only possible, but can also alter increase OFC activation, as indicated by the red arrow; b) contaminacognitive states that are related to hyperactivations in the region. tion related image with instruction to decrease OFC activation; and c) In this context, neurofeedback protocols have widespread clinineutral image accompanied with instruction to bring OFC activity level cal applications. It should be noted, however, this study recruited to baseline, as indicated by the white arrow. As well, the time course plot healthy participants with contamination related anxiety. The next beneath the image indicates neurofeedback efficacy in OFC activation stage of this study should assess the efficacy of neurofeedback (red line) and deactivation (blue line) trials. therapy in a clinical population of obsessive compulsive patients. In the fourth stage, participants receiving neurofeed- Some of the limitations of this study should be addressed. back viewed stimuli similar to that of stage three, with instruc- As Hampson and colleagues mentioned, the neurofeedback prototions to increase, decrease or rest OFC activity. Neurofeedback col did not help all participants to reduce anxiety. The effectivewas shown below the contamination image (Fig.1c) as a time ness of neurofeedback is dependent on the ability of each subject to course plot. The color of the time course plot corresponded to learn explicit control of the brain region of interest and the cognithe direction of OFC activation and plot amplitude is represen- tive strategies implemented during the biofeedback sessions. Thus, tative of OFC activation level. Trials that instructed participants neurofeedback based therapeutic approaches may not be effective in to increase, decrease or relax were colored red, blue and white, all subjects. More research to develop appropriate cognitive straterespectively. Thus, with successful utilization of neurofeedback to gies need to be conducted to increase the efficacy of neurofeedback control OFC levels, participants should see increased plot ampli- psychotherapy. The implementation of neurofeedback also requires tudes on red segments and decreased amplitudes on blue segments that the psychiatric illness is related to a specific brain region of (Fig. 1c). For participants assigned to receive sham feedback, an interest, which was the OFC in this study. This condition may not OFC activation plot was also shown on the screen, indicating suc- be applicable for all mental illnesses. Future studies on neurofeedcessful OFC control; however, these results are copied from an- back should also assess the longevity of the treatment’s effects. other subject and do not reflect the participant’s performance. In
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Limitations and Future Directions Since rt-fMRI share the same basic neuroimaging data acquisition process as fMRI, it also shares some of fMRI’s major limitations. Functional MRI predicts neuronal activity based on the BOLD response, which can be an imprecise indicator of actual neuronal activity14. Furthermore, although rtfMRI is a promising technique for facilitating BMIs, fMRI scanners are bulky and lack portability. Thus, the availability and portability of fMRI technology need to be addressed in order for rt-fMRI applications to actualize their full potential. Nevertheless, there are diverse future applications of rt-fMRI technology that have the potential to revolutionize the field of neuroscience. The study by Hampson and colleagues is just one amongst a multitude of reports that demonstrate the utility of neurofeedback in modulating cognitive processes12,10. Neurofeedback based therapeutic interventions can be used in conjunction with cognitive behavioral therapy and pharmaceutical interventions to treat mental illnesses. Real-time fMRI also enables us to perform on-line monitoring of brain states, which can be used for lie detection and communication with locked-in patients15. With rt-fMRI enabled brain state detection, a database of neuroactivation patterns can be generated in the future, with each activation pattern corresponding to a specific motor or cognitive activity. As suggested by DeCharms (2008), if motor learning can take place through mimicry, cognitive learning can also take place through the mimicry of brain states. Current research already provides preliminary evidence that selective activation of brain regions can result in improved behavioral responses16. Thus, rt-fMRI enabled brain state monitoring and mimicry can also change the process of learning in the future. With increased availability of rt-fMRI, the future of neuroscience can move from brain imaging to brain activation decoding, which can have widespread clinical and psychosocial ramifications.
13. Hampson, M. et al. Real-time fMRI Biofeedback Targeting the Orbitofrontal Cortex for Contamination Anxiety. Journal of visualized experiments : JoVE (2012).doi:10.3791/3535 14. Sirotin, Y.B. & Das, A. Anticipatory haemodynamic signals in sensory cortex not predicted by local neuronal activity. Nature 457, 475-479 (2009). 15. deCharms, R.C. Applications of real-time fMRI. Nature Reviews Neuroscience 9, 720-729 (2008). 16. Rota, G., Handjaras, G., Sitaram, R., Birbaumer, N. & Dogil, G. Reorganization of functional and effective connectivity during real-time fMRI-BCI modulation of prosody processing. Brain and language 117, 123-32 (2011).
1. Cox, R.W., Jesmanowicz, A. & Hyde, J.S. Real-time functional magnetic resonance imaging. Methods 33, 230-236 (1995). 2. Hinds, O. et al. Computing moment-to-moment BOLD activation for real-time neurofeedback. NeuroImage 54, 361-368 (2011). 3. Weiskopf, N. et al. Real-time functional magnetic resonance imaging: methods and applications. Magnetic Resonance Imaging 25, 989-1003 (2007). 4. Cox, D.D. & Savoy, R.L. Functional magnetic resonance imaging (fMRI) “brain reading”: detecting and classifying distributed patterns of fMRI activity in human visual cortex. NeuroImage 19, 261-270 (2003). 5. Davatzikos, C. et al. Classifying spatial patterns of brain activity with machine learning methods: application to lie detection. NeuroImage 28, 663-668 (Elsevier: 2005). 6. Feigl, G.C. et al. Real-time 3T fMRI data of brain tumour patients for intra-operative localization of primary motor areas. European journal of surgical oncology : the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 34, 708-15 (2008). 7. Yoo, S.-S. et al. Brain-computer interface using fMRI: spatial navigation by thoughts. Neuroreport 15, 1591-5 (2004). 8. Lee, J.-H., Ryu, J., Jolesz, F.A., Cho, Z.-H. & Yoo, S.-S. Brain-machine interface via real-time fMRI: preliminary study on thought-controlled robotic arm. Neuroscience letters 450, 1-6 (2009). 9. Caria, A. et al. Regulation of anterior insular cortex activity using realtime fMRI. NeuroImage 35, 1238-1246 (2007). 10. Posse, S. Real-time fMRI of temporolimbic regions detects amygdala activation during single-trial self-induced sadness. NeuroImage 18, 760768 (2003). 11.Yoo, S.-S. et al. Increasing cortical activity in auditory areas through neurofeedback functional magnetic resonance imaging. NeuroReport 17, 1273-1278 (2006). 12. LaConte, S.M. et al. Modulating rt-fMRI neurofeedback interfaces via craving and control in chronic smokers. Brain 1-2 (2008).
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Neuroimaging Eavesdropping on the Human Auditory Cortex Cynthia Chan Department of Human Biology, University of Toronto, Toronto, Ontario, Canada M5T 1K5 Understanding the processes involved in the processing of speech within the human brain is a field of study that is still largely in its infancy. From a basic science perspective, uncovering the salient features of auditory processing will give us a deeper understanding of the mechanisms underlying language, and also help us identify which areas might be damaged in individuals who have difficulty either speaking or understanding speech. However, the most exciting potential for this field of research takes its form in the possibility to give the gift of speech to individuals afflicted with diseases that prevent them from producing speech, such as in lockedin syndrome, amyotrophic lateral sclerosis (ALS), stroke, or dysarthria. The platform for this type of technology was recently created by a research group at the University of California Berkeley3. There, Pasley and his colleagues were able to use algorithms to reconstruct and accurately guess words from populations of neural activity. Indeed, through two separate computational models, they were successful in transforming patterns of intracranial recordings into spectrogram representations of individual spoken words that were both accurate and statistically significant. Although the “deconstructed” words were not completely intelligible, progress made thus far is an exciting step towards forming the platform for future technology in this field to convey imagined speech. Major tenets of language and speech processing have been well conserved over time; however, the main details of how speech, which is the combination of frequency and words, is precisely processed within the brain are still largely unknown. Our current understanding stems from the basic principle that sound is transmitted to the cochlea in the inner ear, which then transforms the auditoryassociated vibrations to action potentials that ultimately reach the primary auditory cortex (A1) for initial processing. This processing is believed to be associated with the posterior superior temporal gyrus (pSTG)1, more affectionately known as Wernicke’s area. Somehow, then, the pSTG is extracting features of auditory information from sound; however, it is still largely unknown what auditory features are being extracted and how this is achieved. To elucidate this, Pasley et al. (2012) used a technique known as stimulus reconstruction to test whether they could recapitulate previously heard speech from population neural responses in humans. Their work was based off a previous study conducted in ferrets at the University of Maryland, where these authors used a form of reverse reconstruction in awake ferrets to reconstruct speech from patterns of activity from neurophysiological recordings in the primary auditory cortex2. Similarly, in the study by Pasley et al3., single trial intracranial recordings from the human pSTG were conducted while the subjects listened to various words and pseudo-words. Then, from these recordings of population neural activity, two algorithms were applied to the recordings in an effort to recreate these words.
Normally, experiments aim to understand how a stimulus affects behavioural activity; in this study, however, the authors aimed to recapitulate the stimulus from the neural response. By essentially working “backwards”, they hoped to uncover the salient features of audition that are necessary for speech processing, and therefore, language processing in humans (Figure 1). Altogether, their computational models were successful in transforming patterns of neural activity into spectrogram representations of sound to ultimately recreate the words these subjects had heard earlier. Of the two models, linear models were able to best reconstruct slow temporal fluctuations 2 . while non-linear reconstructions were most successful for fast temporal fluctuations. This “decoding” of speech was accurate and statistically significant, as shown through several tests of mean identification ranks, correlation, and false positive tests. In addition, their construction of spectrograms was able to produce intelligible identification of words. These results, as further explained below, offer exciting possibilities for the foundation of brain-computer interfaces that could be eventually created for individuals with a maintained high cognitive capacity, but inability to speak. Indeed, previous research demonstrating the similarities in neural patterns of activity between heard and imagined words offers an exciting window of opportunity and hope for advancement in this field within the next decade.
Figure 1. Experimental paradigm for spectrogram reconstruction of population neural activity from the pSTG, as demonstrated by Pasley et al. (2012)3. Depending on the level of acoustic modulations, either linear or non-linear reconstruction was used to accurately decode activity.
Speech Processing and the pSTG The decision to measure population neural activity within the posterior superior temporal gyrus (pSTG) was based on several studies within the literature that have independently supported its
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importance in the processing of speech sounds. Indeed, according to several studies, the pSTG is involved in transforming critical aspects of acoustics into pre-lexical depictions within the brain4–7. Moreover, previous research using functional magnetic resonance imaging (fMRI) in humans have demonstrated that this region is active while humans categorize “speech phenomes”8. In particular, they found the left pSTG to be strongly implicated in this processing. Further studies have also demonstrated higher activity in the pSTG during as correlated with predicted identification of sounds (Figure 2)
Figure 2. Axial maps demonstrating BOLD responses of subjects for syllable versus baseline tasks. Results from Binder et al. (2004) in Nature Neuroscience demonstrated higher activity in the pSTG during as correlated with predicted identification of sounds1.
More specific, non-imaging techniques have also been used in tandem to understand the pSTG’s role in speech. By taking electrocorticographic recordings of patients with epilepsy or brain tumours as they listened to speech sounds in different acoustic steps, other studies have been able to conduct experiments with high spatial and temporal resolution (contrary to the imaging)9. By analyzing these local field potentials, they found that the pSTG was involved in the encoding of higher order speech sounds, adding to further evidence for the role of the pSTG in processing of acoustic speech. These results demonstrated its role in extracting the spectro-temporal features of auditory speech from those features that are not important during the processing, thus highlighting the pSTG as a critical site for speech processing. Interestingly, the pSTG has also been shown to respond specifically to speech, and not just simply sounds10, which further highlights its pinnacle role in higher order speech processing. Based on this wealth of evidence, then, research in the study discussed in this review paper focused directly on the role of the pSTG in order to better understand its main role. Of course, despite the importance that research has attributed to the pSTG fMRI and other studies have implicated several other regions in the auditory circuit as being involved in the processing of speech as well9. Stimulus Reconstruction In order to successfully re-create speech representations of the individual words, a technique known as stimulus reconstruction was employed. In short, the aim of the technique is to use various computation algorithms to reconstruct the original stimulus from measurements of neural activity. The concept of stimulus reconstruction has been widely applied to several other sensory systems, as seen in a recent group that used fMRI images of subjects as they watched movie clips to reconstruct the visual images that they had seen during the recordings11.
Figure 3. Spectrogram reconstruction of both words and pseudowords. Reconstruction models were applied to the ECoG recordings (where the most informative recordings were from the pSTG) in order to reconstruct a spectrogram representation closest to the original3.
In the particular paradigm developed by Pasley et al., cortical surface field potentials recorded from the pSTG as subjects listened to pre-recorded words (Figure 3). The ultimate goal of the study was to use these population neural recordings to reconstruct an auditory spectrogram of the word. A spectrogram is a timevarying spectrum of amplitude12; its resulting image is specific for individual phonetic sounds and therefore specific for certain acoustics. This spectrogram, then, can be used to map back to the original sound and be compared with the reconstruction. Moreover, depending on the level of acoustic modulation rates, one of two reconstruction algorithm models were used. Speech Stimuli and Neural Recordings These cortical surface field potentials were obtained from nonpenetrating multi-electrode arrays of electrodes over the pSTG. Subjects consisted of 15 patients, who were already required to have these electrode arrays placed over their left or right frontotemporal lobes as part of their treatment for either brain tumours or epilepsy localized to this region. ECoG signals were recorded as these patients were presented aurally with words (N=10) and sentences (N=5) from either male or female native English speakers. The words presented consisted of either nouns (including proper nouns), verbs, and pseudowords. Spectrogram Reconstruction Models Before applying computation reconstruction algorithms, the authors used group average t values to determine that the pSTG did, indeed, produce the most informative electrode recordings; that is, they had the highest signal-to-noise ratio. This further validated their decision to analyze and “decode” speech from this specific region. Following this, they applied one of two reconstruction models to the set of ECoG neural population recordings, either a: (1) Linear (Spectrogram) Model, or (2) Non-linear (Modulation) Model. Linear Spectrogram Model In general, a linear spectrogram model was used with a leaveone-out cross-validation fitting process, and then later validated through calculating reconstruction accuracy (r2). The linear
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spectrogram model was “envelope-locked”, meaning that it was capable of responding dependably to variations within the stimulus envelope. In addition, to ensure the frequency selection of neurons, estimates of standard temporal receptive fields (STRFs). This allowed the authors to estimate the neural tuning; they found that the recording electrodes were able to detect both low (200Hz) to high (7000Hz) acoustic speech frequencies. Overall, by comparing the reconstruction accuracy of the spectrogram compared to the spectrogram of the original sound, the authors found that the reconstruction of both slow and intermediate modulation rates achieved an accuracy significantly greater than zero.
els that the pSTG responses were subjected to, the audio representation of the reconstructed spectrograms were largely intelligible. Moreover, tests of statistical significance revealed that word identifications from the reconstructed spectrograms were also significant. One primary mode of assessment was via the median identification rank, which displayed a value of 0.89 – a value much greater than the 0.50 chance level (Figure 6A). This value was also statistically significant compared to a randomization test against a null distribution (p<0.0001), thereby confirming the accuracy of the identification rank. Furthermore, by conducting a ROC (Receiver operating characteristic) plot of word identification (Figure 6B), they found that their methods conferred a true positive predictive rate. Finally, in their last assessment of the true accuracy of word identification from audio produced from their reconstructed spectrograms, they confirmed a correlation (r= 0.41) between the reconstructed and actual word similarity (Figure 6D).
Figure 4. Mean reconstruction accuracy of linear (spectrogram-based) and nonlinear (modulation-based) coding of temporal modulation rate. For faster modulation rates, the nonlinear model was the most accurate3.
Non-Linear Modulation-based Model Despite their success with reconstructing slow and medium level temporal modulations, results from the study found that linear spectrogram models were not able to equally reconstruct fast temporal modulation rates. Thus, a model based on envelope-locking was not used. Instead, a non-linear spectrogram model that was phase invariant and dependent on changes in amplitude was employed; this technique is similar to ones used for the cells in the visual system. By using this method, they were able to achieve significantly higher accuracy for fast temporal modulation rates; this increase in accuracy over the use of linear models for fast temporal modulations was also statistically significant (following ANOVA tests). Further tests analyzing group data also confirmed significantly higher accuracy of the non-linear (modulation-based) model versus the linear (spectrogram-based) model, as shown in Figure 4. 5
Figure 5. Spectrogram representations of a series of heard words. Spectrogram reconstructions were based off population neural ECoGs from the pSTG in either linear or nonlinear models. Accuracy of the reconstruction compared to the true spectrogram depended on the temporal modulation rate 6
Accuracy and Identification of Words Altogether, both linear and non-linear reconstruction methods were able to accurately reconstruct the original spectrogram by using only populations of neural recordings from the pSTG (Figure 6). Although the reconstructions varied based on the specific mod-
Figure 6. Tests of accuracy and statistical significance for word identification of the reconstructed spectrogram representations: (A) median identification value is greater than chance and statistically significant (p<0.0001) (B) Receiver operating Characteristic (ROC) plot shows a true positive predictive rate (C) representations of accurate (right) and inaccurate (left) identification (D) correlation of actual and reconstructed word similarity3.
Future Directions and Final Thoughts Altogether, then, their reconstruction of spectrograms was able to produce intelligible identification of words and statistically accurate decoding of the original spectrogram. Although the recapitulated words were not perfectly intelligible and the reconstruction models used did not include higher level encoding models important in the processing of speech, the progress made in this paper is a pinnacle landmark in discovering the key features of speech processing in the pSTG. From a basic science level, understanding the breakdown processing of speech sounds into meaning within these primary auditory regions of the brain can ultimately lead to a better understanding of potential areas of malfunction in those who are born with, or later develop, an inability to speak. The clinical potential for research in this field is vast and exciting. Based on previous evidence that suggests that similar brain regions are activated during “heard” and “imagined” speech3, these results can be further developed to provide the ability to speak to individuals who still have high cognitive capacity, but an inability to speak. Such is the case with many pathologies within society, such as in individuals with ALS (Amyotrophic Lateral Sclerosis),
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dysarthria, victims of stroke, and those with Locked-in-Syndrome. Although there are obviously considerable ethical concerns to be taken into consideration with these applications of this research, the benefits that they could provide in the clinic are immense. True, advancing these techniques could create issues revolving around an individual’s right to privacy and “mind- reading”; however, having a foundation to potentially help society’s ill, but not developing in fear of invading these “rights” is perhaps more of an ethical concern. Can society justify having the knowledge and not applying it to help those who cannot speak? Within the next 10 years, the implications of further studying and progressing this field will definitely be an area of large debate and possibly resistance. Despite this, the advancements developed in the paper by Pasely et al. have the immensely exciting potential to form the groundwork for further breakthrough studies in providing the ability to speak to the many who are unable. 1. Binder, J.R., Liebenthal, E., Possing, E.T., Medler, D. a & Ward, B.D. Neural correlates of sensory and decision processes in auditory object identification. Nature neuroscience 7, 295-301 (2004). 2. Mesgarani, N., David, S.V., Fritz, J.B. & Shamma, S.A. Influence of context and behavior on stimulus reconstruction from neural activity in primary auditory cortex. Journal of neurophysiology 102, 3329-39 (2009). 3. Pasley, B.N. et al. Reconstructing speech from human auditory cortex. PLoS biology 10, e1001251 (2012). 4. Romanski, L.M. & Averbeck, B.B. The primate cortical auditory system and neural representation of conspecific vocalizations. Annual review of neuroscience 32, 315-46 (2009). 5. Steinschneider, M. Unlocking the role of the superior temporal gyrus for speech sound categorization. Journal of neurophysiology 105, 2631-3 (2011). 6. Rauschecker, J.P. & Scott, S.K. Maps and streams in the auditory cortex: nonhuman primates illuminate human speech processing. Nature neuroscience 12, 718-24 (2009). 7. Hickok, G. & Poeppel, D. The cortical organization of speech processing. Nature reviews. Neuroscience 8, 393-402 (2007). 8. Desai, R., Liebenthal, E., Waldron, E. & Binder, J.R. Left posterior temporal regions are sensitive to auditory categorization. Journal of cognitive neuroscience 20, 1174-88 (2008). 9. Chang, E.F. et al. Categorical speech representation in human superior temporal gyrus. Nature neuroscience 13, 1428-32 (2010). 10. Vouloumanos, A., Kiehl, K.A., Werker, J.F. & Liddle, P.F. Detection of sounds in the auditory stream: event-related fMRI evidence for differential activation to speech and 7
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Neuroimaging Harnessing the power of electrical activity in the brain Farah Din Human Biology Department, University of Toronto Technology now exists that merges the activity of the brain with machines in order to provide assistance to patients. These devices are known as Brain-Machine Interfaces (BMI) and they ultimately provide a source for alternative communication between affected patients and their surroundings. Since the emergence of this technology, many advances and developments have been made, shining light onto the variety of realms benefiting from this device. Significant work by Carmena et al. (2008) has shown the use of BMI devices to be successfully employed in controlling robotic devices to perform simple movements. Although many drawbacks have been noted regarding the operation of BMI systems, recent work has taken a fresh approach in order to tackle these issues. Continual work by Carmena et al. has paved the way for BMI devices to be employed in humans, and to elaborate on the brains plasticity in order to incorporate prosthetic devices in a natural, effortless way. Future work has utilized the ability of these devices to control objects in the external world, and applied them to realms outside of rehabilitation. Such discoveries can allow us to consider the vast capacity of BMI systems to contribute to our advancing future. I. BACKGROUND The first account of electrical activity of the brain being recorded was by Hans Berger over 80 years ago1. He recorded electrical activity non-invasively using electrodes on the surface of the scalp and identified brain wave rhythms of 10hz in response to light1. His observations showed that EEG’s could be used as an index of the gross state of the brain. Technology now exists that merges the activity of the brain with machines in order to provide assistance to patients. These devices are known as BrainMachine Interfaces (BMI) and they ultimately provide a source for alternative communication between affected patients and their surroundings1,3. The electrical activity produced by the brain is detected from the scalp and sent to computers to be analyzed and processed2. This electrical activity is translated into commands and controls used by the machine to perform actions, without the use of peripheral nerves or muscles2. The ability of BMI technology to be independent of neuromuscular control allows an opportunity for patients suffering from neuromuscular disorders or injuries to communicate with their environment based on their intent1,2. Since the emergence of this technology, impressive advances and developments have been made; Vidal et al. (1977), was the first to coin the term BMI and used the system based on visual evoked potentials2. A similar experiment was seen earlier in 1967 by Dewan who also used explicit eye movements in order to modulate brain waves1,3. These findings did not seem to be practical from a clinical standpoint, as they required actual muscle movement and control; the EEG was simply used to reflect the gaze direction. In 1995, Farwell and Donchin were the first to use and
EEG-base device that used P300 evoked potentials4. A flash on the object of their focused attention elicited a P300 evoked potential that was detected by the BMI system in order to recognize the users desired selection4. This was considered to be a significant advance as the P300 evoked potentials used in this system were reflections of the users attention, rather than gaze direction that required neuromuscular control. Wolpaw et al. (2004)5, made further developments on this method and was the first to report use of sensorimotor rhythms as signals for BMI’s. Sensorimotor rhythms occur spontaneously and can be modulated by the users imagination. Users have been trained to use such electrical brain activity to control cursors. These initial results using P300 and SMR’s have shown the ability of non-invasive procedures to act as assistive technology3; these results have been extended to applications in multidimensional domains. II. BMI FOR ROBOTICS BMI’s have provided significant advances in the field of robotics; they have provided control signals used by robotic and prosthetic device to enhance their ability as assistive devices for patients3,6. Chapin et al. (1999), was the first to demonstrate the use of brain signals to control a robotic device8. They were able to train rats to position a robotic arm and obtain water by pressing a lever. Populations of neurons were recorded simultaneously and used in real time to control the robotic device.
Figure 1 An illustration of the basic components of a BMI3.
Carmena et al. (2008), was also able to demonstrate the use of BMIs to control robotic devices6; they trained monkeys to position a cursor over a target by operating a lever to obtain a juice reward. The initial phase consisted of recording brain signals from the motor cortex, which were later used by the monkeys to perform the task6. It was later reported that with time, the neuronal activity became more representative of the robotic arm movements as opposed to the actual arm, a result of brain plasticity6. Researchers studying BMI devices have used both invasive and non-invasive techniques to demonstrate the ef-
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fects of these devices3,9. In 2006, Hochberg et al. trained a human user with a cortical implant, who had suffered a high-spinal cord injury, to open and close a prosthetic hand13. The injury precluded his training with BMI signals produced during actual hand movements. Within 10 minutes, the subject was able to control the robotic hand in two-dimensional movement13. However, these studies have only been demonstrations of the potential use of BMI technology for robotic and prosthetic applications. III. COMPLEXITIES IN OPERATION Although the BMI system has undergone many advances, researchers from all over the world are still struggling to concur some basic critical problems associated with such devices. The use of BMI devices has led to many users experiencing fatigue due to the continuous concentration required10; prolonged use has also led to these symptoms as BMI devices have a very low information transfer rate and require continuous use3,10. For both invasive and non-invasive procedures, improvements in recording methods could be beneficial. A main concern for invasive procedures focuses on maintaining the reliability of the implant placed inside the brain2; concerns’ regarding its recording abilities are frequently discussed and is currently being investigated7. Limitations regarding the complexities of robotic prosthesis are also under consideration. Robotic prosthesis’ have limited ranges, incomparable to those seen in humans12; this limits the implants ability to control the robotic prosthesis in the real world6,7,8. Complex bionic prosthesis with multiple degrees of freedom3 would allow further advancements and allow users to perform dexterous tasks. To date, BMI devices still rely on a variety of subsystems in order to perform. For users, the reliability of these subsystems is crucial in ensuring correct performance; this increases discrepancies and errors in the execution abilities of such devices9,3. Present day BMI devices have only been explored in a laboratory setting, in order for these devices to be relevant clinically, the prototypes currently developed need to be transformed into clinically relevant technology. IV. FRESH APPROACHES At the University of California, Berkeley, The Carmena Lab is currently using a fresh approach when considering prosthetic devices and the use of BMI technology6. Previous research on BMI devices focused on implants tapping into specific neural circuits, or cortical motor maps already present in the patient to control the prosthetic arm3. By tapping into the cortical map associated with the human arm, the prosthetic arm was able to function. New findings from Rhesus monkeys have shown the brains ability to develop entirely new neural circuits dedicated to the prosthetic device6. Rhesus monkeys were implanted with electrodes and trained to use their thoughts to control a computer cursor6. After training, they monkeys mastered the task, and were able to repeat the movement day after day. This was an unprecedented finding in the field of BMI research; the Rhesus monkeys illustrated an ability to develop a motor memory for controlling the virtual device, similar to memories created for controlling parts of the animal’s body6. This new study provides hope that physically disabled people may one day be able to operate advanced prosthetic devices in a natural, effort-
less way. As a result of this newly discovered extraordinary plasticity of the brain, researchers can exploit this knowledge towards BMI advances. New BMI advances focus on developing new hybrid nervous systems that span biological and artificial components6. Rebsamen et al. (2010) attempt to tackle some of the issues presented by BMI devices using brain controlled wheelchairs (BCW)11. Using a similar P300 based system, users are able to navigate the BCW through an environment using predefined locations. A slow, but reliable P300-based BMI system was used to navigate the device, however a quicker BMI system was employed when users wanted to stop the wheelchair11. This increases the safety and efficiency associated with BMI systems, however this has not yet been tested in a clinical setting. Liao et al. have also attempted to eliminate some of the drawbacks associated with BMI devices while applying these advances to a realm outside of rehabilitation10. Traditional noninvasive BMI devices have uncomfortable sensors requiring conductive gel and skin preparation on the user; Liao et al. have developed a novel BMI device allowing the acquisition of EEG signals to happen in a comfortable and convenient manner. A wireless, portable, dry, foam-based EEG sensor can be worn around the users head in order to develop signals used for BMI devices10. This novel device has shown good conductivity and has been applied to gaming consoles used for entertainment purposes. This device has the shown the ability to apply real-time cognitive stage detection towards gaming control10. This allows us to consider possibilities of BMI providing effective controlof the outside world, aside from rehabilitation engineering. V. FUTURE DIRECTIONS As BMI advances progress at a rapid pace, the extent of its growth is technology is up for much consideration. How far this technology will develop and how useful it will become depends on the success and focus of future research. Possibilities of BMI can be applied to developing ‘Smart’ Homes, which use the power of BMI to control electronically driven homes. BMI technology can also enhance our own abilities as healthy humans; being able to see infrared spectrum, and downloading information straight into our cerebral cortex are realms that have yet to be explored, however recently have come become more plausible as these devices continue to be explored. By investigating the increasing ways in which this source of BMI power can be utilized, a greater understanding of the brain can be obtained. This also creates a great deal of concern and sets a precedent for a big ethical debate. 1. Daly, J.J. and Wolpaw, J.R. 2008. Brain-computer interfaces in neurological rehabilitation. Lancet Neurology. 7: 1032-1043. 2. Wolpaw et al. 2002. Brain–computer interfaces for communication and control. Clinical Neurophysiology. 113: 767-791. 3. McFarland, D.J and Wolpaw, J.R. 2010. Brain-Computer Interfaces for the Operation of Robotic and Prosthetic Devices. Advances in Computers. 79: 169-187. 4. Farwell, L.A and Donchin, E. 1995. A mental prosthesis using event related brain potentials. Electroencephalography Clinical Neurophysiology. 70(6):510-523. 5. Wolpaw et al. 2004. BCI2000: A general-purpose braincomputer interface (BCI) system. Trans Biomedical Engineering. 51(6): 1034-1043. 6. Carmena et al.(in press). Redundant information encoding in primary motor cortex during natural and prosthetic motor control. Journal of Computational Neuroscience.
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7. Cincotti et al. 2008. Non-invasive brain–computer interface system: Towards its application as assistive technology. Brain Research Bulletin. 75: 796-803 8. Chapmin et al. 1999. Real-time control of a robot arm using simultaneously recorded neurons in the motor cortex. Nature Neuroscience. 2(7): 664-670 9. Jin et al. 2012. A combined brain–computer interface based on P300 potentials and motion-onset visual evoked potentials. Journal of Neuroscience Methods. 205:265-276. 10. Liao et al. 2012. Gaming control using a wearable and wireless EEGbased brain-computer interface device with novel dry foam-based sensors. Journal of NeuroEngineering and Rehabilitation. 9(5) 11. Resamen et al. 2010. A Brain Controlled Wheelchair to Navigate in Familiar Environments. IEEE Transcations On Neural Systems and Rehabilitation Engineering. 18(6): 590-598 12. Kubler, A. and Neumann, N. 2005. Brain-computer interfaces—the key for the conscious brain locked into a paralyzed body. Progress in Brain Research. 150 (35): 513-525. 13. Hochberg et al. 2006. Neuronal ensemble control of prosthetic devices by a human with tetraplegia. Nature. 442(7099): 164-171.
Neurosurgery Fluorescence labelling and imaging in the operating room Aslan Efendizade Human Biology Department, University of Toronto, St. George Various reports analysing the incidence of medical errors have shown, amongst other things, that they account for approximately 100,000 deaths-per-year in the United States. A significant portion of these errors are surgical in nature, which, according to one study, attending surgeons account for 96% of. Some of the consequences of these surgical errors include tissue and nerve transections, the effects of which range from numbness and chronic pain to urinary incontinence (in radical prostatectomies). Current nerve labelling techniques do not sufficiently provide a contrast ratio that allows for discernment of deep and some superficial nerve fibers. Furthermore, they have several drawbacks such as slow uptake, localized individual nerve fiber labelling, and the lack of the ability to label transected or injured nerves. A recent labelling technique developed by a group from University of California in San Diego, uses a fluorescent peptide that binds preferentially to the connective tissue of peripheral nerve fiber tracts and arborizations. This technique provides many advantages over traditional techniques, and confers benefits in areas that the others lack. Preliminary in vitro crosssectional imaging of human laryngeal tissue shows evidence for the peptides efficacy in human peripheral tissue as well. Introduction In 2000, the US Institute of Medicine published a much cited, and disturbing study, in which they analyzed the amount of injury and death that was caused by malpractice (including drug dosage, mixed prescriptions, misdiagnosis, and surgical error). From the data they obtained, it was determined that medical error is the eight leading cause of death in the United States, at a stag-
gering 100,000 deaths-per-year1. To put that number into perspective, a Boeing 747 typically seats about 524 passengers2. Doing the complicated calculations, one quickly finds that is the equivalent of about 190 full-capacity Boeing 747‘s crashes, every year! Nearly a decade after, it seems that not much has changed in terms of patient safety. An organization called the Safe Patient Project has conducted follow-up analyses3 using data from various organizations, including the Centre for Disease Control, and has concluded that the past ten years has been a failure in terms of reducing patient death due to medical error, and that the 100,000 deaths-per-year estimate is now considered conservative. In a similar pursuit, Dr. Mark Bernstein (a renowned neurosurgeon operating out of Toronto Western Hospital) performed an observational study on records of his surgical patients from 2000 to 20064. He found that over the course of his practice, of the 1108 patients he operated on, there were a reported 2684 errors. Even more disturbing, Regenbogen et al conducted an analysis of 133 surgical error-related malpractice lawsuits in 2007, and found that of the 140 technical manual errors (which they defined as a manual surgical error, including tissue and nerve transections), 96% of these errors involved the attending surgeon, with only 4% attributal to the errors of residents and fellows alone5. What is worse, is of the preventable adverse errors, they report that one-half to two-thirds was attributable to surgical errors. The Problem Although medical errors encompass various issues, such as drug dosage and technical error, this paper’s prime focus is on surgical nerve transections. However, the techniques for solving this issue have implications for solving many other surgical problems.
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Accidental nerve transection is a major problem for neurosurgeons, oncologists, and orthopaedic surgeons, the result of which is an increased post-operative morbidity. In the most simple terms, the resection of a sensory neuron can result in chronic pain or numbness, and the transection of a motor neuron can result in muscle paralysis. However, any given type of surgery has various complications (too many to be reviewed in this paper). One common complication arises in patients with prostate cancer. Radical prostatectomies are often accompanied by urinary incontinence or erectile dysfunction6. To put this into perspective, prostate cancer is just below lung cancer as the most prevalent form of cancer in men7. Furthermore, according to one of the most comprehensive post-prostatectomy analyses performed by Penson et al, six months after the operation, about 80% of participants reported problems with urinary control8. Although acute incontinence may not seem like a major issue, Strothers et al estimated that the economic burden of this degree of prevalence is about $18.8 billion per year9. Clearly, the surgical profession is in dire need of a technique that can provide the same level of imaging for nerve fibers that Egas Moniz provided for imaging of cerebral blood vessels decades ago. Current Techniques Currently, there are various methods used to image nerve fiber tracts for surgeries. For example, the retrograde and anterograde axonal transport present in neurons is used to label nerve fibers using fluorescent dyes10. However, this technique is not without its drawbacks. A major concern is the slow uptake and labelling process, which can take days to occur, and if during the proceding operation a surgeon finds that it did not properly label the fiber, then what? Another issue is that this technique can only be used to label nerve fibers individually, which as one might imagine is a very tedious and time-consuming effort. Another common technique is electromyography, which involves the insertion of an electrode into a muscle or nerve fiber tract, and the subsequent distal muscle twitches are observed to identify relationships between nerve fibers and the muscles they innervate11. The biggest issue with this technique is that it only works for motor pathways. No sensory tracts can be identified. Furthermore, if neurotransmission is blocked for any reason (including nerve compression, trauma, or tumor invasion), there will be no distal muscle twitch observed, so no link can be made in identifying a fiber tractâ€™s targets. A New Technique A group out of the University of California in San Diego attempted to design a peptide that preferentially bound to peripheral nerve tissue. Whitney et al (2011) used a phage display12 technique to develop peptides labelled with a fluorophore which could overcome much of the drawbacks outlined above. They developed several different peptides and after rigorous testing determined that the FAM-NP41 peptide (Fig.1) sequence labelled all peripheral nerves and arborizations with the greatest nerve-to-muscle contrast ratio. Furthermore, they report that this peptide binds both motor and sensory nerve fibers. This labelling technique is claimed to confer many advantages over traditional techniques. For example, according to time-course analysis (Fig.2) of the peptide injection into the sciatic nerve region of aenesthetised mice, the authors found that seonds after the injection they could visualize fluorescence leakage from
the associated capillaries and within minutes nerve fluorescence was significant. However, useful contrast between the nerve fiber and tissue was observable at aroud two hours post-injection, and lasted for several hours thereafter. Furthermore, within 24 hours of the injection, fluorescence had dissipated, and subsequent urine and kidney clearance analysis had shown that the peptides were effectively metabolized and excreted in the urine.
Figure 1 | FAM-NP41 Peptide - due to size, it is suspected that the labelling peptide cannot cross the blood-brain barrier. (Whitney et al, 2011)
Another advantage of this technique is in the imaging of neurons in which neurotransmission has been blocked. As mentioned, electromyography (and in some cases, even axonal transport methods) loses its power if, for example, the nerve has been transected. In testing FAM-NP41 labelling of injured nerves, the authors found that immediately one day after the injury, fluorescence was the same as a control. They did, however, observe a decrease of about 40% three days after, and then regained the loss seven days after. They discovered that this was not because the peptide failed to bind to injured nerves, but because there was a loss of vasculature surrounding the site of injury, thus the delivery of the label was the issue. To test this hypothesis, in one mouse they found that devascularizaiton significantly reduced fluorescence without nerve injury, thus confirming their suspicions. Figure 2 | Fluorescence images of Sciatic nerve and surrounding tissue - Images taken over course of injection show immediately visible leakage from capillaries, and peak contrast at about 3 hours. Fluorescence still present at 5.5 hours post-injection. Label was cleared from tissue within 24 hours. (Whitney et al, 2011, supplementary article)
Human Application and Future Directioins Currently, the only live tests of this labelling technique have been performed on mice. However, the authors also managed to perform some preliminary studies on human tissue. They obtained the resected tissue from a laryngectomy and incubated the nerves and the surrounding muscle tissue with FAM-NP41. Subsequent examination of the tissue revealed that the peptide successfully labelled the connective tissue of nerve fibers (epineurium, perineurium, and endoneurium).
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The next steps in this line of research would be to test it on live human patients. Considering its lack of pharmacological effect and toxicity, as well as its clearance and metabolism, it seems that such clinical tests could be performed in the near future. Furthermore, the size of the peptide seems to prevent it from crossing the blood-brain barrier, so cross-reaction and adverse effects in the central nervous system are not expected.
5 Regenbogen, S.E., Greenberg, C.C., Studdert, D.M., Lipsitz, S.R., Zinner, M.J., Gawande, A.A. (2007). Patterns of technical error among surgical malpractice claims: an analysis of strategies to prevent injury to surgical patients. Annals of Surgery, 246: 705-711. 6 Smith, J.E. (2010). Post-Prostatectomy: Implications for Home Health Clinicians. Home Healthcare Nurse, 28(9): 542-548. 7 American Cancer Society. (2010). Estimated new cancer cases and deaths by sex, US, 2008. Retrieved from http://www.cancer.org/.docroot/CRI/content/CRI_2_2_1X_How_many_ men_get_prostate 8 Penson, D. F., McLerran, D., Feng, Z., Li, L., Albertsen,P. C., Gilliand, F. D., et al. (2005). 5-year urinary and sexual outcomes after radical prostatectomy: Results from the Prostate Cancer Outcomes Study. Journal of Urology, 173(5), 1701-1705. 9 Strothers, L., Thom, D., & Calhoun, E. (2005). Urologic diseases in America project: Urinary incontinence in males—demographics and economic burden. The Journal of Urology, 173(4), 1302-1308. 10 Kobbert, C. et al. Currents concepts in neuroanatomical tracing. Prog. Neurobiol. 62, 327–351 (2000). 11 Davis, W.E., Rea, J.L. & Templer, J. Recurrent laryngeal nerve localization using a microlaryngeal electrode. Otolaryngol. Head Neck Surg. 87, 330–333 (1979). 12 Whitney, M.A, Crisp, J.L., Nguyen, L.T., Friedman, B., Gross, L.A et al. (2011). Fluorescent peptides highlight peripheral nerves during surgery in mice. Nature Biotech. 29(4): 352-358. 13 Shibata, H., Heo, Y.J., Okitsu, t., Matsunaga, Y., Kawanishi T. & Takeuchi S. (2010). Injectable hydrogel microbeads for fluorescence-based in vivo continuous glucose monitoring. PNAS, 107(42): 17894-17898.
Figure 3 | Human never labelling with FAM-NP41 - Cross-sections of human recurrent laryngeal nerve showing fluorescence of epineurium (E,F), and endoneurium (G,H). (Whitney et al, 2011, supplementary article)
The advantages of this technique over other methods of fluorescence labelling are clear. For one, the labelling effects of this technique are not limited to localized areas of tissue, and it also provides visualization of nerve fibers with a significantly higher contrast ratio that allows an observer to discern deep-tissue nerve roots from surrounding muscle and tumours. Secondly, the peptide only binds to connective tissue of nerve fibers (not myelin or axolemma), which may account for the lack of pharmacological or toxic effects on the mice. Third, the time it takes for the peptide to take effect, and its half-life in the tissue makes it a reasonable choice for operations of various lengths. Finally, the ability to label and visualize damaged nerves provides an opportunity for repair of already damaged fibers (either by trauma or surgical error, for example). Although the focus here was mainly on nerve fiber visualization, the technique employed to create the labelling peptide can also be used to create peptides that target other tissue, such as tumours. Furthermore, the use of fluorescence itself can serve many different biomedical purposes, such as the creation of glucosebinding hydrogel beads, which can be used to monitor the concentration of glucose in the blood13. Thus, it seems that something as simple as fluorescence can be strategically and pragmatically implemented to solve and prevent many surgical and medical issues. 1 Kohn, L.T., Corrigan, J.M. & Donaldson, M.S. (2000). To Err is Human: Building a Safer Health System. Institute of Medicine, National Academy Press: Washington D.C. 2 http://www.boeing.com/commercial/airports/misc/A20WE-Rev-45.pdf 3 http://safepatientproject.org/safepatientproject.org/pdf/safepatientproject.org-ToDelayIsDeadly.pdf 4 Stone, S., Bernstein, M. (2007). Prospective Error Recording in Surgery: An Analysis of 1108 Elective Neurosurgical Cases. Neurosurgery, 60: 10751082.
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Neurophysiology Spinal cord injury: Current Breakthroughs and Future Promises in Therapy to avoid rejection of the implanted foreign tissue . Finally, em(7)
Daniel Etarsky Department of Human Biology, University of Toronto Introduction Over 12,000 individuals sustain a spinal cord injury (SCI) every year in the United States, with a total of approximately 260,000 individuals living with an SCI currently (1). It is a devastating condition with drastic consequences to an individualâ€™s life, most commonly occurring as a result of a motor vehicle accident or sports injury. SCI causes the loss of spinal neurons, astroglia, and oligodendroglia (2). Resulting deficits depend highly on the location and extent of the damage, and may range from minor sensory or motor impairment in one or more limbs (incomplete SCI), to the full loss of sensation and voluntary motor function throughout the body (complete SCI). Generally, in both complete and incomplete SCI, some living neurons persist at the site of the lesion however their function is greatly degraded because of extensive demyelination surrounding the lesion site that typically occurs (3). Despite the prevalence of the condition and its severity, current treatment options are very limited. Current clinical therapies are generally limited to the prevention of additional injury and focus on rehabilitative efforts. The efficacy of such therapy is quite limited, and most of the recovery seen can be attributed to the limited endogenous plasticity of surviving neurons at the lesion site rather than direct effects of treatment (3). In addition to limited clinical treatment options, the human body has a very limited natural capacity for regeneration of the damage inflicted by SCI for a variety of reasons. First, at the site of the injury, scar tissue formation may physically obstruct any potential axonal or glial regrowth. Further, the CNS lacks various neurotrophic and growth factors that are conducive to the regeneration of CNS tissue. Furthermore, numerous inhibitory factors, such as some extracellular matrix components and chemorepellants, are found throughout the CNS which actively impede regeneration such as axonal regrowth or remyelination (3). Due to the combination of these factors, the central nervous system is unable to repair damaged areas effectively, unlike the peripheral nervous system. Modern research into SCI therapy thus focuses on methods via which CNS regeneration at the lesion site may be induced, and inhibitory factors overcome. One promising area of research, which will be the focus of this review, is the use of neural stem cell (NSC) grafts to promote repair (4). Such research has had remarkable success in the past within animal models of SCI. The successful and safe restoration of some sensation and motor function by numerous centers around the world in animals such as mice and rats shows great promise (5) (6). Building off of past achievements, a new wave of research with human subjects is just now getting underway. However, such research has long been hindered by lingering ethical issues surrounding the use of embryonic and fetal stem cells. In addition, the use of foreign stem cells for therapeutic purposes requires long-term, intensive immunosuppression
bryonic and fetal stem cell techniques suffer from poor derivation, differentiation, and survival rates stemming in part from low homogeneity of such cell cultures. To overcome many of these issues, researchers have recently turned to induced pluripotent stem cells. These cells can be made from adult tissues, and as the name implies, be induced to a pluripotent state, capable of differentiating into various cell types including neurons, and astrocytes. In the following sections some recent literature on an animal SCI model will be presented, followed by a look at limitations and future directions, and conclued with the possible human applications of this field. Novel Research â€“ Human Induced Pluripotent Cells in Mouse SCI In March of 2012, Fujimoto et al. reported on the treatment of a mouse model of SCI using human induced pluripotent stem (iPS) cells (7). The authors lesioned the spinal cord of NOD-SCID mice at the thoracic vertebra, leading to paralysis of the hind limbs. Seven days post-lesion, the mice received transplants of the human iPS cells into the injury site. Motor function of the hind limbs was monitored for up to ten weeks post-injury and assessed using the Basso Mouse Scale (BMS) locomotor rating scale. After twelve weeks, the capacity of the injured spinal cord to conduct signals was assessed via measurement of motor evoked potentials (MEPs). The MEPs were measured by placing stimulating electrodes into the animalsâ€™ hind limb motor cortex, stimulating the region, and recording the potential which reached the rear hamstring muscle. Numerous tracers, markers, and viruses were employed by the authors in order to determine the fate and differentiated state of the transplanted iPS cells as well as their role in the regeneration of synaptic pathways. Before transplanting the human iPS cells into the mice, the authors tested their differentiation ability in vitro following labelling with GFP. The labelled iPS cells were found to have the same differentiation potential into neurons and astrocytes as unlabelled iPS cells and thus suitable for transplantation. Following this benchmark, the authors injected the iPS cells into the mice 7 days post-injury. As positive and negative controls, the authors injected groups of animals with medium only, and with fetal NSCs (since NSCs have been shown to have therapeutic effects in such mouse SCI models before). BMS scores were tracked for a period of at least 8 weeks, and the results can be seen in Figure 1. As shown, the negative control mice showed significantly less improvement in himb limb motor function as compared to the NSC and iPS cell transplant mice suggesting that the cell transplant is having a direct effect on functional recovery. Further, these results show that iPS cells have a similar effect on recovery as NSCs have been shown to have in the past. Twelve weeks post-injury, the mice had their MEPs recorded. As seen in Figure 1, control mice had significantly reduced MEPs as compared to the iPS transplanted mice, again suggesting that the iPS cells are
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contributing to spinal cord regeneration and functional recovery.
to the lesion site. This result, taken together with the BDA labelling result, indicates that an intact, multi-synaptic “relay” pathway is generated following iPS cell injection in the SCI mice – potentially contributing to the functional recovery seen in these mice. Finally, in an attempt to establish some measure of causation in their results, the authors specifically ablated the injected human iPS cells and their progeny using diphtheria toxin – a toxin selectively lethal for human cell types. Following ablation, the BMS scores of injected mice fell to levels approximate to those seen in SCI control mice – strongly suggesting that the injected iPS cells and their progeny were directly contributing to and causing the functional recovery seen in the animals. Interestingly, though the BMS scores of the ablated iPS injected mice were lower than non-ablated iPS injected mice – they were not significantly so. This suggests that the functional recovery introduced by the iPS cell injection was due in part to some effect of these injected cells on endogenous mouse neurons – perhaps increased neuronal survival and plasticity resulting from increased neurotrophic factor release from the iPSC-derived progeny.
Figure 1. (Nakashima et al.) Panel A - BMS score comparison of treated and control mice. Panel B - MEP measurement of control and treated mice.
In addition to the evidence for functional recovery, the authors wanted to shed light on the mechanism by which the iPS cells were promoting such recovery. Using immunohistochemistry, the authors found that their injected GFP labelled iPS cells had migrated both rostrally and caudally along the lesion site in the mouse spinal cord. These iPS cells also were found to have extended processes into surrounding parenchyma. In addition to migrating along the lesion site, the iPS cells were found to be actively differentiating into various cell types – mainly neurons (expressing Tuj1), astrocytes (expressing GFAP), and rarely oligodendrocytes. The author’s data suggests that the injected iPS cells actively regenerate lost neuronal and astrocytic tissue at the lesion site. Since it is now known that the iPS cells both migrate and differentiate post-injection, the authors wanted to know what impact the injected cells had on existing neurons in the injured spinal cord. By injecting an axonal tracer (biotinylated dextran amine [BDA] – which is not transported across synapses) into the motor cortex of the mice, the authors traced existing neurons from the cortex to the site of the lesion. As compared to healthy control mice, iPS injected and SCI control mice showed significantly less BDA labelling caudal to the injury site – indicating that axonal regrowth was not taking place. Thus, the functional recovery seen is likely not a result of the regrowth of axons damaged by the SCI. If axonal regrowth is not causing the functional recovery, then what is? The authors next investigated the possibility that the injected iPS cells were differentiating into neurons, forming synapses with remaining neurons, and then acting as “relays”, thereby repairing the signal conducting capacity of the spinal cord. To test this hypothesis, the authors again injected the motor cortex of the mice with a tracer. Wheat germ agglutinin (WGA) adenoviruses were used, as they are transported through axons and across synapses as well. The authors found that, compared to SCI control mice, the iPS injected mice showed greater WGA labelling caudal
Conclusions & Future Directions Fujimoto et al.’s study on the use of human iPS cell transplants in SCI injury is one of the first on this topic. It is an exciting development in the field of SCI therapy for numerous reasons. First, the use of iPS cells rather than embryonic or fetal NSCs means that a patient`s own cells could potentially be used – eliminating the need for long term immunosuppression associated with the use of donor embryonic and fetal NSCs. This has the potential to greatly reduce the harmful side effects of SCI therapy and promises to make the use of this therapy in human patients more practical. Further, the use of iPS cells from adult tissue avoids the controversial ethical issues surrounding the use of embryonic and fetal stem cells. These ethical issues have proven to be great barriers to research in this topic (as funding may be difficult to come by). By avoiding these issues altogether, research may be able to proceed more swiftly and thus lead to applicable clinical strategies years sooner, helping many more individuals with SCI. Finally, as the authors mention in their paper, the techniques used to derive stable lines of iPS cells differ from those used to derive NSCs. Techniques involved in the creation of iPS cell lines tend to create more homogenous cultures with greater rates of differentiation capacity and survival. These benefits of iPS cells over NSCs promise to make therapies based off of such methods cheaper and more accessible to those needing it, as well as more effective. Despite all the promising results, many limitations still exist in the field. One of the major obstacles that remain to be overcome is the great risk of tumor formation that is associated with iPS cell grafts. Due to their inherent pluripotent characteristics, iPS cells pose a significant risk of causing tumors in patients. A delicate balance has to be reached between promoting iPS cell proliferation and differentiation, and inhibiting excessive, uncontrolled growth. Techniques which will allow scientists and clinicians of the future to do so must still be elucidated. In addition, though iPS cell grafts do show therapeutic potential, it is far from complete. Lesioned mice never regain their full motor and sensory capacity following iPS transplantation, and as mentioned earlier, damaged axons have not been shown to regrow. Clearly, much work remains to be done to evaluate the mechanisms at play preventing full recovery and axonal regeneration. Furthermore, thus
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far the vast majority of such work has been done in animal models. Research in humans is only now entering its nascent stages. Also, due to its nature, research in humans is likely to progress much more slowly. At present it is unknown if the early successes seen in animal models can be duplicated in humans. As of the time this review is being written, only one clinical trial is in progress testing the efficacy of NSC transplants in humans (8). With some fortune, results from the first human trials may be seen in the next five years, with clinical applications coming in the next decade or two. Moving forward, there is great hope in the field of SCI therapy. Future therapies incorporating iPS cell grafts, combined with neurotrophic factors and other CNS regeneration promoters, may eventually lead to curative therapy for SCI. It is not difficult to imagine a future where paralyzed individuals are able to walk again, or the damage inflicted by degenerating conditions such as amyotrophic lateral sclerosis or multiple sclerosis can be halted and even reversed (9). Lessons learned from research in this topic may have applications in various other fields as well, including research into blindness, dementia, or Parkinson`s. Indeed, nearly every field dealing with CNS damage or degeneration could benefit from advances in SCI therapy as all share common obstacles such as how to induce growth and repair within the CNS.
1. Foundation for Spinal Cord Injury Prevention, Care & Cure. Spinal Cord Injury Facts. Spinal Cord Injury Statistics. [Online] June 2009. http://www.fscip.org/ facts.htm. 2. Neural Stem Cells for Spinal Cord Repair. Sandner, B, et al., et al. February 2012, Cell & Tissue Research, pp. 1-14. 3. Chronic Spinal Cord Injury. Ditunno, JF and Formal, CS. 1994, New England Journal of Medicine, pp. 550-556. 4. Regeneration-Based Therapies for Spinal Cord Injuries. Okano, H, et al., et al. 2007, Neurochem Int, pp. 68-73. 5. Human Neural Stem Cells Differentiate and Promote Locomotor Recovery in an Early Chronic Spinal Cord Injury NOD-SCID Mouse Model. Salazar, D, et al., et al. 2010, PLoS One, pp. 1-15. 6. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. Ogawa, Y, Sawamoto, K and Miyata, T. 2002, J Neruosci Res, pp. 925-933. 7. Treatment of a Mouse Model of Spinal Cord Injury by Transplantation of Human iPS Cell-Derived Long-Term Self-Renewing Neuroepithelial-Like Stem Cells. Fujimoto, Y, et al., et al. 2012, Stem Cells, pp. 1-24. 8. Stem Cells Inc. . Clinical Development Programs. Cinical Trials. [Online] March 2012. http://www.stemcellsciences.com/Therapeutic-Programs/ClinicalTrials.htm. 9. Lumbar Intraspinal Injection of Neural Stem Cells in Patients with ALS: Results of a Phase 1 Trial in 12 Patients. Glass, J, et al., et al. 2012, Stem Cells, pp. 1-17.
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Neuropharmacology Your attention, please: On methylphenidate helping us transcend our boundaries Athena Hau Human Biology Program, University of Toronto, St. George Campus, Toronto, Ontario, Canada The debate around using drugs for enhancement purposes is gaining attention. Methylphenidate (MPH) is one of the most commonly prescribed psychostimulant to help treat ADHD, and is being considered as a neuroenhancer for attention, motivation, and wakefulness. Recent studies have implicated a difference between taking a drug as a treatment and as an enhancement by investigating the effects felt by people with and without ADHD to methylphenidate. The differences in drug response between the two populations and similarities in cognitive benefits imparted by MPH indicate a promising start to MPH being seriously considered as a possible candidate for attentional enhancement. Issues that remain to be resolved include the mechanisms and targets of action, as well as an ethical guideline to define usage. A more attentive person? Pharmaceutical research for drugs has largely been intended for treatment purposes. However, possibilities of augmenting human performance with those very drugs as enhancers have come into heated debate within the past decade. For example, how much attention is enough attention? DSM-IV lists Attention Deficit/Hyperactive Disorder (ADHD) as a disorder that involves the individual being abnormally inattentive, hyperactive, and/or impulsive.1 It affects a large number of people across all ages.2,3 ADHD is hypothesized to be caused by a breakdown in dopaminergic transmission between the cortex and the striatum, which results in the deficits in executive function.4 Methylphenidate (MPH), also known by its trade name Ritalin, is one of the most commonly prescribed psychostimulants to treat ADHD and narcolepsy. Like the rest of the amphetamine family, MPH is a dopamine (DA) and noradrenaline (NA) reuptake inhibitor; by blocking their respective transporters, MPH increases DA and NA levels in the synaptic cleft.4,5 As DA modulates attention and motivation and NA modulates wakefulness amongst other systems, tasks that trigger DA or NA release would show performance improvements. Extensive evidence that drugs used to treat ADHD are being abused had been documented well over a decade ago. Setlik et al. published a study in 2009 investigating the recent statistics of teenage prescription drug intentional abuse and misuse. They analyzed the American national poison data system for poison control centre calls from just before the turn of the 21st century onwards, and found that calls related to prescription ADHD medication abuse had since risen 76%, which was faster than the rate of increase of teenage substance abuse, or non-age-restricted substance abuse in general. Teenage MPH abuse and misuse had risen over 50%, and abuse and misuse of MPH in conjunction with amphetamine, an-
other drug used to treat ADHD, had risen 80%. In contrast, the frequency of MPH being prescribed for treating ADHD had fallen almost 10% to 56%; exposure to MPH itself had fallen from 78% to 30%. In other words, the increase of drug abuse and/or misuse couldn’t be related to the frequency of prescription or exposure. When compared to sales of ADHD medication, the disproportionate increase of prescription ADHD medication-related calls was reflective of accessibility. Severity of cases had escalated over time, and the increase was broad and consistent. All of this suggested a growing problem of teenage abuse and misuse of ADHD medication. What the article termed as abuse and misuse was non-medical usage of prescription medicine. Getting high was, interestingly, only one of the teenagers’ end goals of misuse. Other goals included heightening concentration and alertness, which were precisely the improvements intended for people prescribed with psychostimulant medication. As nonmedical usage of drugs for performance enhancement increase, the underlying implicit question becomes louder. Do using the drugs as enhancers really cause harm to a healthy person? And if moderate use doesn’t cause harm, should enhancers be used to improve performance? The difference between the ‘normal’ brain and the ‘abnormal’ brain One of the crucial distinctions between using drugs as treatments and using drugs as enhancements is the state of the brains upon which they act. Treatment implies that the some part of the brain, whether it be neurobiological or neurochemical, is abnormal or deficient, and the drug is to help restore or bring the brain to a socially normalized level of performance to compensate for that deficit. Enhancement, on the other hand, implies that the brain is normal and the drug is to boost normal performance to superior levels. As such, the neurobiological and neurochemical differences between the healthy and unhealthy brains must be accounted for. Kollins et al. published a study on the effects of MPH on people without ADHD and people with ADHD in 2009. Participants with ADHD and participants without psychiatric diagnoses were scheduled to attend 4 sessions, where they were given, in randomized order, either a placebo or oral MPH in doses of 20mg, 40mg, or 60mg. They were then asked to complete a progressive ratio task, where they would click x number of times to earn a part of the dosage for the next session. The number of completed ratios were measured against the participant’s subjective effects, which indicated how they’d felt with the placebo or dose of MPH. The number of completed ratios was also measured against reported reinforcing effects, which indicated to what extent MPH would be self-administered; this served as a measure of abuse potential.
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The MPH’s subjective effect was found to affect concentration and arousal in both ADHD and nonADHD subjects. The MPH’s reinforcing effect, on the other hand, was found to be significant only in the ADHD population. This implied that brains in different states have different networks of activation when exposed to the same drug. In 2010, Agay et al. published the first study that showed empirical evidence of MPH-induced cognitive enhancements not specific to ADHD. Adults with ADHD and controls were divided into placebo and MPH groups. Once a dose of placebo or 60 mg MPH was administered, they were asked to complete several cognitive tasks that tested working memory, decision making, and risk taking. Working memory is shown to improve in performance across both adults with ADHD and adults without ADHD. Interestingly, decision making was unaffected across both populations. In risk-taking tasks, adults with ADHD routinely took significantly more risks than adults without ADHD, regardless whether they’d received the placebo or MPH. Thus, the study furthered the progression of MPH as a viable candidate for use as an enhancer by collecting empirical evidence that MPH can improve the performance of healthy individuals. Salient concerns and future implications What tempers the experimental confirmations of MPH having a different effect and definite benefit to a healthy individual is the fact that such conclusions are not sufficient to encourage MPH usage as an enhancer. For one, the lack of long-term studies on the effect of MPH on both healthy and ill populations is a serious hurdle.11 Current scientific literature on MPH is centred around acute, short-term therapeutic effects. More must be known about how the brain changes and adapts to drug use over a long period of time before safe recommendations can be offered. Surprisingly, MPH may not be ideal for treating the two classic primary deficits in ADHD.10 ADHD is known to affect prolonged attention and risk taking.10 Working memory is thought of as a secondary deficit of ADHD, if it is indeed a deficit at all; many papers with conflicting conclusions have been published and the matter still has yet to be resolved. Because psychostimulants work on DA levels in the brain, and DA also governs reward, psychostimulants have high abuse risk potentials.4 Unlike amphetamine, though, MPH does not boost DA levels through numerous multiple other means and rarely triggers euphoria at prescription-strength concentrations it, which gives MPH a slightly lower abuse risk profile and may render it more suitable as an enhancer candidate. The fact that MPH does not trigger DA production could also be indicative of a lower effectiveness; however, there is insufficient research comparing drug performances to conclusively maintain a stance on the drugs’ respective efficacies. The abuse potential of MPH is also influenced by the rate of drug delivery.13 As the studies were carried out with short-acting instant-release MPH, more research into the different methods of dose delivery is needed to clarify the difference. In addition, if the psychological effects felt by people who are mentally healthy and people who have ADHD from taking MPH are different9, then there is reason to suspect that MPH taken as an enhancement would subject the user to a different level of abuse potential that to MPH’s level as a treatment. Both studies were noticeably cautious in endorsing MPH as a potential drug for enhancement, and they were right to do so.
MPH has been experimentally determined to benefit only those whose regions of the brain that are active during attentional tasks are not optimally focused; MPH could actually be detrimental to those whose regions are already functioning at optimal levels.14 What the two studies did not show is a conclusive answer to MPH’s benefit in the current dosages that are used for treatment for ADHD. Agay et al.’s study in particular had illustrated the critical need to tease apart the various augmentations that different drugs offer in their mechanisms of action, and in different dosages with different methods of administration. Effectively providing treatment for an illness without fully understanding the drug’s mechanism of action is one matter. Providing enhancement without knowing the drug’s mechanism of action is another matter entirely; one of the differences is in ethics. Treatment implies that there is a duty on part of the physician to improve the patient’s wellbeing. This duty is manifested in legal and moral claims. So far, enhancements do not offer that same claim of duty; the physician is not morally bound to provide enhancements. More than the issue of whether a drug should be used or not, though, is the simple question of what should be done.15 If these drugs are to be legalize as safe for use as enhancements, how do we design the dosage? How should a limit to its use be implemented? Where should the line between beneficial use and dependence be drawn? Though science does not normally play a direct role in policy, policy will ultimately reflect on scientific consideration. For example, if enhancers do become legal for use, how will enhanced performance affect the scales used to evaluate spectrum disorders and disabilities such as autism and ADHD? Professional athletic sports have banned the use of drugs to improve performance on the basis of a loss of the meaning of fair competition. How does that reasoning measure up against banning drugs to improve mental performance, and where should the line be drawn? The fine line between enhancers like caffeine, nicotine, sugar and sleep and enhancers like MPH may very well render the argument weak. And if using MPH does not cause sufficient harm to discourage use, is a person morally obligated to use them to better themselves? Without controlled studies being run to conform MPH’s long-term effects, sufficient knowledge of MPH’s effects on the rest of the brain when taken, and ethics to guide the use, using the drug as an enhancer is not seem feasible. Benchside research needs to collect enough data first, before MPH can proceed to a clinical trial as an effective neuroenhancer. Focusing on the future: Next steps The possibilities offered by neuroenhancements are endless. Soldiers could temporarily overcome the effects of fatigue and sleep deprivation in adverse conditions with huge amounts of distractions. Surgeons could perform for longer periods of time. Pilots and drivers could drastically reduce the amount of errors being committed due to distraction or sleepiness. However, as current studies with MPH as a potential enhancer suggest, the story is not so simple. The two studies have revealed telling factors that differentiate between healthy states and abnormal states, and hinted at the pathways that MPH affect within the brain. However, even though more questions now have to be answered to proceed, cautious optimism can be had in approaching the next few experiments involving different population subgroups and an expanded range of drug dosages. The story of neuroenhancers has only just started. There-
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fore, when more long-term studies on drug epidemiology and pathway of action come to light, perhaps enough details and inferences can be drawn to fully realize the possibility of using MPH, or indeed any drug, to help people exceed their limits. 1. American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision. Washington, DC, American Psychiatric Association, 2000. 2. Faraone SV, Sergeant J, Gillberg C, Biederman J. (2003). “The worldwide prevalence of ADHD: is it an American condition?” World Psychiatry. 2 (2): 104 –113. 3. Kessler RC, Adler L, Ames M, et al. (2005). “The prevalence and effects of adult attention deficit/ hyperactivity disorder on work performance in a nationally representative sample of workers.” J Occup Environ Med, 47 (6): 565–572. 4. Grace AA. (2001) Psychostimulant actions on dopamine and limbic system function: Relevance to the pathophysiology and treatment of ADHD. In: Solanto MV, Arnsten AFT, Castellanos FX (eds) Stimulant Drugs & ADHD: Basic and Clinical Neuroscience. Oxford University Press, Oxford, pp 134–157. 5. Spencer TJ, Biederman J, Madras BK, Faraone SV, Dougherty DD, Bonab AA, Fischman AJ. (2005). “In vivo neuroreceptor imaging in attention-deficit/hyperactivity disorder: a focus on the dopamine transporter”. Biological Psychiatry, 57: 1293–1300. 6. Volkow ND, Wang GJ, Fowler JS, Telang F, Maynard L, Logan J, Gatley SJ, Pappas N, Wong C, Vaska P, Zhu W, Swanson JM. (2004) “Evidence that methylphenidate enhances the saliency of a mathematical task by increasing dopamine in the human brain”. American Journal of Psychiatry, 161: 1173–1180. 7. Ding Y-S, Hannestad J, Planeta-Wilson B, Gallezot, J-D , Lin S-F, Ropchan J, Labaree D, et al. (2009). “Oral methylphenidate (Ritalin) displays high potency to block norepinephrine transporters: a PET study with (S,S)-[ 11C]MRB in healthy subjects”. Journal of Cerebral Blood Flow & Metabolism,29: S63. 8. Setlik J, Bond GR, & Ho M. (2009). “Adolescent Prescription ADHD Medication Abuse Is Rising Along With Prescriptions for These Medications”. Pediatrics, 124 (3): 875-890. 9. Kollins SH, English J, Robinson R, Hallyburton M, & Chrisman AK. (2009). “Reinforcing and subjective effects of methylphenidate in adults with and without attention deficit hyperactivity disorder (ADHD)”. Psychopharmacology, 204: 73–83. 10. Agay N., Yechiam E., Carmel Z. & Levkovitz Y. (2010). “Non-specific effects of methylphenidate (Ritalin) on cognitive ability and decision-making of ADHD and healthy adults”. Psychopharmacology, 210: 511–519. 11. King S, Griffin S, Hodges Z. (2006). “A systematic review and economic model of the effectiveness and cost-effectiveness of methylphenidate, dexamfetamine and atomoxetine for the treatment of attention deficit hyperactivity disorder in children and adolescents”. Health Technology Assessment, 10 (23): iii–iv, xiii–146. 12. Madras BK, Miller GM, Fischman AJ. (2005). “The dopamine transporter and attention-deficit/hyperactivity disorder”. Biological Psychiatry, 57: 1397–1409. 13. Spencer TJ, Biederman J, Ciccone PE, Madras BK,Dougherty DD, Bonab AA, Livni E, et al. (2006). “PET Study Examining Pharmacokinetics, Detection and Likeability, and Dopamine Transporter Receptor Occupancy of Short- and Long-Acting Oral Methylphenidate”. American Journal of Psychiatry, 163: 387–395. 14. Volkow ND, Fowler JS, Wang G-J, Telang F, Logan J, Wong C, Ma J, Pradhan K, Benveniste H, Swanson JM. (2008) “Methylphenidate decreased the amount of glucose needed by the brain to perform a cognitive task”. PLoS ONE 3:e2017. 15. Greely H, Sahakian B, Harris J, Kessler RC, Gazzaniga M, Campbell P, and Farah NJ. (2008). “Towards responsible use of cognitive-enhancing drugs by the healthy”. Nature, 456: 702–705.
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Personality Disorders The behavioural disorder of the future: Internet addiction disorder Nicola Hyslop Human Biology Department, University of Toronto, Toronto, ON 60101 Internet Addiction Disorder, also known as excessive Internet use or problematic Internet use, is a relatively new phenomen of maladaptive internet use. It typically displays as either an additction to cyber-sex, cyber-relations, or online gaming. Due to a lack of standard diagnostic criteria of the disorder, it is roughly estimated that between 1.5 and 8.2% of the population as the disorder. In the most extreme of cases, the disorder presents with withdrawal and tolerance symptoms as well as the breakdown of social relationships. Considerable gray and white matter reductions are seen in the orbitofrontal cortex, rostral anterior cingulate cortex, and supplementary motor area. All of these are areas where comparable reductions are seen in drug-addicted patients. There is currently no standardized treatment of the disorder though psychosocial treatments like cognitive behavioural therapy, family talk therapy, and group therapy are common. There is some evidence to suggest pharmacological treatment of IAD is efficient. The inclusion of the disorder in the upcoming DSM-V is contended by many, who worry Internet addiction is merely a symptom of the comorbid disorders. However, DSM-V inclusion would lead to a standardized, generally accepted set of diagnostic criteria which would thereby make all future studies comparable and conclusive. It would also lead to better treatment for those suffering from Internet addiction, be it a true disorder or not, and would increase research dollars in the field. It is therefore proposed that Internet Addiction Disorder be including in the DSM-V, and later removed from the revised DSM-V if further research proves it is not a disorder in itself. I. BACKGROUND Recent advances in computer technology have meant that the Internet has become a key part of daily life for many. In 2009, there were over 1.5 billion Internet users, a number that is expected to have risen significantly in the past three years1. With the ever-increasing number of Internet users has come the phenomenon of maladaptive Internet use. The United States Federal Communications Commission declared the online role-playing game, World of Warcraft, to be one of the top reasons students fail out of university1. Perhaps some of the most extreme cases of excessive Internet use have been in South Korea, where ten cardiopulmonary deaths occurred in Internet cafes after unrelenting internet use2. Accordingly, the sudden rise in pathological internet use has led to the proposal of a new compulsive-impulsive/ addiction disorder, Internet Addiction Disorder (IAD)3. Because of its relatively new status, IAD is unique in having no set diagnostic criteria, but is generally defined as “internet use leading to clinically significant impairment or distress”1.
IAD patients generally present with one of three phenomenological subtypes: excessive gaming, cyber-relational addiction (that is, chat rooms, social networking), and cyber-sexual addiction3. These three sub-types are all purported to show the four components of stereotypical addiction disorders: excessive use, withdrawal, tolerance, and negative repercussions2. While anecdotal evidence supports the case for withdrawal and tolerance phenomena (for example, nausea and tremor), there are currently no empirical studies proving such to be true2,4. As such, the inclusion of IAD in the DSM-V is quite contentious. There is considerable debate as to the nature and pathogenesis of the disorder. Some are even questioning whether IAD is a disorder in itself and not simply a manifestation of another5. This is further complicated by the high frequency of comorbid disorders presented alongside IAD3. This review will examine the merits and fall backs of the recognition of IAD as an independent disorder by the DSM-V by focusing on diagnosis, epidemiology, anatomical brain changes, and treatment options. II. PROPOSED RESEARCH - Diagnosis and Epidemiology As there is no generally agreed upon diagnostic criteria for IAD, researchers in different countries and use entirely different sets of diagnostic criteria. The most commonly used questionnaire, Young’s Internet Addiction Scale, was modified from the DSM-IV diagnostic criteria of pathological gambling3,6. According to this test, an IAD patient exhibits five of eight putative symptoms: preoccupation with computers, cravings, failure to stop computer use, depressive feelings, lack of timemanagement, breakdown of social relationships, hiding internet use to family, using the Internet to escape negative feelings1. Other diagnostic questionnaires include the Chen Internet Addiction Scale, the Questionnaire of Experiences related to Internet, and the Compulsive Internet Use Scale3. Different countries have validated different questionnaires. For example, while Spain uses the Compulsive Internet Use Scale, the United Kingdom and Korea use Young’s scale3. Since different questionnaires are based on different theories of Internet addiction, they do not form comparable diagnoses. This causes a gross discrepancy between countries in terms of epidemiology and a total inability to draw conclusions or comparisons from studies produced with different diagnostic criteria3. As a result, international IAD prevalence rates are extremely variable—between 1.5-8.2% of the global population3. For example, numbers of IAD patients in the United States have been estimated to be between 0.7-6% as compared to those in Taiwan which are expected to be around 17.9%3. Rather interestingly, 86% of IAD cases present with some other DSM-IV recognized disorder such as ADHD, major depressive
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disorder, and social phobia6. The staggering percentage of comorbidity has led many to question whether IAD is merely a symptom of an underlying disorder or a psychiatric disorder in its own right. While the bi-directionality of IAD and its comorbidities has not yet been addressed (i.e. are patients depressed because of their intense internet use or do they use the internet because they are depressed), there is evidence to suggest excessive use is more than just a symptom. IAD makes co-morbid disorders much less responsive to therapy, unless the IAD is treated directly as its own disease2. Neuroimaging While IAD neuroimaging studies are extremely limited, two studies have shown white and gray matter changes in patients. Decreased gray matter in dorsolateral prefrontal cortex, supplementary motor area, anterior cingulate cortex, cerebellum, and orbitofrontal cortex were noted in patients with severe IAD using voxel-based morphometry7. The degree of gray matter atrophy was strongly correlated to the duration of Internet addiction. Interestingly, areas like the rostral anterior cingulate cortex and the orbitofrontal cortex have shown similar atrophy in severe cases of heroin-dependence and cocaine use7. While interesting, the results are derived from extremely small sample sizes (n=18). They are also purely correlational and do not indicate whether internet addiction causes these brain changes or if a reduction in gray matter predisposes someone to internet addiction. White matter changes have also been examined in several studies, though the results are somewhat spurious. Diffusion tensor imaging showed a reduction in white matter of orbito-frontal white matter, corpus callosum, cingulum, and internal and external capsules in IAD patients8. This is in stark contrast to another study, also using DTI analysis, which found an increase in internal capsule white matter7. Clearly, further imaging studies are much warranted considering the polar-opposite findings between studies. Treatment In part because of the lack of DSM recognition, IAD currently has no standardized course of treatment1. Existing treatment is largely modeled on that of substance abuse disorders3. Psychosocial treatment is quite common, especially cognitive behavioural therapy (CBT). A study on 114 IAD patients receiving CBT showed patients significantly improve by controlling their online time by eight therapy sessions3. CBT can be supplemented with group or family therapy to overcome social phobia and deal with the social strains seen with chronic Internet abuse. Pharmacological intervention is, of course, also used. However, there are next to no empirical studies following the course of treatment to show efficacy of the medication. One study tested the efficacy of the SSRI escitalopram in patients with IAD9. Ten-weeks post-treatment initiation, Internet usage had decrease significantly, with approximately 64.7% of patients feeling “very improved”. Since IAD frequently presents with another psychiatric disorder, treatment of the comorbid disorder should also occur. One study had 62 children with ADHD and simultaneous IAD treated with methylphendate (MPH). MPH-treated patients had a reduction in Internet use after 8 weeks of treatment10. This notion that treatment of an underlying disorder can abolish IAD presentation may support IAD’s exclusion from the DSM-V. Of course, there are more radical therapies in place. Transcutaneous Electrical Nerve Stimulation has been used on IAD patients to effectively reduce time on the Internet11. Additionally, The Gen-
eral Hospital of Beijing Military Region has implemented a military-like training camp, in which patients undergo intensive physical training, drills, and schedules alongside standard treatments with apparent success (though no empirical data was available)11. Future Directions Considering IAD is a global phenomenon observed in almost every developed country, there is a clear argument that it merits further research. Currently, IAD is not recognized by any official diagnostic system like the DSM. Official recognition is the clear next step for further investigation into the pathogenesis and treatment of the disorder. Set diagnostic criteria would not only provide clear statistical data regarding the international and national prevalence of the disorder, but would also ensure every study used comparable selection criteria for its subjects. Comparable studies would mean the scientific community would have a clearer, more reproducible idea of the mechanism of IAD action. DSM recognition would also mean psychiatrists would be trained in the diagnosis and treatment of the disorder, thus serving IAD patients much better. More funding and research would be channeled towards the disorder4. Of course, there are staunch opponents of the inclusion of IAD in the DSM-V. As of now, it is not clear whether IAD is, in fact, a disorder or whether it is just a symptom of another disorder. There are no empirical data to prove withdrawal or tolerance to the Internet exists and most data is from case studies or intervention studies with incredibly small sample sizes. It is frequently argued that by classifying IAD as a disorder when it is ultimately not one, we are pathologize-ing normal behavior and undermining the legitimacy of psychiatry 4. Undoubtedly, further research—including neuroimaging studies—with larger sample sizes are needed to legimatize the IAD diagnosis. Somewhat ironically, this can only be done through official DSM-V recognition. It is thus proposed that IAD be recognized in the DSM-V. If further research ultimately negates the definition of IAD as an independent disorder, IAD can be removed from the revised DSM-V4 1. C. Flisher, Getting plugged in: An overview of Internet Addiction. Journal of Paediatrics and Child Health. 46, 557-559 (2010). 2. J.J. Block, Issues for DSM-V: Internet Addiction. American Journal of Psychiatry. 165, 306-307 (2008). 3. A. Weinstein, M. Lejoeux, Internet Addiction or Excessive Internet Use. 36, 277-283 (2010). 4. R. Pies. Should DSM-V designate “Internet Addiction” a mental disorder? Psychiatry. 6, 31-37 (2009). 5. S. Bernardi, S. Pallanti. Internet addiction: a descriptive clinical study focusing on comorbidities and dissociative symptoms. Comprehensive Psychiatry. 50, 510-516 (2009). 6. C.H. Ko, J.Y. Yen, C.F. Yen, C.S. Chen, C.C. Chen. The association between Internet addiction and psychiatric disorder: A review of the literature. European Psychiatry. 27, 1-8 (2012). 7. K. Yuan, W. Qin, G. Wang, F. Zeng, L. Zhao, X. Yang, P. Liu, J. Liu, J. Sun, K.M. von Deneen, Q. Gong, Y. Liu, J. Tien. Microstructure abnormalities in adolescents with Internet Addiction Disorder. PLoS ONE. 6, e20708 (2011). 8. F. Lin, Y. Zhou, Y. Du, L. Qin, Z. Zhao, J. Xu, H. Lei. Abnormal white matter integrity in adolescents with Internet Addiction Disorder: A tract-based spatial statistics study. PLoS ONE, 7, e30253 (2012). 9. B. Dell’Osso, S.J. Hadley, A. Allen. Escitalopram in the treatment of impulsive-compulsive internet usage disorder: an open-label trial followed by a double-blind discontinuation phase. Journal of Clinical Psychiatry. 69, 452-456 (2008).
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10. D.H. Han, Y.S. Lee, C. Na. The effect of methylphenidate on Internet video game play in children with attention-deficit/hyperactivity disorder. Compr Psychiatry. 50, 251-256 (2009). 11. X. Huang, M. Li, R. Tao. Treatment of Internet Addiction. Curr Psyciatry Rep. 12, 462-470 (2010).
Neurophysiology Treating strokes with music Falisha Karpati Human Biology Department, University of Toronto, Toronto, ON M5S1A1 Stroke, an interruption to blood flow in the brain, affects thousands of people each year and can cause a wide variety of impairments including loss of motor and cognitive functioning. Music therapy has recently begun to be used in treatment for ischemic stroke. Although a standardized protocol for music therapy implementation is yet to be developed, common methods include instrumental performance therapy using a specialized drum set and keyboard as well as vocal performance therapy. These types of therapy have been shown to lead to increases in fine and gross motor skills of the upper limb as well as improved dysphagia following a stroke. Music listening therapy is correlated with improved attention and memory as well as reduced depression and confusion poststroke. Rhythmic auditory stimulation is a therapy method combining music performance and listening, and is correlated with improved mood and interpersonal relationships following a stroke. The behavioural effects of music therapy are supported by neurological evidence, including increased activation in motor regions, greater cortical connectivity and increased mismatch negativity response. Music therapy is effective in improving the motor functioning, cognitive functioning and mood in individuals who have experienced ischemic stroke. Future directions include standardizing a treatment protocol, and conducting imaging studies to determine the brain areas affected by various types and aspects of music therapy in order to be able to select specific treatments for individuals based on their lesion pattern. In the future, music therapy will likely be implemented in regular clinical practice in stroke treatment. I. BACKGROUND - Stroke Stroke is a condition caused by an interruption to the blood flow in the brain. It affects approximately 40000-50000 Canadians each year, and at any given time, approximately 300000 Canadians are living with the effects of stroke.1 There are two main types of stroke, hemorrhagic and ischemic. Hemorrhagic strokes are caused by excessive bleeding in the brain, likely due to a ruptured blood vessel. Ischemic strokes constitute about 80% of all strokes, and will be the focus of this paper. They are caused by a blockage of a blood vessel in the brain, reducing or eliminating blood flow to certain brain areas.1 The specific areas damaged depends on which blood vessel is blocked, and the result-
ing impairments depend on which brain areas are damaged. Motor impairments are very common after stroke, occurring in 90% of patients. These can occur throughout the body, commonly in the upper and lower limbs, and are often severe enough to prevent participation in work and other daily activities.2 Both gross and fine motor skills can be affected. Motor impairments are often observed in the muscles of the mouth and throat, leading to difficulties in speech and swallowing. Approximately 67% of individuals experience dysphagia (difficulties in swallowing) post-stroke.3 Cognitive impairments are also common following a stroke, including difficulties in attention, memory, language comprehension and production, as well as executive functions.4 Mood disturbances are also observed in stroke patients, likely due to a combination of brain lesions as well as the shock and stress experienced from the traumatic event of stroke. Approximately 35% of stroke patients experience depression and 25% experience anxiety.5 The costs of stroke are very high, both in the impairments it causes for individuals as well as economic costs to society. Stroke costs approximately $3.6 billion per year in Canada, to cover medical treatment and the loss of productivity from stroke patients.1 The use of music therapy in stroke treatment can help alleviate these costs, by improving the motor functioning, cognitive functioning and mood of stroke patients using a relatively simple and inexpensive treatment. The method of music therapy will now be discussed. Music Therapy As it is a recently developed therapy, there is no standardized treatment protocol for music therapy. Various groups perform various types of music therapy, but they have all shown to have very significant positive effects on individuals who have experienced stroke. The main types of music therapy are instrumental performance therapy, vocal performance therapy, listening therapy and rhythmic auditory stimulation. Instrumental performance therapy involves the use of a specialized drum set consisting of 8 drums in a semi-circle, and/ or a small keyboard with 8 keys. A series of tones is played on the drums or keyboard by the therapist and the patient is instructed to repeat it, and is given feedback on their performance by the therapist. These tone series are worked on for the duration of the session. The patient uses their full hand to hit the drums, and individual fingers to press the keys of the keyboard. The series of tones can vary in
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length, speed and complexity depending on the level of the patient.2 In vocal performance therapy, the therapist works with the patient to learn to sing series of notes or songs. Respiration and phonation exercises are also conducted.5 The frequency and length of music performance therapy varies by therapist, expressing the need for a standardized treatment protocol. Music listening therapy involves listening to oneâ€™s music of choice for one hour per day for two months.6 Rhythmic auditory stimulation (RAS) combines music performance and listening. Music with a strong rhythm is played, and patients engage in repeated behaviours such as sitting and standing to the rhythm of the music. They also play percussion instruments such as drums to the beat of the background music. RAS is commonly conducted in groups, for two hours a week for 8 weeks.7 A literature search for studies describing the effects of music therapy on stroke patients was conducted, and the results are summarized below. II. RESULTS - Motor Effects Instrumental performance therapy has been shown to increase both gross and fine motor skills in the upper limbs of stroke patients.2,8 Compared to those who received constraint-induced therapy, those who received instrumental performance therapy showed increased frequency, velocity and smoothness of finger and hand tapping bilaterally.2 They also showed increase in performance of standardized motor tests, such as the box and block test, 9-hole peg test and the Action Research Arm Test, which test skills such as reaching for, grasping and moving objects.8 Vocal performance therapy has been shown to improve dysphagia and motor speech production following stroke.3 Improvements were observed in cough and swallow reflexes, resting and speech respiration, and increased motor control of the larynx.3 RAS is correlated with increased range of motion in the ankle and shoulder on the side affected by the stroke, as well as increased flexibility of the affected arm.7 These effects are supported by neurological evidence. Instrumental performance therapy is correlated with a decrease in power of the event-related desynchronization response bilaterally before movement onset as measured by EEG. In contrast, patients who did not receive music therapy did not show this decrease.9 Decrease in power of the event-related desynchronization response can be interpreted as increase in activation of the motor regions of the brain.9 Increased cortical connectivity is also observed after instrumental performance therapy. EEG measurement has shown greater co-activation between electrodes placed over various brain areas after music therapy as compared to those who did not receive this therapy.9 The specific brain areas involved have not been extensively studied, but it has been demonstrated that the overall amount of communication between brain areas is increased after music therapy.9 Cognitive Effects Music listening therapy has been shown to increase cognitive functioning post-stroke more than those who listened to audiobooks or received no listening intervention. Specifically, increases were observed in verbal memory, as measured by the ability to recall word lists, and focused attention, measured by reaction time in mental arithmetic and the Stroop test.4 The neurological correlate of this increase in cognitive function is an increase in the frequency and amplitude of the mismatch negativity (MMN) response as measured by MEG (Figure 3). MMN is a response observed when a violation in a repeated
auditory pattern is perceived.4 MMN has previously been shown to correlate with increases in cognitive functioning, and this finding was supported by the observation that the level of MMN increase of an individual correlated with their level of cognitive improvement.4 Mood Effects Several types of music therapy have effects on patientsâ€™ moods. Music listening therapy is correlated with decreased depression and confusion following stroke, as compared to those who listened to audiobooks or did not receive listening material.4 Vocal performance therapy has also been shown to reduce depression and anxiety, as measured by the Beck Inventories before and after treatment.5 RAS led to increased reports of positive mood, which was correlated with reports of increased quality and frequency of interpersonal relationships following treatment.7 III. Disucssion The results described above suggest that music therapy can improve individualsâ€™ motor function, cognitive function and mood following a stroke. They suggest that there is something specific to music-based treatment that is causing these improvements, not just a massed-practice structure or general auditory stimulation. Individuals receiving constraint-induced therapy (which also uses a massed-practice structure) or who listened to audiobooks did not show the improvements observed in those who received music performance or listening therapy.2,4 There are several possibilities for what causes musicbased therapies to show more improvements than related nonmusic-based therapies. Music listening has been shown to activate many of the same brain areas that are affected by middle cerebral artery stroke4, which was a common type of stroke in the participants of the above-described studies. By listening to music, either as part of music listening therapy or in performance therapy sessions, neurons and connections in these areas are being activated. This likely leads to long-term potentiation, strengthening these connections and make them more easily activated in subsequent situations. Listening to music containing both instruments and lyrics activates the brain bilaterally, whereas listening to purely verbal material activates mainly the left hemisphere. This bilateral activation strengthens connections on both sides of the brain, therefore it can be effective regardless of the side of the lesion.4 Another aspect unique to music therapy, mainly performance therapy and RAS, is auditory-sensorimotor coupling. This is supported by the increase in cortical coherence correlated with music therapy as described above.9 When one is playing music or coordinating motor behaviours with music, brain regions involved in auditory processing must be activated. The activation of motor areas would be temporally coupled with this in order to plan and execute the next required action based on the auditory stimulus that was heard. Activation of frontal areas at this time is also likely occurring, in order to decide if the previous action was correct. A third contributing factor to the effectiveness of music therapy is its inherently rewarding and motivating nature.2 Patients who have received the therapy rated it as enjoyable and effective, and would recommend it to others.7 This enjoyable nature of the treatment likely assists in the mood improvement observed following the treatment, as well as motivation to comply with the music therapy protocol as well as other treatment types such as medication. Improved compliance then likely leads to optimal benefit obtainment from treatment.
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Although not yet studied in humans, animal studies provide a suggestion for another potential reason for the benefits of music therapy. They have shown that exposure to frequent complex auditory stimulation was correlated with increased neurogenesis and increased glutamate transmission in several brain areas, mainly the anterior cingulate cortex.4 As this area is involved in emotion and cognitive functioning such as attention, functions observed to be affected by music therapy, it is feasible to hypothesize that these mechanisms may be at work in humans. Significance Music therapy leads to improvement in a wide variety of impairments caused by stroke, which is supported by both behavioural and neurological evidence. It is also inexpensive, portable, and requires minimal human and material resources. There are also no known side effects to this treatment. Therefore, music therapy should be developed more extensively then implemented in regular clinical practice in stroke treatment. Future Directions Future directions to move towards implementation of music therapy in regular clinical treatment for stroke include developing a standardized treatment protocol and determining the brain areas affected by various types and aspects of music therapy. Currently, the length of therapy sessions, frequency of sessions and length of treatment of music performance therapy is extremely variable depending on the therapist or research group. The protocols of music listening therapy and RAS are consistent, but comparisons of different protocols have not been conducted so it is unsure if the one used is the most beneficial. Future studies comparing different treatment schedules should be conducted for each type of music therapy. Neuroimaging studies should also be conducted to determine which brain areas are affected by different types of music therapy. This could consist of structural imaging before and after treatment, or functional imaging during a treatment session. This would allow medical professionals to choose a music therapy program for a patient based on which pattern of brain structure/ function improvements matches best with the patientâ€™s lesion pattern. The future of music therapy can include a standardized treatment protocol that can be personalized to patientâ€™s needs.
com/site/c.ikIQLcMWJtE/b.3483991/k.34A8/Statistics.htm (2011). 2. Schneider, S., Munte, T., Rodriguez-Fornells, A., Sailor, M. & AltenmĂźller, E. Music-supported training is more efficient than functional motor training for recovery of fine motor skills in stroke patients. Music Percept 27, 271-280 (2010). 3. Kim, S.J. Music therapy protocol development to enhance swallowing training for stroke patients with dysphagia. J Music Ther, 47, 102-119 (2010). 4. Sarkamo, T. et al. Music listening enhances cognitive recovery and mood after middle cerebral artery stroke. Brain, 131, 866-876 (2008). 5. Kim, D.S. et al. Effects of music therapy on mood in stroke patients. Yonsei Med J, 52, 977-981 (2011). 6. Sarkamo, T. et al. Music and speech listening enhance the recovery of early sensory processing after stroke. J Cognitive Neurosci, 22, 2716-2727 (2010). 7. Jeong , S. & Kim, M. Effects of a theory-driven music and movement program for stroke survivors in a community setting. Appl Nurs Res, 20, 125-131 (2007). 8. Schneider, S., Schonle, P. W., Altenmuller, E. & Munte, T. F. Using musical instruments to improve motor skill recovery following a stroke. J Neurol, 254, 1339-1346 (2007). 9. Altenmuller, E., Marco-Pallares, J., Munte, T. F. & Schneider, S. Neural reorganization underlies improvement in stroke-induced motor dysfunction by music-supported therapy. Ann NY Acad Sci, 1169, 395-405 (2009).
1. Heart and Stroke Foundation. Statistics. http://www.heartandstroke.
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Neuroimaging Decoding the brain: Reconstruction of visual experiences and the future of mind-reading Priscilla Kwan Neuroimaging has revealed a great deal about brainenvironment interactions through studies of stimuli-evoked activation. An emerging trend in the field is the technique of “inverse” imaging, which uses brain activation patterns to reconstruct stimuli in their original form. Recent successes in the field of visual stimuli reconstruction have drawn huge interest regarding “mind-reading” machines, whereby brain-computer interfaces allow for the projection or communication of visual imagery, thoughts, or intentions. Here we present the results of a landmark study published last fall, where researchers at UC Berkeley successfully reconstructed dynamic natural movies from visual cortex activity detected by fMRI. The use of a new motion-energy encoding model signals rapid advancements in the technological sphere, as well as more innovative techniques in decoding the brain. These results have enormous implications for the future of neuroimaging, and are potentially highly significant in the realm of brain-machine interfaces and neuroethics. I. BACKGROUND The last three decades have brought on immense progress in the understanding of cortical representations and brain activity. Advancements in neuroimaging have allowed us to see into the brain, track its activation and correlate magnetic or hemodynamic signals with very specific task-related activity. Scientists are now interested in modeling brain activity in a realistic manner, using current neuroimaging techniques to determine precisely how stimuli are coded within the brain. This decoding initiative most typically involves stimulus reconstruction – a process by which researchers attempt to recreate stimuli by their elicited patterns of brain activity. This paper will focus on visual stimulus reconstruction as it is a relatively more studied area and because the visual cortex has been comprehensively mapped for studies of this nature. The concept of reconstructing stimuli is not in itself a novel one; several breakthroughs had been made leading up to the seminal results of the paper being presented. In 2006, Thirion and colleagues successfully achieved what they termed “inverse retinotopy” – using fMRI-evoked activity in the primary visual cortex to infer properties about static (2-D) visual stimuli (Thiron et al, 2006). The experiment was able to achieve a higher level of accuracy than any previous work due to alterations in the stimulusactivation computational method. It also confirmed that mental imagery (of visual stimuli) causes pattern-specific activation in other visual areas. This was followed up by a 2009 study published in Nature, detailing the results of an experiment that used fMRI-detected brain activation from the visual cortex to interpret how subjects oriented objects in their visual working memory (Harrison & Tong, 2009). This was a significant breakthrough
as it confirmed that feature information is held in visual memory using predictable cortical representations - the contents of which can be decoded even when no stimulus is actually present. This has since been replicated in a study which used fMRI to detect subjective mental states (Tamaki & Kamitani, 2011). Two major gaps exist in the literature leading up to the present study. The first is that no experiments have successfully been able to reconstruct dynamic, moving stimuli in the form of movies; this is partially because computational and software-based technology has not yet advanced enough to process such rapid changes in brain activity. The ability to reconstruct visual experiences, as opposed to predicting imagery or mental states, could play an important role both in expanding the current understanding of visual processing and in applying this technology in a therapeutic setting. Here, we present the results of a 2011 study that addresses both these challenges and, from the preliminary results, appears to overcome them in the form of a new motion-energy encoding model that allows for the capturing of fMRI signals as subjects are processing movie clips. The result – a successful, 1-minute reconstruction clip of dynamic, moving images – has striking implications for researchers in this field, as well as longer-term considerations in areas of neuroimaging, therapy, and neuroethics. II. PROPOSED RESEARCH - Materials and Methods The present study by Nishimoto et al, part of Jack Gallant’s lab at the University of California Berkeley, aimed to create the first “quantitative model of dynamic mental events,” using fMRI-evoked brain activity to reconstruct movie clips. A brief review of the methods follows, as well as presentation of major results. Three healthy human subjects viewed a series of clips from colour movies (each clip between 10 and 20 s in length) while functional MRI scans were taken. Only data from the primary visual cortex was analyzed for this experiment, as this area deals only with surface features of visual stimuli. Data was fed into a computing system that mapped out each three-dimensional voxel in the primary visual cortex and created regression models that matched frames from each video clip with patterns of activation in each voxel. Notable was the use of a new motionenergy encoding model that uncoupled the rapidly changing visual stimuli from the slower hemodynamic mechanisms that are the basis for BOLD signals in fMRI scanning. The model was first tested for its accuracy in predicting BOLD signals. Stimuli pass through filters that create an output (predicted BOLD signal), which could then be compared to the actual BOLD signal to measure prediction accuracy (see Figure 1). The model was found to produce higher prediction accuracy than previous static models.
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Results The resulting video reconstruction – in which presented clips and reconstructed clips are showed side by side for comparison – can be seen online at doi:10.1016/j.cub.2011.08.031. This was the first instance of a dynamic visual reconstruction from fMRI-evoked brain activity. Moreover, it demonstrated several important insights about visual activity and the strengths of this new model. Firstly, it confirmed the relationship between specific activity in the primary visual cortex and the stimulus features they represent. The authors note a second established relationship between eccentricity (how clear the stimulus is) and optimal speed of eye movement. The reconstruction was notably done to a higher level of accuracy than had previously been achieved.
lus coding may not be as superficial in its activation of areas responsible for more complex stimulus processing (Tong, 2011). Nonetheless, these results are potentially highly significant to the neuroscientific community, and may have wider implications for society further down the line. This is the first achievement of dynamic stimulus reconstruction from brain activity alone; the advancement of computational processes (in the form of the motionenergy encoding model) is also a notable step forward in overcoming past limitations of fMRI BOLD signals. In a larger sense, decoding the brain has enormous potential to further our understanding of neurobiology, and to help transform that knowledge into therapeutically significant treatments. As the rates of neurodegenerative disease continue to rise in North America, one can imagine the
Figure 1. Reconstructions of Natural Movies Using fMRI-detected Brain Activity. (Taken from Nishimoto et al, 2011) For each block (A, B, C): The top row shows the video clips viewed by each subject. The second through sixth rows show the five frames from previously viewed clips that produce the closest-matched activation in the visual cortex. Reconstructed movies are shown in the bottom row of images (as screenshots). A video of the final reconstruction is available at doi:10.1016/j. cub.2011.08.031. Right: a graph showing the relative accuracy of the reconstruction for both maximum a posteriori (MAP) and averaged high posterior (AHP) reconstruction. Both measures are significant for all three subjects (S1-3), with AHP reconstruction found to be more accurate than MAP.
Discussion The use of neuroimaging technology to decode the brain has so far taken the form of stimulus reconstruction, particularly in the visual modality. This review presents the results of an fMRI experiment in which dynamic visual experiences were reconstructed – in the form of movie clips – for the first time. Using fMRI-evoked primary visual cortex activity and a new, advanced encoding model for generating predicted BOLD signals, researchers were able to successfully reconstruct primitive movies in a 1-minute video clip. The authors note several limitations that must be addressed in the discussion of this study. The first concerns sample size: only 3 subjects were used for the reconstruction, and each viewed only a limited number of clips with which the computer reconstructed their visual activity. The strength of a dictionary, in a sense, is limited by the number of terms it contains; in this regard, a larger sample size (number of video clips) would be expected to produce more accurate reconstructions. Gallant and colleagues emphasize the preliminary nature of this type of study, and place an emphasis on replication and accuracy in future experiments. Some have cautioned against the generalization of this technique to higher-order visual areas, arguing that stimu-
benefits of such an instrument for the communication-impaired. If inverse imaging helps decode the brain, the next logical step forward is to apply that code in meaningful ways; this is the field of brain-machine interfaces (BMI), which aims to connect the brain with an external device, harnessing the power of brain activity to manipulate the environment, independent of human motor systems. Already, researchers are experimenting with brain-computer interfaces that can interpret intention and subsequently execute a function. One recent study used real-time fMRI to decode signals in the visual cortex in response to directed visual attention (Andersson, 2011). Further, providing subjects with real-time fMRI feedback helped train them to direct their visual focus, raising intriguing possibilities for intracranial BCI implants with similar functions. A running undercurrent of this discussion that must not be ignored concerns the relevance of these results in the larger realm of society. The emerging field of neuroethics, which is primarily interested in the proper use of neuroscientific information and technology, looks to play a major role in the future of neuroscience. Shortly after being published, the results of this study along with the reconstruction video were the subject of
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much fervour in the popular press. The use of phrases like “mindreading” in this context is not technically accurate, yet the reality proposed by such technology is urgently in need of discussion from a neuroethical perspective. It is vital that researchers, physicians, and policy-makers establish open channels of communication to ensure that these techniques are integrated into the public in a controlled and regulated manner, taking into accounts issues of privacy, education, and empirically valid data.
Andersson P, Pluim JP, Siero JC, Klein S, Viergever MA, Ramsey NF. Realtime decoding of brain responses to visuospatial attention using 7T fMRI. PLoS One, 6 (2011).
Future Directions As stated before, these results represent a major landmark in the neurobiological study of visual perception and in the development of neuroimaging as a research field. The immediate future for researchers is to replicate the results of this study, with a view towards increasing accuracy and scope of visual reconstructions. Advancements in fMRI – particularly in spatial and temporal resolution – will bring much-needed strength and validity to this technique, and may eventually enable accurate “readings” of dynamic mental events, such as thoughts, intentions, memories, or dreams. The future is brimming with possibility and intrigue in the face of these results; however, the scope of their implications should not be underestimated. Discussions on the neuroethics of “brain reading,” as the development of similar technologies continues to progress, should be opened sooner rather than later.
Tamaki M, Kamitani Y. Decoding subjective mental states from FMRI activity patterns. Brain Nerve 63, 1331-8 (2011).
Harrison, S.A., and Tong, F. Decoding reveals the contents of visual working memory in early visual areas. Nature, 458, 632–635 (2009). Nishimoto, S., Vu, A.T., Naselaris, T., Benjamini, B.Y., & Gallant, J.L. Reconstructing visual experiences from brain activity evoked by natural movies. Current Biology, 21, 1641-7 (2011).
Thirion, B., Duchesnay, E., Hubbard, E., Dubois, J., Poline, J.B., Lebihan, D., and Dehaene, S. Inverse retinotopy: inferring the visual content of images from brain activation patterns. Neuroimage, 33, 1104–1116 (2006). Tong, F. Aligning Brains and Minds. Neuron, 72, 199-201 (2011).
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Nutrition Stay Hungry, Stay Foolish? A review of the Neuroprotective properties of Caloric Restriction Ceilidh MacPhail Rodents suffering from diet induced insulin resistance and obesity exhibit cognitive deficits that intensify with increasing age as compared to controls. Epidemiological data and clinical studies in humans supports the theory that excessive dietary energy intake and insulin resistance can impair cognition. On the other hand, there is a growing body of evidence suggesting caloric restriction (CR), can enhance neural plasticity and reduce the risk of the brain succumbing to age related disease. CR may exert beneficial effects on the brain by triggering adaptive cellular stress response pathways that shift gene expression profiles supporting neural plasticity and cell survival. This article will review the evidence in rodent, non-human primates and human studies focusing on neuroprotective properties of CR. There are physiological and psychological obstacles to adopting CR diet. However, the traditional lifelong commitment to CR may not be necessary to receive the neuroprotective benefits. Short term CR, alternate day fasting or novel CR mimetics may all activate the adaptive stress response pathways and preserve cognitive function. This article will close with a summary of the cognitive benefits of 2 promising CR mimetics. Key words: Caloric Restriction (CR), Ageing, Mimetics, Neurodegenerative, Cogntive Decline, Sirtuins, Stress Response. I. BACKGROUND Brain senescence is associated with increased oxidative stress, impaired energy metabolism, altered cellular signaling and pathological accumulation of proteins1. Cognitive decline is an accepted part of the ageing process along with a host of neurodegenerative disease like Alzheimerâ€™s or Parkinsonâ€™s. We know there are important factors that influence senescence such as diet, exercise and genetics. Yet the mechanism behind senescence is not completely understood nor how exercise, diet and genetics offer neuroprotective effects. This review will investigate taking common-sense interventions one step further. A better understanding of the molecular mechanisms mediating neuronal survival and health may be an avenue to new pharmacological interventions that exploit neuroprotective mechanisms. The relationship between dietary intake and the ageing process holds relevance to the current public health landscape, with rising global obesity rates and strong trends towards a greying population. The ultimate goal would be a pharmacological intervention that promotes a quality of life through the maintenance of cognitive faculties. In other words increasing the healthy life span of individual not just their life span. Any discussion on increasing healthy life span must start with Caloric Restriction (CR). CR is the only successful known intervention that can extend life in yeast, C-elegans, Flies, Mice and possibly primates and humans.2 Caloric Restriction refers to an overall reduced food intake to 60-75% of recommended daily intake with maintenance of nutritional (vitamins, min-
erals) intake. There are 2 strategies to achieve this intake reduction, dietary restriction with a daily lowered food intake or alternate day fasting. In addition to increased longevity CR regimes are associated with lowered prevalence of pathology including age related disease. This article will review evidence in animals and humans of the neuroprotective role of CR There are very real barriers to Caloric restriction regimes wide acceptance. A minority of individuals have demonstrated longterm commitment to a reduced diet however the global obesity trends suggest that the majority of people find such efforts challenging. An alternative solution could be a CR mimetic, a pharmacological intervention that could reproduce the metabolic effects of Caloric restriction without the dietary changes. The second half of the paper will outline possible CR mimetic candidates II. Neuroprotective properties of Caloric Restriction Non-Human Primates Studies There are 3 University groups investigating CR in nonhuman primates including a study at the University of Wisconsin ongoing for 25 years. Colman et al published in their 20 year preliminary results a 30% increase in survival for the CR rhesus monkeys as compared to controls3. They reported a 50% reduction of malignant neoplasms and cardiovascular disease. 37% of control animals succumbed to an age related disease as compared to 13% of the CR monkeys. Reduced brain atrophy was reported for CR animals in brain areas involved in motor control and higher cognitive function. Grey matter measurements revealed a significant protective effect of CR on the mid cingulate cortex, lateral temporal cortex and right dorsolateral frontal lobe. Finally, the CR monkeys appear physically more youthful, Studies in Nonhuman primates consistently report improved insulin sensitivity and lowered fasting glucose compared to controls.4 It is important to note improvements in fasting glucose and insulin sensitivity was seen in 6 month long CR intervention suggesting life long CR is not necessary to receive benefits of the intervention.5 Rodents Studies Early Studies on dietary restriction indicated life long caloric restriction (alternate day fasting) with improved learning in complex maze situation. On the other hand, there is evidence to suggest that diets high in fat and sugar impair spatial learning in water 6, Y 7 and complex mazes8. Fasting regimes in young mice improved memory, and increased LTP and neurogenesis possibly through a brain-derived neurotrophic (BDNF) factor dependent mechanism. 9 Mice models of Alzheimerâ€™s disease with mutations amyloid precursor protein (APP) and presenilin 1 (PS1) were protected under a CR diet.10 The APP and PS1 mutants in middle age reduced their diet to 60% of their recommended intake for 18 weeks. Amyloid beta plaques by a third as compared to control mice. The brain requires a reliable blood supply to function. Reduced inflammation and cardiovascular benefits of CR supports healthy brain ageing in the long run. Stroke-prone hypertensive
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mice experienced on a CR regime experienced a delayed onset of stroke, with no change in blood pressure compared to controls.11 The protective effect may have been from the reduced inflammatory cytokine levels resulting from the adipose tissue reduction. More recently a study investigated the relationship between brain plasticity and CR.12 After birth rats had amblyopia induced after birth. Once the rats reached adulthood their normal eye was covered and the amblyopic eye uncovered. The rats were divided into either an alternate day fasting regime or control adlibitum. The fasting rats experienced restored vision in deprived eye as measured by visual acuity and. (figure 2) Reduced levels of GABA and GAD 65 were reported indicating reduced intracortical inhibition in the food restricted rats. In addition long term potentiation was stronger in the visual cortex compared to the control animals. The increase in plasticity may not be restricted to the visual cortex as the CA1 of the hippocampus exhibited the increase in LTP and reduction in intra-cortical inhibition
CR regimes and has a reduced expression of Sirt-1. CR has a profound impact on all levels of the transcriptome. There will be a trade off between the benefits and risks of such a regime. CR mice have been observed to have reduced immunity and wound healing.5 Too severe CR regimes may have adverse impact on cognition Human Studies Human studies of CR have obvious ethical, biological and logistical limitations. Human studies use biomarkers of longevity as most studies are between 6-12 months. The Comprehensive Assessment of Long term Effects of Reducing Intake of Energy (CALERIE) study is an ongoing randomized controlled trial investigating the effects of CR over 2 years. Part 1 results reported CR subjects had lower fasting glucose , leptin and insulin15. Similar studies in diabetic16, obese and the elderly17 report the expected benefits in cardiovascular health and improvement in insulin sensitivity. Few studies have investigate the impact of CR on cognition directly. However Knecht et al reported enhanced verbal memory performance in elderly individuals on a CR regimen 18 III. Candidate Caloric Restriction Mimetics The cellular mechanisms mediating the beneficial effects of CR are slowly being unraveled. 4 metabolic pathways have been associated with the effects of CR; (1) insulin/ insulin-like growth factor-1 (IGF-1), (2) sirtuins, (3) target of rapamycin (TOR or mTOR in mammals), and (4) 5’ adenosine monophosphateactivated protein kinase.19 CR is a crude intervention that will have a dramatic impact on many metabolic pathways. It is likely all 4 pathways mediate different benefits of the CR response.
Table 1. Results form the study by Spolidoro et al 12. A, Graph shows the decrease in cortical inhibition due to the reduction of GABA in the visual cortex of the food restricted mice show food restricted mice. Measurement of GABA release in the visual cortex of food-restricted rats (FR = 0.51 +/- 0.12 pmol n = 7; t-test P < 0.001) with respect to controls (CTL = 2.94 0.33 pmol ; n = 5) using in vivo brain microdialysis. B, GAD65, an enzyme responsible for GABA synthesis, expression was quantified through immunohistochemistry in the visual cortex of FR rats (n = 6) and in CTLs (n = 6; t-test P = 0.001). D, Recovery of Visual acuity in the formerly deprived eye was evaluated in Revere Sutured, food restricted rats (1.01 +/- 0.05 versus 1.02 +/- 0.07 ; n = 5; paired t-test P = 0.851) and in Reverse sutured control animals (0.72 +/- 0.03 versus 1.03 +/- 0.02; n = 4; paired t-test P = 0.004). Error bars represent s.e.m.; asterisks
Fusco et al built on the knowledge that cAMP responsive element binding (CREB) has a role in nutrient sensing in the brain and interacts with the CR associated sirtuin-113. CREB is intimately involved with neuronal plasticity, which CR may be able to enhance. Mice deficient in forebrain CREB did not respond to
2 promising CR mimetics that have been studied are resveratrol, metformin. Metformin is commonly known as a medication for type II diabetes. Metformin acts on Liver Kinase B1 to reduce blood glucose levels by phosphorylating adenosine monophosphate activated protein kinase (AMPK). Metformin interacts with 2 of the identified CR metabolic pathways supporting it as a candidate CR mimetic. In rats on a CR diet AMPK activity is down regulated in the liver 20 . (To, Shimokawa, 2007)A micro array study found that the metformin induced a expression profile similar to rat on CR diet.(Higami, Shimokawa, 2006) Sirtuin Activators are another group of candidate compounds for a CR mimetics. Sirtuins are an evolutionary conserved family of NAD dependent Histone Deacetlyase There are 5 human sirtuins and they mediate the stress response when there are limited resources. Sirtuins have been linked to longevity in animals in humans. Horwitz et al screened for compounds that could activate Sirt1 and identified, Resveratrol a polyphenol found in the skin of grapes. 22 Subsequent studies have shown that in obese mice resveratrol administrations prevents insulin resistance, restores a life span and replicates transcriptional changes seen under a CR regime.23 At the cognitive level resveratrol may improve motor coordination in mice. Resveratrol administration prevents neurodegeneration in models of Alzheimer’s disease. 24 In non-human primates 18 months of oral resveratrol supplementation improved spatial memory more so than a 70% CR diet.25 Human studies of resveratrol supplementation (150mg) for 30 days with obese subjects reported a shift toward a CR gene expression profile.26 Increased expression of
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Sirtuin 1, AMPK, while decreasing circulating glucose and inflammation markers was observed among other changes. Contradictory evidence exists linking sirtuins to the benefits of CR. 27 More research is needed to definitively establish the role of the sirtuins in CR. The other members of the sirtuin family and their respective activators hold promise for CR mimetics. For example genetic polymorphism of the Sirtuin3 promoter have been associated with longevity in the Italian population. 28 IV. Conclusion Short and long term studies have demonstrated in rodents and non- human primates the neuroprotective and systemic benefits of various forms of CR interventions. Human studies have yet to test rigorously the neuroprotective aspect of CR however the improvements in cardiovascular and glucose regulation have been documented. The intimate relationship between cardiovascular health, glucose regulation and the brain suggests that there are likely neuroprotective benefits to CR interventions in humans. Currently evidence regarding CR mimetics is scattered and contradictory. While resveratrol appears promising in the studies discussed such investigations must be replicated and expanded upon. 1. Stranahan, A.M. & Mattson, M.P. Recruiting adaptive cellular stress responses for successful brain ageing. Nature reviews. Neuroscience 13, 209-16 (2012). 2. Roth, L.W. & Polotsky, A.J. Can we live longer by eating less? A review of caloric restriction and longevity. Maturitas 71, 315-9 (2012). 3. Colman, R.J. et al. Caloric restriction delays disease onset and mortality in rhesus monkeys. Science (New York, N.Y.) 325, 201-4 (2009). 4. Colman, R.J. & Anderson, R.M. Nonhuman primate calorie restriction. Antioxidants & redox signaling 14, 229-39 (2011). 5. Mercken, E.M., Carboneau, B. a, Krzysik-Walker, S.M. & de Cabo, R. Of mice and men: The benefits of caloric restriction, exercise, and mimetics. Ageing research reviews 1-9 (2011).doi:10.1016/j.arr.2011.11.005 6. Molteni, R., Barnard, R. J., Ying, Z., Roberts, C. K. & Gómez-Pinilla, F.A. High-fat, refined sugar diet reduces hippocampal brain-derived neurotrophic factor, neuronal plasticity, and learning. Neuroscience 112, 803–814 (2002). 7. McNay, E.C. et al Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance. Neurobiol. Learn. Mem 93, 546–553 (2010). 8. Morrison, C.D. et al. H. High fat diet increases hippocampal oxidative stress and cognitive impairment in aged mice: implications for decreased Nrf2 signaling. Journal of neurochemistry 114, 1581–1589 (2010). 9. Lee, J., Duan, W. & Mattson, M.P. Evidence that brain-derived neurotrophic factor is required for basal neurogenesis and mediates, in part, the enhancement of neurogenesis by dietary restriction in the hippocampus of adult mice. Journal of neurochemistry 82, (2002). 10. Mouton, P.R., Chachich, M.E., Quigley, C., Spangler, E. & Ingram, D.K. Caloric restriction attenuates amyloid deposition in middle-aged APP/ PS1 mice. October 464, 184-187 (2009). 11. Chiba, T. & Ezaki, O. Dietary restriction suppresses inflammation and delays the onset of stroke in stroke-prone spontaneously hypertensive rats. Biochemical and Biophysical Research Communications 399, 98–103 (2010). 12.Spolidoro, M. et al. Food restriction enhances visual cortex plasticity in adulthood. Nature communications 2, 320 (2011). 13. Fusco, S. et al. A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction. Proceedings of the National Academy of Sciences of the United States of America 109, 621-6 (2012). 14. Yanai, S., Okaichi, Y., and Okaichi, H. Long-term dietary restriction causes negative effects on cognitive functions in rats. Neurobiolgy of Ageing 25, 325332 (2004). 15. Meydani, M., Das, S., Band, M., Epstein, S. & Roberts, S. CALORIC RESTRICTION AND GLYCEMIC LOAD ON MEASURES OF OXIDATIVE STRESS AND ANTIOXIDANTS THE EFFECT OF CALORIC RESTRICTION AND GLYCEMIC LOAD ON MEASURES OF OXIDATIVE STRESS AND ANTIOXIDANTS IN HUMANS : RESULTS FROM THE CALERIE TRIAL OF HUMAN CALORIC RESTRIC. Health (San Francisco) 15, 456-460 (2011). 16. Skrha J, Kunesova M, Hilgertova J, Weiserova H, Krizova J, K.E. Short-term very low calorie diet reduces oxidative stress in obese type 2 diabetic patients. Physiology Research 54, 33-39. (2005).
17. Willcox BJ, Willcox DC, Todoriki H, Fujiyoshi A, Yano K, He Q, Curb JD, S.M. Caloric restriction, the traditional Okinawan diet, and healthy aging: the diet of the world’s longest-lived people and its potential impact on morbidity and life span. Ann N Y Acad Sci 1114, 434-455 (2007). 18. Witte AV, Fobker M, Gellner R, Knecht S, F.A. Caloric restriction improves memory in elderly humans. Proceedings of the National Academy of Sciences 106, 1255-1260 (2009). 19. Fontana, L., Partridge, L. & Longo, V.D. Extending healthy life span--from yeast to humans. Science (New York, N.Y.) 328, 321-6 (2010). 20. To, K.; Yamaza, H.; Komatsu, T.; Hayashida, T.; Hayashi, H.; Toyama, H.; Chiba, T.; Higami, Y.; Shimokawa, I. Down- regulation of AMP-activated protein kinase by calorie restriction in rat liver. Experimental Gerontology 42, 1063-1071. (2007). 21. Higami, Y.; Tsuchiya, T.; Chiba, T.; Yamaza, H.; Muraoka, I.; Hirose, M.; Komatsu, T.; Shimokawa, I. Hepatic gene expression profile of lipid metabolism in rats: Impact of caloric restriction and growth hormone/insulin-like growth factor-1 suppression. Jounral of Gerontology 61, 1099-1110. (2006). 22. Howitz, K.T., Bitterman, K.J., Cohen, H.Y., Lamming, D.W., Lavu, S., Wood, J.G., Zipkin, R.E., Chung, P., Kisielewski, A., Zhang, L.L., et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425, 191–196. (2003). 23. Baur JA, Pearson KJ, Price NL, et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444, 337–342 (2006). 24. Kim, D., Nguyen, M.D., Dobbin, M.M., Fischer, A., Sananbenesi, F., Rodgers, J.T., Delalle, I., Baur, J.A., Sui, G., Armour, S.M., Puigserver, P., Sinclair, D.A., Tsai, L.H. SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer’s disease and amyotrophic lateral sclerosis. EMBO 26, 3169–3179 (2007). 25. Dal-Pan, A., Pifferi, F., Marchal, J., Picq, J.-L. & Aujard, F. Cognitive performances are selectively enhanced during chronic caloric restriction or resveratrol supplementation in a primate. PloS one 6, e16581 (2011). 26. Timmers, S. et al. Calorie restriction-like effects of 30 days of resveratrol supplementation on energy metabolism and metabolic profile in obese humans. Cell metabolism 14, 612-22 (2011). 27. Guarente, L. Sirtuins, Aging, and Medicine. New England Journal of Medicine 2235-2244 (2011). 28. Rose G, Dato S, Altomare K, et al. Variability of the SIRT3 gene, human silent information regulator Sir2 homo logue, and survivorship in the elderly. Experimental Gerontology 1065-70. (2003).
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Structural Imaging Visualizing concussions: DTI and H-MRS in diagnosis and return-to-play decisions Monica Maher Human Biology Department, The University of Toronto. Toronto, Ontario CA Mild traumatic brain injury, or more commonly known as concussion, represents s significant proportion of traumatic brain injuries occurring every year, however medical opinion of this pathology has previously been somewhat casual, with the recommended recovery timeline being a mere 2-10 days. There are a range of sequelae associated with concussions, including memory impairments, balance disturbances, visual and auditory problems, confusion, headaches and dizziness, in addition to very persistent metabolic and structural abnormalities. Current methods of diagnosis and monitoring involve neuropsychological testing batteries, which are adequate but are rather limited in terms of informing physicians of what is occurring within the brain itself. Two imaging technologies can recently taken the stage, offering increased sensitivity to microstructural damage and metabolic disturbances within the central nervous system that CT and functional MRI cannot compare to. These technologies are Diffusion Tensor Imaging (DTI) and Proton Magnetic Resonance Spectroscopy (H-MRS). DTI uses measurements of Fractional Anisotropy and Mean Diffusivity to glean insight into the structural integrity of white matter tracts and the mobility of water molecules within the brain, which informs of the overall structural heath of the brain. H-MRS focuses on the changes in important metabolite concentrations within specific brain regions of interest, particularly N-acetylaspartate which is a marker of neuronal health and energy levels. These methods could potentially be used in conjunction to improve diagnosis of the severity of mTBI as well as offering a superior method for monitoring the recovery of patients, to ensure they do not return to sport while their brain is structurally and metabolically vulnerable, regardless of neuropsychological test results. I. BACKGROUND Concussion, or mild traumatic brain injury, is the most common type of traumatic brain injury with an estimated 1.6 to 3.8 million sports-related concussions occurring per year in the United States – with the vast majority of these going undiagnosed or untreated since many individuals do not seek medical attention.1 Hallmark symptoms of a mild traumatic brain injury quite diverse and unspecific, with patients presenting with such symptoms as confusion, headaches, dizziness, nausea, memory impairments and disturbances in balance, vision and hearing – with this sequelae being termed “Post-Concussive Syndrome”. Concussion usually involves the rapid onset of these generally short-term neurological impairments, which typically resolve spontaneously.2 In the concussions – particularly those sustain during athletic competition – have not been given as much attention clinically as other forms
of traumatic brain injury as it was not considered as severe, with the typical recovery timeline given by physicians ranging from a mere 2 to 10 days.3 However in recent time, in light of recent injuries sustained by several high-profile athletes, it is becoming apparent that the long-term consequences of mild traumatic brain injury must be explored. Currently concussions are diagnosed and monitored with neuropsychological testing batteries and when athletes return to baseline performance they’re allowed to return to competition, but a significant problem with this method is that mild traumatic brain injury has been found to be associated with metabolic and electrophysiological disturbance that can persist for months, even perhaps years.4 There are also issues of compensation by other brain areas and motivation to perform that must be taken into consideration when basing return-to play decisions on performance on these neuropsychological tests. The techniques of Diffusion Tensor Imaging and Proton Magnetic Resonance Spectroscopy can overcome these limitations and may offer direct insight into what is occurring structurally, functionally and metabolically within the brain during Post-Concussive Syndrome. Diffusion tensor imaging uses Fractional Anisotropy measure to describe the preferred direction of diffusion in the brain and thus inform about fiber directionality as well as determine the integrity of the white matter tracts in the brain.1 Mean diffusivity describes the overall mobility of water in the brain. Fractional Anisotropy generally decreases with mild traumatic brain injury, reflecting the reduction of white matter integrity resulting from axonal injury, while Mean Diffusivity increases and these changes from baseline are indicative of microstructural damage to the white matter. Another promising method of imaging is Proton Magnetic Resonance Imaging (H-MRS), which is a non-invasive technique that allows visualization of metabolic changes occurring within the brain, detecting metabolic disturbances that are not evident in other forms of brain imaging and is particularly sensitive to diffuse axonal injury.5 A key metabolic marker that can be used to assess severity of concussion and traumatic brain injury is N-acetylaspartate, which is the second most common metabolite in the human brain, used as a marker of neuronal cell bodies where it is exclusively found and serves as a sign of neuronal health.3 Levels of NAA drop significantly after traumatic brain injury and correlate well to results of neuropsychological testing batteries, with levels increasing in line with the patient’s recovery. This is consistent with the fact that NAA is involved in energy production and that energy levels in the brain (reflected ATP availability) typically decrease with traumatic brain injury.4 NAA decreases are also seen in any disease of the central nervous system involving neuronal or axonal trauma, hypoxic-ischemic or toxic injury.2
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II. REVIEWED RESEARCH-Materials and Methods The DTI study that this review paper focuses on used a participant pool consisting of 10 college varsity athletes who had sustained mild traumatic brain injury in the context of sports-related concussions. Those scoring between 13-15 on the Glasgow Coma Scale were classified as having mild concussion, moderates score between 9-12 whereas severe TBI corresponded to scores of 8 or less.1 These individuals were matched to 10 healthy controls that were also varsity athletes but had no prior history of concussion, psychiatric illness, neurological disorders, traumatic brain injury or substance abuse.3 Before undergoing imaging, patients in all studies were assessed with the computerized neuropsychological test ImPACT, which is specifically geared towards assessing the cognitive impairments of concussion as compared with baseline testing scores.1,3
and Fractional Anisoptropy within the uncinate fasculus, which is the tract that connects the limbic system to the frontal lobes.1 H-MRS The concussed athletes presented metabolic impairments within the brain, particularly within the prefrontal and motor cortices where N-acetylaspartate levels were significantly lower in this patient group when compared to their controls when MR scanning was completed and assessed at 5 days post-injury (Figure 2). After 6 months, recovery of N-acetylaspartate was seen in the majority of patients and this recovery of metabolite levels corresponded to the subjects’ improved performance on neuropsychological tests.2,3
Diffusion Tensor Imaging The DTI study that this review paper focuses on used a participant pool consisting of 10 college varsity athletes who had sustained mild traumatic brain injury in the context of sports-related concussions. Those scoring between 13-15 on the Glasgow Coma Scale were classified as having mild concussion, moderates score between 9-12 whereas severe TBI corresponded to scores of 8 or less.1 These individuals were matched to 10 healthy controls that were also varsity athletes but had no prior history of concussion, psychiatric illness, neurological disorders, traumatic brain injury or substance abuse.3 Before undergoing imaging, patients in all studies were assessed with the computerized neuropsychological test ImPACT, which is specifically geared towards assessing the cognitive impairments of concussion as compared with baseline testing scores.1,3 H-Magnetic Resonance Spectroscopy A Siemens’s 3.0T whole body MRI machine was used for this procedure and regions of interest were positioned with a precise anatomical localization procedure. Single-voxel 1H-MRS spectroscopy measures were completed using a Point RESolved Spectroscopy sequence with a 12-channel head coil. Metabolite concentrations were estimated with the Linear Combination model spectral analysis software and statistical analyses were conducted using SPSS.3 Participants were scanned 5 days post-injury and again at 6 months. Results - DTI With Diffusion Tensor Imaging there were several clusters of significant voxels, which exhibited increased mean diffusivity and decreased Fractional Anisotrophy (see Figure 1). These clusters were found predominantly within the left hemisphere of the concussed patients, compared with their age- and sex-matched controls. The main white matter tracts that were significantly affected in all patients were the inferior fronto-occipital fasciculus within the saggital stratum, the superior longitudinal fasciculus and the uncinate fasciculus. These areas have thus been suggested to be particularly vulnerable to concussions because of their centralized location. Mean diffusivity and Fractional Anisotropy values corresponded to the severity of the concussion, with more mild concussions having significantly smaller deviations from baseline compared to those of moderate and then severe concussions, although these deviations were still significantly different from baseline. Correlations are also seen between memory test performance
Figure 1. Mean FA in the various concussed groups (mild, moderate & severe) as compared with controls. Decline in FA corresponds to the severity of the concussion. Taken from Cubon et al., 2011.
Discussion The organic causes of post-concussive symptoms have been greatly debated, however these studies provide evidence that they are rooted in microstructural brain injury – primarily traumatic axonal injury.4 Increased MD values are purported to represent cytotoxic edema and inflammation, while decreases in FA may reflect wallerian degeneration and subsequent cell death.7 Diffusion tensor imaging is a sensitive enough measure to visualize the microstructural damage associated with mild white matter injury.1 Reductions in Fractional Anisotropy are strongly associated with severity of post-concussive symptoms and cognitive dysfunction such as impaired attention, executive functions and working memory. Abnormalities are commonly seen in several key brain regions and the results of the reviewed articles are consistent with previous findings implicating the unicinate fasciculus, inferior fronto-occipital fasciculus and the superior longitudinal fasciculus. Abnormalities within the uncinate fasciculus, which connects the limbic system to the frontal lobes, are typically seen in such pathologies as Alzheimer’s disease and schizophrenia. Impaired memory performance in concussed individuals correlates to decreases in Fractional Anisotropy in the uncinate fasciculus.1 When completing measures of working memory, those individuals with more severe
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post-concussive symptoms also show increased recruitment of the working memory networks in addition to further recruitment of regions outside this network â€“ demonstrating attempts to compensate for deficits.4 The functions of the inferior fronto-occipital fasciculus are fairly poorly understood but this tract is hypothesized to be involved in attention shifting, which is well aligned with the post concussive symptom experienced by many patients involving difficulties with attention. These impairments are usually subtle and only noticeable with neuropsychological tests, however they flare up when patients return to the more cognitively demanding environments of school or work.4 Some other DTI studies have also demonstrated very long-term shear-related changes in these identified regions up to 5.7 years after the concussion was sustained.5
Figure 2. H-MRS spectra for NAA. The top spectrum shows the high single peak corresponding to high normal NAA levels in controls. The spectrum on the bottom (A) displays the severe decreases in NAA after concussion. Taken from Kubal, 2012.
Proton Magnetic Resonance imaging has proven itself to be a valuable technique for gathering information pertaining to the metabolic state changes occurring in the brain after mild traumatic brain injury. N-acetylaspartate is the metabolite given the most attention in concussion-related H-MRS as it is consistently depressed in concussed patients, usually by approximately 19% at 3 days after the initial insult.6 NAA levels in M1 are critical for distinguishing between healthy controls and concussed patients at all time points up until recovery, however sustaining further subconcussive
blows during the acute phase can further the insult to the brain and delay metabolic recovery.7 Declines in NAA are associated with decreased levels of brain ATP and the more significant the initial decrease in NAA, the slower the subsequent metabolic recovery of both these compounds will be. There are also observed decreases detected by H-MRS in normal glutamate metabolism with concussed individuals, which is not surprising given the hypoglycolytic state induced by closed-head injury and this drop in glutamate corresponds to injury severity and persists for 2-4 weeks. The time course for the recovery of N-acetylaspartate levels in the subjects in the study detailed by Henry et al. (2011) was slightly different from those seen in other H-MRS studies, as the levels of this metabolite seemed to remain depressed for a longer period of time than expected. It was proposed that this might perhaps be a result of the use of a unique patient population of student athletes. Although these patients follow the same recovery protocol for return-to-play as professional athletes commonly studied, they are still required to attend classes and in many cases they are expected by coaches to resume physical activity within a week so as not to lose fitness. The cognitive and physical demands placed on many of these athletes perhaps preclude faster recovery. Similar decreases in NAA are also seen in such brain pathologies as tumor, epilepsy, stroke, dementia, multiple sclerosis and hypoxia.2 This impaired metabolic state detailed by H-MRS imaging also represents a period in which the brain in particularly sensitive and vulnerable to a second injury. Significance Diffusion tensor imaging and Proton Magnetic Resonance Spectroscopy represent a superior means of diagnosing and assessing rehabilitation of concussed individuals as compared with neuropsychological testing batteries. There are several confounds involved in basing recovery progress on neuropsychological tests that are not concerns with these imaging techniques. Motivational issues are particularly salient, since athletes know that the one obstacle preventing them from getting back to sport is their performance on these tests, so they are particularly strongly motivated to perform well. Furthermore, there is the confound of compensatory activity in other brain areas that might give the impression on these tests that the patient has recovered more than they actually have. DTI and H-MRS are techniques that allow direct visualization of how the brain is recovery, both structurally and metabolically. Another important application of DTI and H-MRS in monitoring recovery is in preventing the occurrence of SecondImpact Syndrome. There exists a post-concussive window of vulnerability where the brain is metabolically compromised and during which time patients are at a 3 times greater risk of sustaining a second, more severe concussion from what would typically be considered a subconcussive blow.6 The resulting metabolic insult â€“ as determined by assessing NAA levels through H-MRS â€“ stemming from this second injury is very much inline with what would be expected from a severe traumatic brain injury, such as with a serious car accident or hemorrhagic stroke. Furthermore, recurrent concussions can have a cumulative effect and are responsible for myriad cognitive impairments including early-onset memory disturbances, depression and dementia.2 The serious possibility of Second-Impact Syndrome emphasizes the importance of identifying and monitoring concussions with DTI and H-MRS to ensure that athletes are not being allowed to return to competition or training while still within this window of sensitivity.
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Future Directions While DTI and H-MRS look very promising as imaging techniques for concussive injury, the main limitation to overcome is cost. MRI machines are very expensive to both purchase as well as the cost per subject scanned, so currently it is impractical to use these techniques for each and every case of concussion, however this is something that could be overcome with time or perhaps changes to healthcare coverage. In the future it would be very sensible for clinics in high performance athletic centers or university and college sports clinics to be equipped with DTI and H-MRS in order to quickly diagnose athletes immediately after a potential concussive insult occurs as well as make it more easily accessible for athletes whoâ€™s recovery needs to be closely monitored. While research currently is able to clearly diagnose concussions with these imaging techniques, focus should now shift towards using them to make optimal return-to-play decisions. Questions that should be asked should related to whether metabolic and structural changes need to completely return to baseline before patients are allowed to compete and if once reaching baseline they could immediately get back into sport or wait a certain period of time to ensure metabolite levels and structural ameliorations are stabilized. Another possible future experiment could investigate perhaps whether there is a window of NAA levels, FA and MD values in which it would be acceptable to return to play, rather than waiting until baseline is again reached. Finally, a newly emerging area of sport-related concussion is pediatric concussions. Currently, the pathophysiology of sports-related concussions in children is poorly understood but is highly prevalent.8 Results attempting to characterize pediatric sports-related concussions have thus far been mixed, with some studies suggesting that they do not produce as serious metabolic and structural disturbances, whereas others posit the opposite â€“ that they are far more serious, posing more severe long-term consequences.
1. Cubon, V.A., Putukian, M., Boyer, C. & Dettwiler A. A Diffusion Tensor Imaging Study on the White Matter Skeleton in Individuals with SportsRelated Concussion. J Neurotrauma 28,189-201 (2011). 2. Signoretti, S., Lazzarino, G., Tavazzi, B. & Vagnozzi, R. The pathophysiology of concussion. PMR. 2011;3:s359-S368. 3. Smits, M. et al. Microstructural brain injury in post-concussion syndrome after minor head injury. Neuroradiology 53, 553-63 (2011). 4. Henry, L.C. et al. Metabolic changes in concussed American football players during the acute and chronic post-injury phases. BMC Neurol 11, 105 (2011). 5. Gonzalez, P.G. & Walker, M.T. Imaging modalities in mild traumatic brain injury and sports concussion. PMR 3, S413-24 (2011). 6. Kubal, W.S. Updated imaging of traumatic brain injury. Radiol Clin North Am 50, 15-41 (2012). 7, Henry, L.C. et al. Acute and chronic changes in diffusivity measures after sports concussion. J Neurotrauma 28, 2049-59 (2011). 8. Maugans, T. a, Farley, C., Altaye, M., Leach, J. & Cecil, K.M. Pediatric sports-related concussion produces cerebral blood flow alterations. Pediatrics 129, 28-37 (2012).
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Neuroimaging A Portable Approach to Neuroimaging: Putting the Fun Back into Functional Near-Infrared Spectroscopy Alexander Mihaescu Psychology Department, University of Toronto, Toronto, ON M5S 2E9 Current neuroimaging techniques lack a way to reliably measure brain activity while moving or walking. Functional near-infrared spectroscopy (fNIRS) is an emerging new form of neuroimaging technology based on the hemodynamic response which has exciting new applications that can add to the current body of neuroimaging techniques. Oxygenated and deoxygenated blood reflect a different amount of light back based on the wavelength of light. This signal discrepancy between two wavelengths can be measured to create a voxel map of brain activation based on the amount of oxygenated blood in the brain areas. Compared to the gold standard of fMRI, fNIRS has poorer spatial resolution but better temporal resolution. One of the biggest benefits of fNIRS over traditional fMRI is how portable fNIRS is, allowing studies to be performed on participants who are moving. Current fNIRS research is dominated by motor and language studies which hope to take advantage of the portability of the fNIRS device. Motors system research has benefited from fNIRS as real-time complex movements such as locomotion can be visualized in the brain. Language studies with fNIRS is preferable because the imagining technique is resistant to artifacts caused by facial movements while speaking. fNIRS has been used to study the brain development of babies, suggesting a potential for bedside clinical screening. More research needs to be done on fNIRS to improve itâ€™s spatial resolution and to integrate its data with other forms of neuroimaging. Future studies will try to eliminate confounds and improve optode positioning.
Functional near-infrared spectroscopy (fNIRS) is an emerging new form of neuroimaging technology1. Similar to the commonly known fMRI method, fNIRS uses the level of blood oxygenation in the brain as an indirect measure brain activity1. NIRS technology has been applied medically for many years, with its most common use being the pulse oximeters which measure the bodyâ€™s blood oxygenation through the fingertips2. fNIRS is based on the hemodynamic response of neurovascular tissue, which relies on the assumption that more brain activity leads to greater blood volume in the brain as neurons send out signals that they are oxygen starved1. However the brain region will not be able to utilize all of the brought in oxygen, resulting in an accumulation of oxygenated blood1. Active brain tissue will have a greater concentration of oxygenated blood1. Oxygenated and deoxygenated blood reflect a different amount of light back at different wavelengths1. Near-infrared light (750-950 nm) can pass through the skull to reach the cerebral cortex1. fNIRS can be used to measure up to 3cm deep in the cortex1. Reflected light is computed through a variation of the Beer-Lambert law, which is used to calculate the light absorption
value when passing through a medium to find the concentration of oxygenated blood compared to deoxygenated blood2. Two different wavelengths of light along the NIR spectrum are emitted and the reflected light from the two sources is compared with each other to find the difference in absorption, thus calculating blood oxygenation1.
Figure 1. fNIRS device setup with the location of the optodes. The array of light sources and detectors is used to create the fNIRS map of brain activity. Comparing the two light source signals reveals a map of brain activity that can be visually represented3.
fNIRS devices are arranged in a checkered manner, with alternating bands of light sources and light detectors1. These light sources emit light into the skull at the NIR wavelengths1. The light is reflected back out of the skull in a predictable banana-shaped arc that is picked up by the row of detectors2. These detectors measure the amount of light that is reflected from the different input wavelengths. The light that is reflected back can be analyzed to determine the concentration of deoxygenated hemoglobin compared to oxygenated hemoglobin1. The use of multiple channels of light source and detector create a map of the brain with multiple voxels measuring localized brain activity1. Concentration data can be turned into a familiar pictorial representations of brain activity1. Advances in fNIRS technology have made the headpiece much smaller, where it is now resembles a blindfold that is slipped over the forehead2. The data from the light detectors is beamed wirelessly to the computer for analysis, making the fNIRS device very portable2. II. PROPOSED RESEARCH COMPARISON OF NEUROIMAGING fNIRS neuroimaging offers a unique addition to the current body of neuroimaging techniques which can be use to assess new questions in neuroscience that current methods are unable to address. Compared to the gold standard of fMRI, fNIRS has poor-
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er spatial resolution, with 1 cm2 sized voxels2. fMRI has spatial resolution in the order of millimeters2. However, fNIRS has better temporal resolution than fMRI2. Additionally, fNIRS is far cheaper and more portable than an fMRI machine2. MRI machines can cost upwards of 1.7 million dollars to purchase2. In contrast, the price of fNIRS devices are range from $250,000 USD to as little as $25,0002. fMRI studies are impractical for all but the wealthiest of researchers, while fNIRS can study the brain for less, making NIRS research more accessible. fNIRS are also much more portable in comparison to fMRI machines2. Currently, wireless models exist which the participants only has to wear an electronic headband in order to get tested2. fNIRS is quieter than an MRI machine2. This has implications in many audio based studies where the background rumble of the fMRI machine is a confound. fNIRS can be used while patients perform a variety of postures and movements2. In contrast to this, fMRIs require participants to be completely still and lying down, limiting their use when studies wish to study the relation between movement and brain activity imaging2. Compared to MEG and EEG, fNIRS has better spatial resolution, but poorer temporal resolution2. fNIRS are in the middle between fMRI and EEG, having midway temporal and spatial resolution between these two methods2.
The use of fNIRS on infants has allowed scientists to track the developmental changes in brain tissue5. Specific tissues types can be tracked throughout the developmental lifespan5. Brain regions associated with particular higher cognitive functions can be identified and observed how they changed over time5. This creates an invaluable means of observing first-hand the development of the infant brain that has been difficult to visualize in vivo until now5. i. LANGUAGE SYSTEM Talking while undergoing an fMRI produces movement artifacts in the image. These types of movements can be performed while performing fNIRS because the portability of the device makes it resistant to movement artifacts5
Figure 3. Results for Jasinka et al. (2011). fNIRS images showing the significant differences in brain activation of the dyads for the different communication tasks. Gender differences were observed only across these dyads, not individually. Males are in the left column, females are on the right6. Figure 2. Table comparing the properties of various neuroimaging techniques relative to one another. OI is Optical Imaging (fNIRS)2.
RESULTS AND DISCUSSION fNIRS applications are exciting for how potentially revolutionary they could be to the field of neuroscience. Current research has adopted fNIRS in language and motor studies largely due to the specific benefit of portability4,5. fNIRS can be used to validate realistic tasks performed outside a laboratory setting as they are the first truly portable type of neuroimaging. fNIRS studies can be performed just about anywhere, increasing the ecologic validity of the experiments2. Complex tasks can be performed by patients while undergoing testing2. It allows for neuroimaging to occur while a movement is being performed, even as complex as walking and talking4. fNIRS has also been readily adopted by developmental biologists who wish to study the young developing brain of babies5. Because no harmful radiation is involved in the method, fNIRS is safe to use on infants without fear of damaging the delicate growing brain5. The portability of the device once again comes in handy, allowing for the free movement of the participants and minimizing the discomfort of extended use5. This allows for the use of fNIRS on babies as young as 1 Â˝ without too many complications5.
ii. MOTOR SYSTEM fNIRS imaging has been used to study the neural correlates of movements which cannot be studied with traditional fMRI7. Hatakenaka et alâ€™s 2007 study used fNIRS to show the precise differentiation of brain areas that activate sequentially in a complex motor task7. Male participants repeatedly performed a task with their right hands while undergoing fNIRS imaging7. The motor task was to keep a metal stylus touching an object while it spun on a disk7. Participants improved at this motor skill task as they repeated the trials, and this improvement in performance was matched with increases in oxygen levels in several the sensorimotor cortex (SMC), premotor cortex (PMC) and prefrontal regions7. fNIRS data showed that there was an increase in oxygenation in the preSMA during the first three trials, after which the oxygenation fell back down to baseline7. Conversely, SMA activity was low in the first few trials, then steadily increased until the fifth trial where it hit a high activity plateau7. This data suggests that the preSMA plays an important role in the early phase of motor learning while the SMA might be more involved in the later learning phases of the motor skill7. The spatial resolution on the fNIRS imaging was enough to differentiate specific brain regions and identify their individual activation patterns over the course of the trials7. This level of online active imaging was previously not possible with traditional fMRI7.
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Holtzer et al. (2011) explored fNIRS potential in mobile imaging by examining the cognitive cortical mechanisms used to control gait while walking8. The relationship between gait control and functional correlates of cognition is very poorly understood8. This experiment used wireless fNIRS devices to capture brain activity while the participants were walking8. Participants were divided into two groups of young and old who then had to perform two tasks of walking while talking (WWT) and walking without talking (NW)8. It was found that was increased activation in 14 of the 16 voxels measuring the pre-frontal cortex (PFC) in the WWT condition compared to the NW condition8. Younger participants also showed an increase in oxygenation in the PFC in the WWT condition, compared to the older participants8. This study showed that there was increased activity in the PFC while walking and talking, compared to just walking8. The decrease in PFC activity in older participants in WWT suggests that the PFC is underutilized with age in these locomotive tasks requiring attention8. The neural correlates of gait control were demonstrated with this experiment, showing that attention and the PFC may play a role in complex motor tasks, but not in simple locomotive tasks8. SIGNIFICANCE OF THE WORK Functional Near-Infrared Spectroscopy offers many benefits over fMRI which will attract much attention in the future from the neuroscience community2. fNIRS is cheaper and less constraining than fMRI2. Smaller labs can afford to buy an fNIRS machine, making fNIRS research easily reproducible2. fNIRS has good ecologic validity because it allows research to be done outside a lab due to the portability of the device2. New avenues of research can be explored outside the laboratory setting to confirm hypotheses in a real world situation2. Complex behavioral tasks is believed to be one of the biggest areas of future fNIRS study2. Rather than competing with fMRI, fNIRS should be used to address issues that fMRI is incapable of. Its portability and resistance to movement artifacts puts fNIRS in the perfect position to explore complex tasks involving movement and language2.
machine means it can readily be deployed for the mass testing of children in a hospital. fNIRS testing could become a routine part of the medical care used to ensure the healthy growth of children6. Bedside fNIRS testing of language and motor development can be used to detect abnormal development6. With fNIRS, children as young as 1 are able to be scanned, enabling for early detection of abnormal brain development which will mean the possibility of having early interventions if these problems are caught early6.
1. Irani, F., Platek, S. M., Bunce, S., Ruocco, A. C., & Chute, D. Functional Near Infrared Spectroscopy (fNIRS): An Emerging Neuroimaging Technology with Important Applications for the Study of Brain Disorders. The Clinical Neuropsychologist, 21(1) 9-37 (2007). 2. Irani, F. (2011). Functional Near Infrared Spectroscopy In Cohen, R., & Sweet, L. (Eds.), Brain Imaging in Behavioral Medicine and Clinical Neuroscience and Behavior (93-103). New York: Springer. 3. Gandjbakhche, A. H. Quantitative Biophotonics for Tissue Characterization and Function. Online Source 2010. 4. Rossi, S., Telkemeyer, S., Wartenburger, I., & Obrig, H. Shedding light on words and sentences: Near-infrared spectroscopy in language research. Brain and Language, Available online (2011). 5. Petitto, L. A. Coritical images of early language and phonetic development using Near Infrared Spectroscopy. In K. Fischer & A. Battro (Eds.), The Educated Brain. England: Cambridge University Press 213-232 (2007). 6. Jasinska, K. K., Jowkar-Baniani, G., Ahmed, F., Forster, E., Bhasin-Lacemen, S., Naimi, A., Petitto, A. L. & Dunbar, K. N. (2011). Simultaneous imaging of Neural activations of women and men in real-time conversation using fNIRS. Hall A-C 7. Hatakenaka, M, Miyai, I, Mihara, M, Sakoda, S, & Kubota, K. Frontal regions involved in learning of motor skillâ€”a functional NIRS study. Neuroimage, 34 109-17 (2007). 8. Holtzer, R., Mahoney, J. R., Izzetoglu, M., Izzetoglu, K., Onaral, B., & Verghese, J. fNIRS study of walking and walking while talking in young and old individuals. The journals of gerontology Series, 77(8) 879-887 (2011). 9. Steinbrink, J., Villringer, A., Kempf, F., Haux, D., Boden, S & Obrig, H. Illuminating the BOLD signal: combined fMRIâ€“fNIRS studies. Brain and Language Published Online (2011).
FUTURE DIRECTIONS fNIRS is a new technology with improvements to its design constantly being proposed. Current research is focusing on finding a way to integrate fMRI and fNIRS voxel readings with each other, to confirm that the hemodynamic (fNIRS) and BOLD (fMRI) response is measuring the same thing9. The different sized voxels between the two neuroimaging techniques is also a problem that must be overcome in order for better correlates to be drawn between the two neuroimaging techniques9. This can be done by either increasing fNIRS spatial resolution or testing out the BOLD signal of a larger brain region9. Because fNIRS research is in its infancy, there is currently very little homogeneity of testing procedures9. Methods regarding optode positioning, dealing with individual differences and correcting errors of skin pigmentation and extracerebral tissue distortion of the light signal will need to be addressed9. One exciting future application for fNIRS technology is in its clinical use, screening babies and young children for brain development abnormalities6. Clinical use of fNIRS has found a niche in testing children who may not be suitable candidates for fMRI due to their young age6. The portability and low cost of an fNIRS
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Mood Disorder MRI-guided accelerated repetitive transcranial stimulation over two days achieves results comparable to full traditional treatment Lily Qiu Human Biology Department, University of Toronto, Toronto, ON, M5S 1J7 Depression is one of the leading causes of increased years lived with disability, and has one the biggest global burdens on people as a disease. The impacts of depression will only increase as time progresses, emphasizing the need for an effective, fast, and accessible treatment for a growing number of people afflicted with the disorder. Although pharmacological solutions are widely used to tackle depression, approximately one third of people become treatment-resistant. The non-pharma-cological alternatives for treatment include brain stimulation of the direct neural targets of depression; however, even these techniques have major disadvantages. Deep brain stimulation is an extremely invasive procedure, requiring surgery; electroconvulsive therapy impairs episodic memory and current rTMS protocol is time intensive and has low effectiveness. rTMS, though, is not invasive, nor does it impair memory, thus given its major advantages over other brain stimulation therapies, work needs to be done to maximize its potential. In this review, three pivotal changes are suggested to increase the effectiveness and accessibility of rTMS treatment for depression. First is a change in treatment targets: traditionally the dlPFC has been used as a brain target for rTMS, however promising research has shown that the dmPFC or vmPFC are also involved in depression. Second, an improvement in specificity is needed, by replacing the 5cm method with MRI-based neuronavigation to locate brain areas to target. Lastly, the time commitment needed for rTMS spans over many weeks and therefore makes treatment impossible for many individuals. Lowering the frequency (Hz) of stimulation, but increasing the amount of stimulation over a shorter period of time, produces comparable results to traditional treatment and requires a much smaller time commitment. If all these factors are successfully amalgamated into a new rTMS treatment protocol, the face of depression treatment could be changed significantly and patients could live the reality of an effective and safe depression treatment that takes as little as a few days.
year), and by 2020, it is projected to be the 2nd leading contributor across all ages and sexes1. At present, the most common treatment is pharmacological, but up to one third of all patients are treatment resistant. Furthermore, the majority of those who do respond to anti-depressants will relapse3. Beyond anti-depressants there are alternative treatments which directly target the neural correlates of depression through stimulation of targeted brain locations. Deep brain stimulation (DBS) is an effective treatment (60% effectiveness) which requires implanting a small, stimulating electrode into the brain. However, this makes the process extremely invasive, requiring neurosurgery and use of anaesthetic. Electroconvulsive therapy (ECT) involves the use of external electrodes to cause a clonic seizure using electrical current. It can be as high as 95% effective in some types of depression but has a significant downfall: ECT disrupts episodic memory, which is the memory that accounts for the events that people experience throughout their lives. A third alternative is repetitive transcranial magnetic stimulation (rTMS) which uses pulsing magnetic fields administered by an external coil to increase, or disrupt, activity in brain targets. Its major advantages over DBS and ECT are that it is completely noninvasive, does not impair memory, and is actually seen to increase cognitive skills4. However, current methodology in rTMS application makes it a time-intensive procedure which has lower effectiveness than either DBS or ECT. Given its advantages over ECT and DBS, rTMS proves to be a treatment option worthy of ameliorating its potential. Although rTMS technology has been present since 1994, three alterations could greatly increase its effectiveness. This review aims to cover the three aspects: alternative treatment areas, increased specificity of brain targets using MRI neuronavigation and changes in frequency and length of treatment.
I.BACKGROUND Depression is characterized by the presence of a depressed mood, feelings of low self-worth and loss of interest or pleasure1. It is the leading cause of years lived with disability2, as it disrupts the normal functioning of an individualâ€™s abilities to regulate his or her everyday responsibilities, spanning social, workplace and interpersonal domains. Currently, depression is the 4th leading contributor to the global burden of disease, as measured by the number of years lost due to depression (disability-adjusted life
A. Incorporation of the dmPFC as a Brain Target Traditionally the dorsolateral prefrontal cortex (dlPFC), which is associated with affect and working memory, among other things, is the brain area that is targeted in rTMS treatment. However, other areas of interest have been circulating in the literature which may be an alternative, or even better, candidate for depression treatment. The dorsal medial prefrontal cortex (dmPFC) is seen as a â€œconvergence pointâ€? where memories and emotion get processed4 and it has been observed to be involved in inducing
II. PROPOSED RESEARCH - Materials and Methods This section will address three facets of the current rTMS treatment protocol which can be improved and altered to increase the effectiveness of rTMS as a treatment for depression.
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depression-like dysphoric moods. Stuferak et al, while stimulating the subthalamic nucleus (STN) using DBS for Parkinson’s treatment on a patient, additionally stimulated areas superior and lateral to the right STN, including the dmPFC. Within 30 seconds of stimulation at 130 Hz, the patient described feelings of intense dysphoria, profound anhedonia, discomfort and shed tears, while stating,”[I] did not care about myself or anything… someone could have come in to shoot me and I could not have cared less.” These effects disappeared upon cessation of stimulation5. Although this is one case study, these drastic results suggest that the dmPFC is certainly involved in the regulation of mood and depression and should be taken into consideration as a candidate for a brain target involved in depression. Additionally, the ventromedial PFC and ventrolateral PFC may also be good candidates, as they are involved in emotional reappraisal3 (Fig. 1).
C. Lower intensity, but Higher Frequency Treatment over a Shorter Period of Time One typical rTMS treatment session consists of 20-75 rTMS trains, of 5-20Hz and 2-10 second duration at an intensity of 100-120% resting motor threshold. Each session lasts approximately one hour and the patient needs to come in for 20-30 treatments. This proves to be extremely time intensive and can be a problem for patients who are limited by their work schedules, travel schedules or accessibility to treatment centres. Holtzheimer et al conducted a study modulating the traditional time scale and intensity of rTMS: 15 rTMS sessions, each lasting 10 minutes, were administered only over 2 days and each 5 second train was of a 1-Hz frequency, at 100% motor threshold intensity, directed at the dlPFC (Holtzheimer). III. EXPECTED RESULTS A. Different Brain Areas Although no studies involving rTMS as depression treatment and brain areas other than the dlPFC have been conducted, unpublished work by Dr. Jonathan Downar has seen promising results 4. Based on past research indicating that patients with damage to the dmPFC often suffer depression-like injuries though, this area of the brain remains a good candidate for incorporation of future standard rTMS treatment protocol. B. MRI-Neuronavigation A study comparing usage of the 5cm method versus MRI-guided rTMS in depressed patients found that 6 weeks posttreatment, those who underwent MRI-guided rTMS resulted in a 15 point decline in their Montgomery-Asberg Depression Rating Scale (MADRS), whereas the 5-cm method group only declined 9 points6 (Fig. 2). Another case study investigating MRIguided rTMS saw a decrease of 24 points in the MADRS after 10 weeks of treatment7. Thus, by using MRI as a basis for location specific targets, the efficiency of rTMS is greatly increased.
Figure 1: Medial and lateral views of the brain showing the dorsolateral prefrontal cortex (DLPFC) which is the traditional target in rTMS depression treatment. Dorsomedial prefrontal cortex (dmPFC) and ventromedial prefrontal cortex (vmPFC) are potential candidate areas10.
B. Use of MRI-Neuronavigation Traditionally, to locate the dlPFC, or Brodmann Areas 9 and 46 (BA 9/46) for rTMS treatment, psychiatrists use the “5 cm method.” This involves locating the motor cortex by stimulating the scalp until a hand muscle (abductor pollicis brevis) twitches, then moving 5 cm in the anterior direction along the saggital plane of the head6.Not surprisingly, this method is often inaccurate, and a study investigating the accuracy of the 5cm method in comparison to MRI localization determined that the 5cm method correctly located BA 9/46 in only 7 of 22 subjects7. An alternative to the 5 cm method is by using MRI images to guide localization of brain areas. Patients would undergo an MRI scan with a number of fiducial markers, such as vitamin E capsules, indicating scalp locations which were then associated with specific brain areas. Matching each fiducial location on the MRI scan with a point on the patient’s external head allows proper placement of the rTMS coil for accurate stimulation of a specific brain area6.
Figure 2: Change in MADRAS score over time for standard 5-cm method patients and MRI-guided rTMS patients6.
C. Changes in Intensity and Frequency of rTMS Results from a study using accelerated rTMS treatment over 2 days found that a significant treatment effect was achieved by day 3, as Hamilton Depression Rating Scale scores decreased by 47%. Furthermore, cognitive skills did not decline, nor were seizures induced. These findings make the results of accelerated rTMS
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comparable to results obtained from a full 4-6 week rTMS treatment, both in terms of safety and quality of results, making the accelerated version of rTMS a more efficient method than traditional treatment9. Discussion Amalgamating all three of these changes into a new rTMS treatment protocol could have major potential in the realm of depression treatment if successful. As depression increases its status as a leading cause of burden to the global population, an effective, fast and personalized treatment is needed. With the exploration of new brain areas, including the dmPFC, depression treatment will become more personalized, as the neural bases of depression is not the same for everybody. In addition to personalizing treatment, information about alternative areas other than the dlPFC will give psychiatrists more options if stimulation of one brain area fails to provide a response. In addition to new candidate brain targets, it is vital for the proper localization of these targets to be accurate for each and every patient who seeks treatment. The 5 cm method only applies to localizing the dlPFC (BA 9/46) and often incorrectly locates the brain target. Incorporating MRI maps of the brain to find brain targets is an easy way to ensure that brain areas are localized accurately and that individual differences in brain structure will not interfere with finding a target, but will actually enhance the process. Furthermore, the specificity of targeting brain areas will increase with MRI-guidance. The major setback of current rTMS treatment today is that it is extremely time intensive, requiring long periods of successive time that a patient must devote. By altering the frequency and intensity of rTMS so that treatment can occur over periods as short as 2 days, this will eliminate the time-commitment problem and make treatment much more accessible for many more patients.
which reaches farther than the limits of just depression treatment. 1. “Depression.” World Health Organization, 2012. Web. 23 March. 2012. http://www.who.int/mental_health/management/depression/definition/ en/index.html 2. “Investing in Mental Health.” Department of Mental Health and Substance Dependence, Noncommunicable Diseases and Mental Health, World Health Organization. 2003. Switzerland. Nove Impression. Web. 23 March. 2012. <http://www.who.int/mental_health/en/investing_in_mnh_ final.pdf> 3. Kennedy SH, Downar J. New device therapies for treating depression. Advocate 18 (1), 6-8 (2011). 4. Downar, Jonathan. Personal interview. 7 March. 2012 5. Stefurak, T et al. Deep brain stimulation for Parkinson’s disease dissociates mood and motor circuits: a functional MRI case study. Movement Disorders 18 (12), 1508-1541 (2003). 6. Fitzgerald, PB et al. A Randomized Trial of rTMS Targeted with MRI Based Neuro-Navigation in Treatment-Resistant Depression. Neuropsychopharmacology 34, 1255–1262 (2009). 7. Herwig U et al. Transcranial magnetic stimulation in therapy studies: examination of the reliability of ‘‘standard’’ coil positioning by neuronavigation. Biol Psychiatry 50, 58–61 (2001). 8. Trojak B, Meille V, Chauvet-Gelinier J-C, Bonin B. Further evidence of the usefulness of MRI-based neuronavigation for the treatment of depression by rTMS. J Neuropsychiatry Clin Neurosci 23 (2), E30-31 (2011). 9. Holtzheimer, PE et al. Accelerated repetitive transcranial magnetic stimulation for treatment-resistant depression. Depression and Anxiety 10, 1-4 (2010). 10. Pizzagalli DA. (2011). Frontocingulate Dysfunction in Depression: Toward Biomarkers of Treatment Response. Neuropsychopharmacology. 36: 183-206. http://www.nature.com/npp/journal/v36/n1/images/npp2010166f2.jpg 10. Pizzagalli DA. (2011). Frontocingulate Dysfunction in Depression: Toward Biomarkers of Treatment Response. Neuropsychopharmacology. 36: 183-206. http://www.nature.com/npp/journal/v36/n1/images/npp2010166f2.jpg
Significance of Work The significance of this work could be potentially huge and revolutionize the way that depression is treated. Previous, but unpublished research, by Dr. Jonathan Downar 4 that uses accelerated MRI-guided rTMS on the dmPFC has resulted in a 55% response rate (50% decrease in depression symptoms) and 3035% remission (effectively no depression symptoms) for patients. This is extremely promising and hopeful for those depression patients for which anti-depressants have consistently failed them. Future Directions The use of an rTMS protocol which combines all of these factors is still in its infancy, so this field holds a lot of potential for further research. Because the studies presented in this review are based on small sample sizes, a key step towards making rTMS a standard depression treatment is to conduct more studies with much larger sample sizes. By doing this, researchers will be able to increase the specificity of brain targets, allocate more candidate areas for stimulation and optimize the time-requirement and strength of rTMS. The possible outcome is a definition of specific stimulation parameters and targets to personalize depression treatment for each individual patient. Dr. Jonathan Downar4 has postulated that due to the shortened time course of accelerated rTMS, one psychiatrist with 3 machines could treat up to 900 patients per year! Additionally, with the refinement of rTMS techniques and inclusion of MRI, rTMS can be ameliorated for some of its current uses, including treatment for Parkinson’s disease, strokes and migraines, giving this research potential
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Neuropathology Inflamed Brain: The role of Inflammation in the Development of Depression Alicia A. Rodrigues Human Biology Department, University of Toronto, Toronto, ON Major depressive disorder (MDD) is a serious psychiatric illness that is highly prevalent. Despite the availability of various antidepressants, treatment outcomes remain poor for many individuals. There is a need for the increased understanding of the mechanisms and processes underlying the manifestation of depression so that ultimately novel and more effective treatments may be developed. A growing body of literature has implicated inflammation in depression. Higher levels of inflammatory markers are found in individuals with major depressive disorder, and medical disorders characterized by inflammation are often associated with depression. Inducing inflammation produces depressive symptoms in individuals, and sickness behavior in response to infection and inflammation shares many similarities with depression. This review will discuss these observations linking inflammatory processes to depression, and discuss the mechanisms by which inflammation may lead to depression, including the effects on neurotransmission and the neuroendocrine system. Finally, the importance of this area of research will be reviewed, with a focus on how this area of research may lead to novel antidepressant treatments. Background Major depressive disorder (MDD) is a severe psychiatric illness that is highly prevalent and associated with significant functional impairment.1 MDD is often recurrent or chronic, and is expected to become the second leading global cause of burden of disease by 2020.2 Despite major advances in research in the field of depression, much of our understanding of the pathophysiology of this disorder remains incomplete. The monoamine hypothesis of depression underlies the mechanism of action of many antidepressants currently used in treatment, however, this focus on neurotransmitters fails to explain all aspects of the disorder, and treatment outcomes are often poor for many individuals.1 This demonstrates the need to increase our understanding of the disorder, and bring these findings together to translate the research into effective treatments.1 In the past twenty years, there has been a growing area of research linking psychiatric disorders, particularly depression, to inflammatory processes. If inflammation plays a role in the onset of depression, it would be predicted that risk factors for developing depression are also associated with elevated levels of inflammatory markers.1,3 Indeed, significant risk factors for MDD, including psychosocial stress, poor diet, obesity, various medical illnesses, lack of sleep, poverty, and social isolation all lead to increased inflammation, suggesting the existence of a common pathway of these factors.1,3 Further evidence comes from studies showing that
levels of inflammatory markers are increased in individuals with MDD, and medications such as interferon that effect the immune system can lead to depressive symptoms. The observation that ‘sickness behaviour,’ which is the body’s response to increased inflammation and cytokines, shares symptoms with depression further delineates this association. Other findings suggest that current antidepressants work by targeting inflammatory mechanisms, and that blocking inflammation may lead to development of novel treatments. These main observations will be discussed in greater detail in this review, along with a look at the possible pathophysiology linking inflammation and depression, and finally a discussion on how these findings may translate to a clinical setting to treat patients effectively. Inflammation Inflammation is a physiological process coordinated by the immune system. Although acute inflammation is localized and short-lasting, chronic inflammation results in increases in systemic circulating concentrations of inflammatory markers and cytokines, and can lead to atherosclerosis, cardiovascular disease, and, possibly, depression.4 Inflammation is characterized by increased mRNA expression of proinflammatory cytokines, such as interleukin 6 (IL-6), interleukin-1β (IL-1β), and tumour necrosis factor α (TNFα), and upregulation of enzymes that encode inflammatory enzymes, such as cyclooxygenase-2 (COX2).2 Cytokines are signalling molecules produced by accessory immune cells at sites of infection, and they communicate with leukocytes to organize and coordinate inflammatory processes in response to pathogens.2,4 These cytokines produced in the periphery can act on the brain, resulting in a local production of proinflammatory cytokines which go on to have effects on pathways involved in cognition and mood, by influencing neurotransmitters, neuroendocrine function, and plasticity.5 MDD is associated with elevated levels of inflammatory markers In individuals with major depressive disorder, the levels of proinflammatory cytokines and inflammatory markers are increased. One meta-analysis found that increased levels of IL-1, IL-6, IL1ra and CRP are positively associated with depression, and this was a dose-response relationship.1 Similarly, Dowlati et al. (2010) included 24 studies in a meta-analysis, looking at the concentration of cytokines in depression.1,2,6 Patients with MDD showed significantly higher serum levels of TNFα and IL6 compared to control patients.1,2,6 Finally, in a recent meta-analysis, IL6, TNFα and sIL-2R were found to be significantly elevated in MDD patients compared to controls.2,7 More convincing evidence comes from studies where these in-
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creased levels are corrected in response to antidepressant treatment.2 In a meta-analysis of twenty-two studies using antidepressant treatments, levels of IL1β and IL6 decreased in response to treatment with SSRIs, along with a reduction of depressive symptoms.2,8 This suggests that inflammatory cytokines contribute to symptoms of depression, and that antidepressant treatments may work by mediating the concentration of these cytokines.2 Inflammatory cytokines as biomarkers Since depression is a heterogenous disorder, developing a biomarker would aid in diagnosis and help to predict treatment response of different individuals.2 Levels of inflammatory cytokines can be used to predict an individual’s risk of developing depression and thus may serve as useful biological markers.2 In one study it was found that increased C-Reactive protein (CRP) and IL6 levels at baseline were associated with depressive symptoms in these individuals 12 years later at follow-up.1 Furthermore, another study found that elevated IL-1ra predicted an increased risk for the presence of depressive symptoms over a 6-year follow-up period.1 Since high CRP levels at baseline is a risk factor for new MDD onset, this suggests that inflammatory cytokines could potentially be useful in identifying at-risk individuals. This promising possibility would enable early intervention and the use of preventative treatments, as well as a method to diagnose individuals in a standardized manner.2 Inflammatory medical disorders are associated with MDD Inflammatory disorders are associated with unusually high rates of depression.9 Within 18 months of experiencing a myocardial infarction, 65% of patients develop depressive episodes, and approximately one-third of stroke patients develop MDD compared to 13% of control individuals.9 Metabolic syndrome (MetS) is a group of related metabolic dysregulations, including hypertension, abdominal obesity, hyperglycemia, insulin-resistance, leptin-resistance, and hypercortisolemia.5 Patients with MetS have an increased prevalence of mood symptoms compared to the general population.5 Inflammation is a significant component of MetS; for example, type 2 diabetes and obesity are characterized by elevated plasma levels of cytokines.5 Together, these findings suggest that various medical conditions can lead secondarily to the development of depression through the common underlying inflammatory processes. Treatment with cytokines is associated with MDD Medications that effect the immune system can lead to depressive symptoms.1,2 Cytokines, such as interferon-α and IL2, are often used in the treatment of disorders such as hepatitis C, multiple sclerosis and cancer.1,2 Individuals undergoing these treatments are at an increased risk of developing symptoms of depression. The inflammatory stimulation induced by IFNα yields a variety of behavioural outcomes, ranging from fatigue and malaise to full blown MDD requiring psychiatric hospitalization.2,10 In one study it was found that within 12 weeks of interferon-α treatment at high doses, about 45% of patients without pretreatment depression developed MDD.2,11 Pretreatment with the SSRI paroxetine was found to reduce the psychological symptoms associated with depression in these patients.10 IFN- α increases levels of the proinflammatory cytokines IL6 and MCP1.1 Furthermore, hepatitis C patients undergoing IFN therapy have reduced levels of peripheral serotonin, and increased depressive symptoms in these individuals
are associated with elevated metabolites of indoleamine deoxygenase enzyme.2 Functional polymorphisms in IL6 and the serotonin transporter (5HTT) affect the risk of developing depression in response to IFN-α treatment.1 These studies that show that inducing inflammation results in depression provide strong evidence for the role of inflammation in depressive symptoms. Sickness behaviour Sickness behaviour is a symptomatology which shares similarities with major depression. This response to infection and inflammation is characterized by fatigue, anhedonia, poor appetite, lack of sleep and social isolation.1,2,10 Sickness behaviour is induced by proinflammatory cytokines (IL1α, IL1β, IL6, TNFα, INFγ), and administration of lipopolysaccharide or proinflammatory cytokines evokes these symptoms of sickness.1,2 This suggests that mediators of sickness behaviour may also play a role in the pathogenesis of depression. Hypothalamus-Pituitary-Adrenal Axis The hypothalamus-pituitary-adrenal (HPA) axis plays an important role in mediating the stress response. Clinical depression is often associated with a hyperactive HPA axis, with increased levels of plasma cortisol, and decreased sensitivity to administered corticotrophin releasing hormone (CRH) or dexmethasone.1,2,10 Proinflammatory cytokines such as IL6 that are significantly elevated in response to chronic stress result in increased release of CRH and adrenocorticotropic hormone (ACTH), which potentiates the HPA axis.10 Thus, the hypercortisolism and decreased sensitivity of the HPA axis seen in depressed individuals may be mediated by proinflammatory cytokines through their actions on the HPA system.1,10 Neurotransmitters Inflammation and the serotonin system seem to interact with each other through a bidirectional relationship. Proinflammatory cytokines, such as TNFα, INFγ, and IL2, activate the kynurenine pathway by inducing indoleamine -2-3-dioxygenase (IDO).1,2 (See Figure 1). IDO is an enzyme that catabolises the metabolism of tryptophan into kynurenine.2 Since tryptophan is a substrate for the synthesis of serotonin, this reduces the availability serotonin. Kynurenine crosses the blood-brain-barrier into the brain where it is metabolized by kynurenine monoxygenase into kynurenic acid (KA), and quinolinic acid (QA), leading to 3-hydroxy kynurenine (3HK).2 The metabolites 3HK and QA are NMDA receptor agonists, and this alteration of the glutamatergic system may play a contributing role in the development of depression.1 IDO enzymatic activity is increased in response to acute or chronic activation of the immune system, such as in conditions like atherosclerosis, obesity, rheumatoid arthritis, and coronary heart disease, and treatment with IFNα increases IDO activity, and elevates levels of kynurenic acid and quinolinic acid.1,10 In patients with MDD, those who had a history of attempted suicide had higher levels of kynurenine compared to those with no history of suicide attempt.1 Other findings show that cytokines may affect neurotransmission of serotonin by mechanisms other than this IDO-mediated pathway. For example, interferon-α has been found to decrease the expression of the serotonin receptor 1A in several non-neuronal cell lines, and this effect is blocked by desipramine and fluoxetine, two antidepressant drugs. 10 Tricyclic antidepressants, as well as
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selective-serotonin/noradrenergic reuptake inhibitors normalize plasma levels of cytokines and inflammatory proteins, including IL-6, IL-1β, TNF-α, INF-γ.1 Besides serotonin, glutamate is another neurotransmitter that has been implicated in inflammation and depression. Glutamate-induced excitotoxicity occurs when overactivation of glutamate receptors leads to excess build up of intracellular calcium, resulting in neuronal death.10 Proinflammatory cytokines mediate increase neurotransmission of glutamate through several different mechanisms. One way this occurs is when cytokines increase levels of the NMDA-agonists kynurenic acid and quinolinic acid, which are neurotoxic metabolites.10 These studies imply that one mechanism by which proinflammatory mechanisms exert effects to result in depression is through alteration of neurotransmission, including the serotonergic and glutamatergic systems.
Figure 1. The kynurenine pathway. Proinflammatory cytokines interferon-γ (IFN-γ) and Tumour necrosis factor-α (TNF-α) induce indoleamine-2-3-dioxygenase (IDO), which reduces serotonergic neurotransmission by decreasing the availability of tryptophan. This pathway produces the metabolites 3-hydroxykynurenine and quinolinic acid, which are agonists for the NMDA receptor. This pathway links inflammation (through the cytokines) to decreased serotonin transmission which has been widely studied in depression. Source: Dantzer, R., et al. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat Rev Neurosci 9, 46-57 (2008).
Treatment Based on animal studies, COX2 inhibitors, which block the production of proinflammatory cytokines and prostaglandin E2, have emerged as a potential treatment.1,2 In patients with MDD treated with reboxetine, the COX2 inhibitor celecoxib significantly improved response to the antidepressant, as measured by response on the Hamilton Depression Scale.2 Another inhibitor of cyclooxygenase, acetylsalicylic acid more commonly known as asprin, accelerates the onset of SSRI antidepressant effects, and improves response rate.1 In a recent study by Wang et al. (2011), treatment resistant depressive rats had significantly improved depressive behaviours and decreased COX2 and prostaglandin E2 levels in the hippocampus when acetylsalicylic acid was used adjunctively with fluoxetine.12 Focusing on diet in future research may provide insight into ways of both preventing and treating depression using readily available therapeutic options. In particular, docosahexaenoic acid is an omega-3 polyunsaturated fatty acid (PUFA) that inhibits production of inflammatory cytokines and has antidepressant effects.9 It has been suggested that omega-3 and omega-6 PUFAs
exert their anti-depressant effects by regulating apoptosis-inducing factors, such as excitotoxicity and free radicals. Cytokines lead to apoptotic cell death, and apoptosis is increased in depressed individuals compared to controls.9 Another potential treatment comes from studies where inhibiting IDO using 1-methyltrytophan has been successful in decreasing depressive symptoms in animal models using inflammatory challenges.1 Lastly, behavioural therapies, such as meditation, physical exercise, and psychotherapy have been found to reduce the peripheral inflammatory response.2,3 Future Directions This burgeoning area of research has shown that there is undoubtedly a link between depression and inflammation. The exact mechanism by which inflammation may lead to the onset of depression is unknown, however there is evidence to show that cytokines may work by affecting the major neurotransmitter systems associated with depression or by activating the HPA axis. Much remains to be understood to clarify the pathophysiology of depression and to elucidate the role of inflammation. Future research will continue to clarify the exact role neuroinflammation plays in depression. This increased understanding of inflammatory processes and the pathophysiology of depression may lead to novel treatments. With increased interest in techniques such as deep brain stimulation, research into inflammation may provide more pharmacological interventions, aimed at decreasing inflammation or the downstream effects. More importantly, future directions may lead to an increased focus on preventative measures, such as exercise and diet, in order to intervene early and decrease the effects of inflammation. Even further, the use of inflammatory markers could offer the potential to have biomarkers identify different subtypes of depression, and whether certain individuals are vulnerable to developing the disorder, allowing intervention and preventative measures to be implemented. Another interesting future direction is the possibility of inflammation providing a valid animal model of depression. A significant future direction will be to study inflammation not only in the context of depression, but related to many other psychiatric or neurological disorders, such as Alzheimer’s Disease, Parkinson’s Disease, Amyotrophic lateral sclerosis, schizophrenia, and anxiety.1 Findings can also expand to the study of conditions such as stroke, rheumatoid arthritis, and metabolic syndrome, to understand the increased risk of developing depression in these patient populations.1,2 Significance and Conclusion Depression is a serious, chronic and potentially debilitating disorder that is highly prevalent. Despite many currently available antidepressants, treatment outcomes remain poor, and patients often experience relapse. Depression affects a wide range of behaviours, from cognition and motivation, to sleep patterns and feeding. This is associated with many effects in the brain, including structural changes, and effects on neurogenesis, neuroendocrine paths, and neurotransmission. This reflects the heterogeneity of depression and the wide range of behavioural phenotypes. The many studies and wide ranging pieces of evidence provided in this review demonstrate an undeniable role of inflammation in depression. This exciting area of research suggests a common or shared pathway whereby stressors and risk factors lead to inflammation,
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resulting in the manifestation of conditions or disorders, such as depression, in predisposed individuals. In particular, inflammation is such a significant field because it ties together many pieces of evidence into a logical story. Stressors lead to inflammation, which leads to changes in the brain, ultimately resulting in the manifestation of different behaviours, including depressive symptoms. We are still a long way off from having a complete understanding of the pathophysiology involved in depression and other psychiatric disorders. However, studying neuroinflammation is an exciting and promising step toward achieving this. More importantly, studying inflammation offers the possibility of developing novel and effective treatments. 1. Raedler, T.J. Inflammatory mechanisms in major depressive disorder. Curr Opin Psychiatry 24 519-525 (2011). 2. Krishnadas, R., & Cavanagh, J. Depression: an inflammatory illness? J Neurol Neurosurg Psychiatry (2012). 3. Raison, C.L, & Miller, A.H. Is Depression an inflammatory disorder? Curr Psychiatry Rep 13, 467-475 (2011). 4. Motivala, S.J. Sleep and Inflammation: Psychoneuroimmunology in the Context of Cardiovascular Disease. Ann Behav Med 42, 141-152 (2011). 5. Dinel, A., et al. Cognitive and Emotional Alterations Are Related to Hippocampal Inflammation in a Mouse Model of Metabolic Syndrome. PLoS ONE 6, e24325 (2011). 6. Dowlati, Y., et al. A meta-analysis of cytokines in major depression. Biol Psychiatry 67, 446-457 (2010). 7. Liu, Y., Ho, R.C., & Mak, A. Interleukin (IL)-6, tumour necrosis factor alpha (TNF-alpha) and soluble interleukin-2 receptors (sIL-2R) are elevated in patients with major depressive disorder: a meta-analysis and meta-regression. J Affect Disord (2011). 8. Hannestad, J., Dellagioia, N., & Bloch, M. The effect of antidepressant medication treatment on serum levels of inflammatory cytokines: a metaanalysis. Neuropsychopharmacology 12, 2452-2459 (2011). 9. Pascoe, M.C., Crewther, S.G., Carey, L.M., & Crewther, D.P. What you eat is what you are- A role for polyunsaturated fatty acids in neuroinflammation induced depression? Clinical Nutrition 30, 407-415 (2011). 10. Dantzer, R., et al. From inflammation to sickness and depression: when the immune system subjugates the brain. Nat Rev Neurosci 9, 46-57 (2008). 11. Musselman, D.L., et al. Paroxetine for the prevention of depression induced by high-dose interferon alfa. N Engl J Med 344, 961-966 (2001). 12. Wang,Y., Yang, F., Liu, Y., Gao, F., Jian, W. Acetylsalicyclic acid as an augmentation agent in fluoxetine treatment resistant depressive rats. Neurosci Lett 499, 74-79 (2011).
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Nanotechnology Targeting system of the future: The evolution of nanotechnology Dushyaan Sri Renganathan University of Toronto Human Biology Department Toronto, Ontario, M5S 3J6 The field of nanotechnology has been growing substantially over the past 10 years, especially in terms of advancements which allow for improved cell targeting and imaging. Over the past decade many different types of nanoprobes have been created for tumor imaging, and this review will examine some of the newer models such as zinc oxide probes, NIRF based probes, MRI based probes, and multiplex brain tumor targeting probes. One type of probe in particular, the NPCP-CTX-Cy5.5, has addressed many of the shortcomings in older models, and it seems to be the first in the line of a new generation of multiplex super probes. Due to the advent of these new advances, nanoprobes are showing a great deal of potential in the field of medicine, where they can be used for varying purposes, from disease monitoring to drug delivery. Background Brain tumors can arise in various forms and pose a serious risk to the patient regardless of their stage. Unfortunately, even though modern approaches are somewhat successful in treating these cancerous growths, they come with their own set of detrimental side effects. Most treatments are either very invasive or subject the body to an onslaught of harsh chemicals and radiation1. Needless to say, the process of treating brain tumors might leave some individuals irreparably damaged. Therefore, there exists a great need for probes that help target and remove these outgrowths noninvasively without compromising accuracy. The process of precisely targeting these biological entities has posed the greatest challenge, due to the lack of specificity of older probes1,2. Nanobiotechnology might provide a solution to this lack of precision3. This field, which arose from the intersection of molecular biology, nanotechnology, and medicine, has grown in the past decade1,3. The ingenuity of nanoprobes lies in their miniature size, which allows them to transverse different biological barriers relatively unencumbered. They have shown great accuracy in detecting biological molecules (eg. DNA, RNA, and proteins) or other factors (eg. cancer, viruses, and bacteria)3. Also, the versatility of these probes is due to their ability to allow the chemical conjugation of different components, which can improve either targeting or barrier crossing1. The potential customizability of nanoprobes has made them a hot topic in areas such as personalized medicine. This review will provide an overview of the advancements in this field by analyzing examples used to image cancer cells both in vitro and in vivo. Near-infrared fluorescence (NIRF) based probes, MRI based probes, zinc oxide probes, and multiplexed probes will be examined for their applicability as potential tumor targeting devices. ZnO light-emitting nanoprobes Zinc oxide nanoprobes are considered one of the most
promising candidates for tumor detection4. They have been selectively engineered to provide stronger emission signals around the purple to UV region, while concurrently reducing absorption by cells and tissues, a common problem faced by older probes, which emit around the yellow to green region4. Zinc oxide is conjugated with metals, such as gold and silver, in order to increase uptake into the cytosol and endosome while minimizing toxicity to the cell4. Chieh Yang and colleagues were the first to conjugate the zinc oxide probe with an antibody for epidermal growth factor receptor (EGFR) in order to targeted squamous cell carcinomas4. These cancerous cells have a high expression of these receptors on their surface, and therefore are easily targetable by these nanoprobes4. (figure 1) In vitro tissue samples show a strong purple emission, which is easily detectable by a normal microscope when the probes are excited; however, when these nanoparticles are administered without the EGFR tag only a low emission is observed. (figure 1) Without the antibody motif, the probes exhibit a low specificity and tumor binding4. Also, when probes conjugated with EGFR were added to Hs68 cells, a low signal was observed once again4. Hs68 cells are derived from human foreskin fibroblasts, and express low levels of EGFR on their surface4. Therefore, even though these probes exhibit targeting specificity, they are limited by the conjugated targeting antibody. Nanoprobe Imaging using MRI MRI has become one of the most fundamental forms of imaging, due to its high temporal and spatial resolution6. It poses little risk to the subject, due to the utilization non-ionizing radiation, and its capacity to track diffusion processes and proton densities make it an ideal technique for monitoring magnetic probes in vivo, given that an appropriate contrast dye is used5,7. Gadolinium (III) is the most commonly used contrast dye, but due to its toxicity, it is normally used in combination with chelators such as DTPA, DOTA, or DTPA-BMEA7. Recently, gadolinium (Gd) has been combined with nanoparticles for imaging fine cell structures, as ultra small Gd oxide nanoparticles (US-Gd2O3),coated with diethylene glycol (DEG), can be easily taken up into cells, and visualized using T1-weighted MRI images6. Fuacher et al. used these probes to image glioblastomas within chick embryos using MRI.(figure 3) These probes were highly sensitive and were able to pick up early stage tumors6. (figure 2) Large amounts of gadolinium were internalized and retained within the cell in order to provide high contrast images5,6. Retention times were determined by measuring gadolinium concentrations at different time points after co-incubation of embryo with probes5. Internalization was also observed using Transmission electron microscopy (TEM)5. These probes have potential as early stage tumor detectors; however, safety concerns regarding Gd poisoning reduces the applicability of this model in humans5,7.
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Figure 1 | Squamous cell carcinomas probed with ZnO Figure a-b depicts squamous cell carcinomas probed with ZnO conjugated with EGFR antibodies. C depicts cancerous cells probed with ZnO alone. A&C were imaged under white light while B was imaged using UV pulsed laser. Modified from REF 4.
Figure 2 MRI image of Glioblastoma within chick embryo. A-d depicts T1-weighted images of unlabeled cells;(f-i) depicts Gd labeled cells where tumor detection occurs earlier. E&J depicts labeled and unlabeled tumors which have been harvested. Modified from REF.6
Near Infrared Fluorescence Imaging (NIRF) Near Infrared Fluorescence Imaging (NIRF), is becoming a common tool for imaging living organisms. It is an ideal candidate for imaging cancer cells because it has low absorption and suffers from virtually no autofluorescence. It also possesses high spatial resolution, high sensitivity, and is safe because it utilizes non-ionizing radiation8,9. Furthermore, this model is very cost effective when it comes to creating new nanoprobes10. NIRF nanoprobes target tumors through three different methods: 1) targeting cancerous material through enhanced permeability and retention (EPR); 2) targeting specific antigens or receptors on the cell surface; 3) and activation by the microenvironment associated with that tumor cell.8,9,10 Tumor cells have a disrupted vasculature, which is created by the secretion of different factors that cause its rapid proliferation. These tumor cells usually have perforated basement membranes, numerous trans-endothelial channels, and poor lymphatic systems.8 These defects can be utilized by nanoprobes to enter the cells through EPR. Small nanoparticles between 10 to 100nm, if retained in circulation for a prolonged period of time, can accumulate within tumor cells9,10. The amount of accumulation depends on the size, half life, and surface properties of the nanoparticles
Figure 3 | Implantation and imaging protocol. Modified from REF.6
in addition to the leakiness of the vasculature8,10. Furthermore, NIRF dyes appear to increase the retention time within tumor cells. However, this targeting method is passive and doesnâ€™t show the same level of specificity as the other two forms10. Conjugating nanoprobes with specific targeting molecules, for the cancerous tissue of interest, is now a common technique employed to increase specificity8. These targeting molecules can range from small peptides to large proteins and antibodies8. Goa et al was one of the first to use quantum dots (QD), a type of nanoprobe, conjugated with a NIRF dye and targeting construct for imaging purposes11. This construct consisted of an arginine, glycine, and aspartic acid residue, which targeted integrin avb3-positive vasculature in mice implanted with human xenografts11. Even though this construct exhibited great specificity, its large size restricted it to the surrounding vasculature preventing it from targeting cells specifically11. Activateable nanoprobes increase their signal in the presence of a biological molecule or microenvironment. These types of probes are considered superior because they have a higher signal to background ratio. The activity of these probes are highly dependent on tumor related enzymes such as cathepsin D and matrix metalloproteinases- 2, which are necessary to cleave different moieties in
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order to activate the NIRF dyes8,9. Therefore, different probes need to be customized to different types of tumors. Currently there are two types of activateable nanoprobes: 1) the conjugation of a NIRF dye to a nanoprobes moiety which inhibits its action; 2) the conjugation of a QD to gold particles where gold plays an inhibitory role8. Lee et al. created gold nanoparticles that had a Cy5.5 conjugated component. These nanoprobes would react with metalloproteinases-2, which would cleave its core releasing the fluorescent dye from the quenching effects of the gold nanoparticles8.(Figure 4) These nanoprobes exhibited very low background signals and fluorescence was only seen in the presence of metalloproteinases-2 (MM-2)8.(figure 4) This same principal is seen when QDs are conjugated with gold nanoparticles where fluoresce is reduce by 70% in the presence of the inhibitor11. When these probes react with the enzyme of interest the fluorescence of the QD is restored. Activatable nanoprobes have a lot of potential, but there are still a few pitfalls that need to be addressed before this work can be done in humans.
Figure 4 | Activation of fluorophore via MM-2 (A) schematic of probe activation . (B) In vitro activation of HT1080 tumor xenografts after co-incubation with MM-2 (left). In vivo visualization of tumor xenografts within mouse mammary fat pad after intravenous injection of probe. Modified from REF.9.
Multiplex Probes Newer probes, which are under development, contain a combination of different components which aid in visualization and targeting. A prime example, is work done by Veiseh and colleagues where medulloblastomas were targeted using a multiplex nanoprobe.
The probe (NPCP-CTX-Cy5.5) consisted of an iron oxide core, which allowed detection using MRI, polyethylene gycol coat, which facilitated barrier crossing, chlorotoxin, a tumor targeting molecule, and a Cy5.5 fluorophore12. The multiplex nature of this probe allowed for better visualization of tumor cells in vivo. MRI imaging of iron levels indicated that probes specifically targeted cerebellar tumors without binding to healthy tissue in regions such as the frontal lobes12.(figure 6) Furthermore, probes that lacked the chlorotoxin targeting motif exhibited little specificity for medulloblastomas12. (figure 6) The specificity of these probes was further demonstrated in the tumor free controls, which exhibited no difference in iron levels between the pre and post injection conditions12. These nanoparticles were able to overcome nonspecific binding; one of the biggest flaws associated with older probes. Fluorescent microscopy depicted similar results as the MRI imaging, where signals from Cy 5.5 were only seen when probes contained the chlorotoxin motif, and these were mainly localized to the cerebellar regions12. (figure 5) The fluorescent images also indicated that the targeting domain increased retention time to 120 hrs, while those lacking this motif only lasted 48hrs post injection within the brain12. (figure 5) These probes were even able to traverse the blood brain barrier (BBB) without causing any damage. This was observed through Gadolinium DTPA imaging12. (figure 7) Though normally the Gd DTPA contrast dye remains within the periphery of the brain, due to the blood brain barrier. When this dye was re-administered, post nanoprobe injection, the Gd DTPA still remained within the peripheral neurovasculature12. (figure 7) Therefore, due to the polyethylene glycol coat, these probes are able to traverse the BBB without disrupting it, a crucial ability if these probes are to be used later for therapeutic purposes12. In addition, the toxicity profiles of these nanoprobes indicated that they pose little to no threat to rodents, though they did have a tendency to accumulate in certain organs such as the liver. (figure 8) However, tests examining liver enzymes, such as alanine aminotransferase, indicated that no cell death or damage was caused by these probes12. (figure 8) Therefore, they can be administered at relatively high doses with minimal consequence to the subject12. NPCP-CTX-Cy5.5 nanoprobes represent the forefront of current nanotechnology12. They are easily able to transverse biological barriers such as the BBB and specifically target tumor tissue12. Furthermore, the activity of these probes can be viewed using dual imaging techniques, and their presence poses little threat to the host12. Figure 5 | Fluorescent imaging of medulloblastomas within the cerebellum (A)2h post injection detection of Cy5.5 levels in mice injected with probe and targeting motif (NPCP-Cy5.5-CTX), probe without targeting motif (NPCPCy5.5-CTX) and no injection. (B) 120h post injection detection of Cy5.5 levels within the same conditions. Bottom brain image shows magnified view of probe distribution within cerebellum for each condition. Imaging also indicated targeting motif increased retention time. Modified from REF. 12.
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Figure 6 | R2 Mapping of Iron levels in tumor and wild-type mice (A) R2 MRI mapping of iron levels pre and post injection of nanoprobes in the frontal lobe and cerebellum within tumor (ND2:SmoA1). Mice were injected with targeting probe (NPCP-CTX) and ones lacking the targeting motif (NPCP). (B) R2 MRI mapping of iron levels pre and post injection of nanoprobes within wild-type mice. (C&D) Difference in iron levels from baseline in the frontal lobe and cerebellum after injection of the two types of probes. Modified from REF. 12.
Future Directions Multiplex probes are an amalgamation of the best features currently possessed by different nanoparticles. They hold enormous therapeutic potential, which has yet to be fully exploited. Veiseh and colleagues are one of the first groups that attempt to use these super probes. They have created a very elegant targeting system, which can be generalized to many other disease models, not just cancer. Provided that the correct targeting motif is used, different brain pathologies such as β-amyloid plaques in Alzheimer’s or dopamine neurons in Parkinson’s can be targeted and imaged12,13. Examining these cells in vivo will provide a much better understanding of the progression of these diseases. Furthermore, this work has huge implications in the field of cancer research. Now that cancer cells can be specifically targeted, systems can be created to deliver therapeutic drugs in order to kill these cells12. These probes also have a lot of potential as imaging tools for post operative diagnostics due to their acute sensitivity. Normally, resected tumors have a high chance of reoccurrence in the first 5-6 years12. Therefore, nanoprobes can now be used as imaging tools to test for reoccurrence during this time frame. Whether the role maybe fin vivo imaging or diagnostic analyses, these probes have wide ranging functionalities making their potential almost limitless.
Conclusion As more research is conducted into the utilization of nanoprobes for imaging and therapeutic purposes, newer designs are slowly being developed. Nanoprobes are being created that exhibit greater specificity and can be visualized through multiple different imaging techniques. The NPCP-CTX-Cy5.5 is just one example of this new generation of super probes, which have multiple functionalities. Nanoprobes are going to be the targeting and delivery system of the future, and we are just starting to unlock the vast potential they hold. 1) Torchilin V.P. Multifunctional nanocarriers, Advanced Drug Delivery Reviews. 58, 1532–1555 (2006). 2) Veiseh et al. Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging. Advanced Drug Delivery. 62, 284-304 (2010). 3) Chi et al. Nanoprobes for in vitro diagnostics of cancer and infectious diseases.33 189-206 (2012). 4) Yang et al. Tumor detection strategy using ZnO light-emitting nanoprobes.23, (2012) 5) Bulte JWM, Kraitchman DL. Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed. 17, 484–499 (2004). 6) Faucher et al. Ultra-small gadolinium oxide nanoparticles to image brain cancer cells in vivo with MRI.6, 209-218(2011). 7) Vuu et al. Gadolinium–rhodamine
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nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging. Bioconjug Chem, 16 995–999 (2005). 8) He et al. Near-infrared fluorescent nanoprobes for cancer molecular imaging: status and challenges. 16, 574-583 (2010). 9) Lee, S. et al. Activatable imaging probes with amplifiedfluorescent signals. Chem. Commun. 4250–4260 (2008). 10) Schellenberger, E. Bioresponsive nanosensors in medical imaging. J. R. Soc. Interface. 7, S83–91(2010). 11) Gao, J.H. et al. Multifunctional magnetic nanoparticles: design, synthesis, and biomedical applications. Acc. Chem. Res. 42, 1097– 1107(2009). 12) Veiseh et al. Specific Targeting of Brain Tumors with an Optical/ Magnetic Resonance Imaging Nanoprobe across the Blood-Brain Barrier. Cancer Research. 69, 6200-6207 (2009). 13) Sun et al. Zhang, Magnetic nanoparticles in MR imaging and drug delivery, Advanced Drug Delivery Reviews. 60, 1252–1265(2008).
Molecular Neuroscience Somatic L1 Retrotransposition in the Human Brain: Generating Somatic Diversity between Neurons Jessica Suddaby Human Biology Department, University of Toronto, Toronto, ON Long interspersed element-1 (LINE-1 or L1) retrotransposons are mobile genetic elements that insert themselves into new locations throughout the genome through a “copy and paste” mechanism. L1s comprise approximately 20% of the human genome. L1 somatic insertions are thought to exert their influence by inducing neuronal diversity and thus creating somatic mosaicism within the mammalian brain. L1-induced neuronal diversity has positive and negative potential consequences including adaptive neural plasticity and behavioural changes, and non-adaptive disease propensity. This potential for positive or negative outcomes may serve as an evolutionary gamble for traits that can ultimately increase fitness by promoting survival or decrease fitness by causing neurological diseases. Future directions should continue to examine the complex effects of neuronal L1 retrotransposition in behavioural genetics as well as attempt to catalogue and characterize new L1 insertion sites within the mammalian brain. Introduction Retrotransposons are mobile genetic elements that use a “copy and paste” mechanism to insert themselves into new locations within the genome of a single cell. Retrotransposons are abundant, comprising at least 50% of the human genome. There are three active families of retrotransposons: long interspersed element-1 (LINE-1 or L1), Alu, and SVA. L1 retrotransposons, which comprise approximately 20% of the human genome, are the most prevalent class of retrotransposons. Although it was previously believed that L1 retrotransposition occurred primarily in the germline, it has become evident that L1 retrotransposition is active in somatic tissues as well, particularly the brain. L1 retrotransposition occurs via an RNA intermediate. A section of the L1 DNA opens, allowing the L1 RNA intermediate to be “copied” from its template. The L1 RNA travels into the cytoplasm where it begins to construct proteins specified by the L1 DNA. The
proteins then proceed to form a molecular complex with the RNA and return to the nucleus. In the nucleus, a double stranded copy of the L1 retrotransposon is produced using the RNA as a template. After an endonuclease nicks specific sites of the genomic DNA, the retrotransposon duplicate is then inserted or “pasted” into the genome where the cut was made (Figure 1). Although most L1 retrotransposons are rendered inactive due to truncations, active L1 retrotransposons can impact the genome by creating insertions, deletions, new splice sites or gene expression fine-tuning1. L1 somatic retrotransposition creates somatic mosaicism within the brain by producing neuron-to-neuron variation in genomic content. In essence, somatic L1 retrotransposition alters the genetic makeup of the brain. It may be one of the contributing variables for individual differences between monozygotic twins2. The extent to which L1 retrotransposition impacts somatic cells, however, is not completely understood. Somatic L1 retrotransposition can have both positive and negative effects on neuronal genomes. The current challenge is to understand how somatic L1 retrotransposition promotes neuronal diversity in individuals, thereby impacting neural plasticity, cognition, behaviour, and disease risk. Figure 1. The mechanism of somatic L1 retrotransposition in neurons. Somatic L1 retrotransposition occurs via a “copy and paste” mechanism whereby segments of DNA mobilize and insert themselves into new locations in the genome
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Somatic L1 Retrotransposition and Neurogenesis L1 elements are mobilized early in life during the embryonic development of the central nervous system and later in life during adult neurogenesis. The development of the human nervous system relies on the diversification of neuronal progenitor cells (NPCs). NPCs give rise to three key lineages of the nervous system: neurons, astrocytes, and oligodendrocytes3. The divergence of NPCs is influenced by a complex combination of genetic and environmental factors, including somatic L1 retrotransposition. A seminal study by Coufal et al. (2005) was able to show that engineered human L1s can retrotranspose in adult rat hippocampus NPCs in vitro and in the mouse brain in vivo4. Building on this, Coufal and colleagues (2009) later conducted a study that demonstrated that NPCs isolated from a fetal human brain and NPCs from human embryonic stem cells (hESCs) both support the retrotransposition of engineered human L1s in vitro. This suggests that somatic L1 retrotransposition occurs in the mammalian, and more specifically the human, brain. It also suggests that somatic L1 retrotransposition contributes to somatic mosaicism, which begins very early in life3. Somatic L1 retrotransposition that occurs during embryogenesis results in subpopulations of cells that carry the same insertion event5. These somatic L1 retrotransposition events that are found in different brain regions compared to somatic L1 retrotransposition events that take place during adult neurogenesis, which are restricted to specific areas of the brain, such as the dentate gyrus of the hippocampus5. L1 retrotransposition also plays an important role in the adult brain, particularly in adult neurogenesis in the hippocampus. A recent study by Baillie et al. (2011) reported that L1 retrotransposition is active in the human brain with over 7,743 somatic L1 insertions in the hippocampus and the caudate nucleus, two brain regions involved in neurogenesis6. These somatic L1 retrotransposition events disproportionately affect protein-coding loci, which further supports the hypothesis that L1 retrotransposition influences neural plasticity and behaviour. Using a quantitative multiplex polymerase chain reaction assay, Coufal and colleagues (2010) found an increase in the copy number of endogenous L1s in the hippocampus compared to the copy number of endogenous L1s in the heart or liver genomic DNA from the same individuals3. This supports the idea that somatic L1 retrotransposition is particularly active in the brain, particularly the hippocampus, as opposed to other somatic tissues. L1 retrotransposition in the hippocampus is thought to be triggered by environmental influences such as nutrition, metabolic state, exercise, stress, and infection5. A study by Muotri et al. (2009) investigated whether there was a link between L1 expression in hippocampal neurons and voluntary exercise. It was found that L1-enhanced green fluorescent protein (L1-EGFP) transgenic mice that voluntarily exercised (runners) had increased somatic L1 retrotransposition in the subgranular layer of the dentate gyrus of the hippocampus compared to L1-EGFP transgenic mice that did not voluntarily exercise (non-runners)7. Running L1-EGFP transgenic mice displayed three times more granular cells expressing EGFP than non-running mice (Figure 2)7. Somatic L1 Retrotransposition and Disease Despite playing a positive role in neurogenesis, L1 retrotransposition has also been found to play a role in human neurological diseases. A study by Muotri et al. (2010) found that L1 retrotransposition in rodents is increased in the absence of the methyl-CpG-binding protein-2 (MeCP2) protein, a protein highly
Figure 2. Quantification of EGFP positive cells in the hippocampus. L1-EGFP transgenic mice that were housed in cages with running wheels (runners) displayed three times more granular cells expressing EGFP in the dentate gyrus of the hippocampus compared to L1-EGFP transgenic mice that were not house in cages with running wheels (nonrunners)7.
involved in DNA methylation and implicated in numerous neurodevelopmental diseases such as Rettâ€™s syndrome, a neurodevelopmental disorder that almost exclusively affects females1. Using NPCs derived from human induced pluripotent stem cells and human tissue, it was shown that patients with Rettâ€™s syndrome carrying MeCP2 mutations have increased L1 retrotransposition (Figure 3)1. The correlation between MeCP2 mutations and somatic L1 retrotransposition in neurons adds another level of complexity to the molecular events that are implicated in neurological and neurodevelopmental disorders. Somatic L1 retrotransposition has also been linked to ataxia telangiectasia, a neurodegenerative disease marked by poor motor coordination and small, dilated blood vessels8. A more recent study by Coufal et al. (2011) has shown that there are increased levels of in vitro L1 retrotransposition in cells lacking or containing severely reduced levels of ataxia telangiectasia mutated (ATM), a serine/threonine kinase involved in DNA damage signalling and neurodegenerative diseases that is implicated in the disease8. In addition, there is supporting evidence that increased levels of L1 retrotransposition are found in ataxia angiectasia patients compared to healthy controls8. Finally, it has recently been found that neuronal L1 retrotransposition can be induced by oxidative DNA damage, which has been associated with numerous diseases including many types of cancer, inflammatory diseases, and neurodegenerative diseases9. A study by Giorgi et al. (2011) showed that BE(2)C neuroblastoma cells, the embryonic precursors to sympathetic neurons, treated with hydrogen peroxide and subjected to an in vitro retrotransposition assay, show an increased expression of endogenous L1 retrotransposition9. This suggests that oxidative stress can result in dysfunctional L1 retrotransposition that can ultimately lead to disease. Although this is the most recent work with respect to neuronal L1 retrotransposition and disease, L1 retrotransposition has also been implicated in other psychiatric diseases including schizophrenia and autism5.
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survival, the mechanism by which the change was made is heritable, and therefore will be passed on to offspring if it was beneficial.
Figure 3. MeCP2 regulates neuronal L1 retrotransposition in vivo. A) EGFP-MeCP2 KO mice visualized significantly more EGFP-L1 retrotransposition in the cerebellum and striatum compared to wild-type controls. B) MeCP2 KO mice showed increased EGFP-L1 retrotransposition in numerous brain regions. C) Representative images from a three-dimensional reconstruction of wild-type and MeCP2 KO with the L1-EGFP transgene.
Evolutionary basis for Somatic L1 Retrotransposition in the Brain One explanation for somatic L1 retrotransposition in neurons is that it is a mechanism for rapidly generating neuronal diversity in response to challenges presented by a constantly changing environment5. A mechanism that generates somatic variability during neurogenesis and allows greater adaptability to fluctuating environments would potentially be favoured by natural selection5. Although somatic L1 retrotransposition does not alter the germline, and are not changes that can be passed on to offspring, the variability it creates does impact fitness, which can be subject to natural selection. Somatic L1 retrotransposon mechanisms producing neuronal variability, however, appear to be random5. Therefore, the genomic shuffle created by somatic L1 retrotransposition may be either beneficial, promoting adult neurogenesis in response to the changing environment, and therefore neural plasticity, or it may be detrimental, causing neurological diseases such as Rettâ€™s syndrome or ataxia telangiectasia. Since these genomic alterations are somatic, however, they will only be present for one generation. Thus, if the somatic L1 retrotransposition leads to a negative outcome, it will not be passed on to future generations. On the other hand, if the somatic L1 retrotransposition event leads to a favourable change that promotes fitness and
Figure 4. Potential mechanisms and repercussions of L1 retrotransposition in the brain . Somatic L1 retrotransposition is thought to occur both during embryogenesis and adulthood, creating a genetic mosaicism within the brain, particularly in areas such as the dentate gyrus of the hippocampus. Environmental influences such as nutrition, metabolic state, exercise, stress, and infection can trigger L1 retrotransposition events. These neuronal L1 retrotransposition events are thought to cause neurological diseases or behavioural changes, which can ultimately serve as evolutionary adaptations.
Conclusion and Future Directions The discovery that L1 retrotransposition is very active in the brain overturns the previously held theory that L1 retrotransposition events only occur in the germline ensuring that the genetic changes made were ultimately passed on. These L1 retrotransposition events appear to occur relatively frequently, which suggests that they may play a significant role in behavioral genetic influences and as such warrant more research. Future research directions include cataloging and characterizing new L1 insertions sites within the mammalian brain in order to gain a more comprehensive understanding of the role of L1 retrotransposition in the brain. With the costs of high-throughput sequencing methods steadily decreasing every year, this goal may be possible to achieve in the distant yet imminent future. Since researchers are just beginning to unravel the extent to which L1 retrotransposition affects neurogenesis, behaviour, and disease, there are many possible avenues of investigation. Although exercise has been identified as an environmental factor that results in increased neural L1 retrotransposition, it would be useful to identify other environmental situations and challenges that prompt L1 retrotransposition and neurogenesis in the brain such as challenging, novel tasks or stress-inducing situations. Given that L1 retrotransposition has been found to be involved in psychiatric disorders such as schizophrenia, future research could examine whether L1 retrotransposition also plays a role in other prevalent psychiatric disorders such as major depres-
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sive disorder and bipolar disorder. Somatic L1 retrotransposition in neurons adds another dimension to the current understanding of the complex, dynamic interactions between genetics and the environment. By creating differences from neuron to neuron, somatic L1 retrotransposition has the potential to create an inexhaustible range of phenotypes even between individuals with identical genomes. This suggests that somatic L1 retrotransposition, among other genetic-environment interactions, has the ability to generate an intangible level of variance from genomes. 1. Muotri, A.R. et al. L1 retrotransposition in neurons is modulated by MeCP2. Nature 468, 443-6 (2010). 2. Kaminsky, Z. a et al. DNA methylation profiles in monozygotic and dizygotic twins. Nature genetics 41, 240-5 (2009). 3. Coufal, N.G. et al. L1 retrotransposition in human neural progenitor cells. Nature 460, 1127-1131 (2010). 4. Muotri, A.R. et al. Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition. Nature 435, 903-10 (2005). 5. Singer, T., McConnell, M.J., Marchetto, M.C.N., Coufal, N.G. & Gage, F.H. LINE-1 retrotransposons: mediators of somatic variation in neuronal genomes? Trends in neurosciences 33, 345-54 (2010). 6. Baillie, J.K. et al. Somatic retrotransposition alters the genetic landscape of the human brain. Nature 479, 534-7 (2011). 7. Muotri, A.R., Zhao, C., Marchetto, M.C.N. & Gage, F.H. Environmental influence on L1 retrotransposons in the adult hippocampus. Hippocampus 19, 1002-7 (2009). 8. Coufal, N.G., Garcia-perez, J.L., Peng, G.E., Marchetto, M.C.N. & Muotri, A.R. Ataxia telangiectasia mutated ( ATM ) modulates long interspersed element-1 ( L1 ) retrotransposition in human neural stem cells. PNAS 1, (2011). 9. Giorgi, G., Marcantonio, P. & Del Re, B. LINE-1 retrotransposition in human neuroblastoma cells is affected by oxidative stress. Cell and tissue research 346, 383-91 (2011).
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Optogenetics Optogenetics and Its Recent Applications Vivek Verma Human Biology Department, University of Toronto, Toronto, ON M5S3J6.
It is important to gain further insights in the pathophysi-
ology of psychiatric and neurological disorders to be able to formulate better experimental interventions for treatment. It is crucial to extend our understanding beyond the neurochemical and structural deficits that contribute to Parkinsonâ€™s disease, anxiety disorders, depression, schizophrenia and drug addiction. We need to identify the specific neural circuits and the causal abnormalities in these circuits that underlie these disorders. However, the traditional manipulation techniques are not sophisticated enough to allow us to study how different neural circuits and specific neurons within the circuits contribute to perception and behaviour. Hence, the temporally and spatially precise control over biological systems provided by optogenetics makes it a revolutionary technique in the field of systems neuroscience. Optogenetics combines optical and genetic methods to allow control of specifically defined events in precisely targeted cells in a freely moving animal1. This technique, which was introduced by Karl Deisseroth, has been highly acclaimed and was recognised as Method of the Year 2010 by Nature Methods. It has also been widely accepted and is being used by thousands of researchers around the world because they can now rapidly activate or inactivate specific population of neurons using pulses of light. Traditional genetic, pharmacological and electrical manipulation methods have certain limitations that have been overcome by optogenetics. Traditional methods either affect non-targeted cells and fibres within the target region, have slow kinetics, or have poor or no reversibility2. Optogenetics, on the other hand, allows cell-specific targeting and has fast on-off kinetics providing temporal precision on millisecond-timescale11,2,3,4. Moreover, the firing pattern of neurons is locked to the frequency of pulses of light. Hence, this technique allows precise control over the identity of targeted cells as well as their pattern of activation or inactivation. Consequently, it has been employed by many researchers to study aberrations in neural circuits that contribute to the genesis of various psychiatric disorders. Method and Technological advances Opsins are membrane-bound proteins, including ion channels, discovered in bacteria and algae2. These proteins are light sensitive as they can incorporate retinal molecules, a vitamin A-related organic photon-absorbing cofactor, and thus, can be activated using pulses of light. Opsins can be selectively expressed in specific neurons using various expression targeting strategies. One of the earliest opsins in the optogenetic toolbox were channelrhodopsin 2 (ChR2), which is a cation channel that can be maximally activated by blue light at 470 nm to elicit action potentials at the frequency of delivered light pulses, and halorhodopsin (NpHR), which is a chloride channel that can be activated by yellow light at 589 nm to hyperpolarize the targeted neurons4. ChR2 and NpHR can be expressed in the same target cell to allow bidirectional
control over modulation. Besides modulation of ion flow, optogenetic tools can also provide control over intracellular biochemical signalling pathways. Proteins known as OptoXRs that have been engineered by replacing intracellular domains of rhodopsins with intracellular domains of G-protein coupled receptors (GPCRs) allow light-mediated initiation of second-messenger signalling cascades4. The recent expansion of the optogenetic toolbox has led to introduction new opsin variants that allow more powerful manipulations in neuronal activity. Now, it is possible to evoke extremely fast firing frequencies using new variants of channelrhodopsin3. Stabilized step-function opsin (SSFO) can be used to generate a long-lasting and subthreshold depolarization to increase the excitability of the target neurons5. The encoding DNA for opsins can be inserted into the target neurons by using: 1) viral expression systems, 2) transgenic animal targeting, 3) circuit targeting and 4) developmental and layer specific targeting3. Viral vectors have been the most popular method of targeting opsin expression as they lead to high expression levels and provide cellular specificity via cell-specific promoters and stereotactic targeting of virus injection. In-utero electroporation and transgenic animal targeting, on the other hand, lead to expression of opsin genes at birth and thus, allow the researchers to conduct electrophysiological experiments in isolated slices at different ages. However, transgenic animals have significantly lower levels of opsin expression compared to viral vectors3. Engineered opsins can traffic down to axons and thus, delivery of light can be specifically targeted at the axons to study specific projections2. Apart from selecting best suited opsins and expression targeting strategies, choosing the appropriate method of light delivery is also crucial in designing optogenetic experiments. The most common method of light delivery to deep cell bodies or axons in behaving animals is using chronically implanted optical fibers coupled with high power lasers and LEDs1. These optical fibers can also be fused with electrodes to form an optrode that can be used for electrophysiological readout from the optogenetically controlled tissue4. The recent refinements and advancements of the optogenetic toolbox have introduced more precise and potent tools to target the delivery of opsin genes and light, making this leadingedge technique even more powerful. Applications in animal studies Optogenetics has been employed by many researchers to study behaviour and variety of psychiatric and neurological disorders in animal models. Experiments in C. elegans, flies, mice, zebrafish and rats have demonstrated that this technique provides extremely precise control over the behaviour of the organism2. The pulses of light can be used to evoke or inhibit a specific behaviour of interest within milliseconds. Zimmerman et al. (2009) manipulated innate escape response in a fly by targeted expression of ChR2 in acj6 neurons and using light to excite these neurons6.
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Karl Deisseroth demonstrated that photoactivation of specific groups of neurons in the right motor cortex of the mouse can induce the animal to move in circles2. He also demonstrated that targeted expression of NpHR in motor neurons of worms allows light-mediated inhibition of motor activity of the organism2. Although the applications of optogenetics are way beyond causing organisms to escape or movie in circles, these fascinating experiments show how the precise and rapid control over the activity of specific neurons translates into precise control over the behaviour of the animal. Optogenetics, due to its high spatial and temporal precision, has transformed the way in which classical behavioural assays are conducted. Elevated plus maze tests that compare a subject’s response in two different conditions can now be completed in one session because of bidirectional control over anxiety imparted by optogenetic control of amygdala circuitry7. Traditional paradigms such as conditioned place preference, which require a pharmacological treatment, usually have multiple sessions. Due to rapid reversibility of optogenetics, the multiple sessions can be avoided by allowing the mouse to move freely in the arena on the conditioning day and delivering the light only when the mouse enters the conditioning chamber8. Moreover, the pattern of stimulation of the target region can be controlled more precisely in comparison to altering drug doses. A study used projection targeting to specifically photostimulate axons of cells in basolateral amygdala (BLA) that project to central nucleus of amygdala (CeA) and found that activation of this pathway produced an anxiolytic effect. However, stimulation of cell bodies in BLA irrespective of their target regions produced an anxiogenic effect7. Thus, specific targeting of the projection from BLA to CeA uncovered a novel anxiolytic pathway, which can be considered as a potential target for developing new drugs for anxiety disorders. Amygdala has been known to play a crucial role in acquisition of association between conditioned and unconditioned stimuli in the fear conditioning paradigm. However, the role of circuits within the amygdala has not been studied yet due to the limitations of classical techniques. Now, the neutral stimulus can be directly paired with the stimulation of specific neurons in lateral amygdala and recent studies have presented evidence of fear conditioning with this approach9. This can lead to resolution of specific network of neurons in the amygdala that contribute to experience and expression of fear. To enhance understanding of circuits that mediate reward, a couple of experiments have investigated if optogenetic activation of specific cell bodies or projections leads to self-stimulation. These studies demonstrated that optogenetic stimulation of dopamine neurons in the ventral tegmental area (VTA)10 or the axons of cells in BLA that project to nucleus accumbens11 produces selfstimulation in rats. Thus, application of optogenetics in studying rewards has validated the existing knowledge as well as presented new insights into the circuits that contribute to reward seeking behaviours. Application with other techniques Deep brain stimulation (DBS), in which surgically implanted electrodes are used to deliver high-frequency pulses to target brain areas, has been used to treat treatment-resistant disorders such as Parkinson’s disease and depression. DBS in subgenual cingulate cortex in humans has led to anti-depressive effects, whereas
DBS in subthalamic nucleus (STN) has had profound therapeutic benefits for Parkinson’s disease patients3. However, the mechanism of action of this therapy is not clearly understood yet. It is not known whether the therapeutic effects are due to direct effect on cell bodies in the target region or the axons passing through this region. To understand the mechanisms of DBS, optogenetics was used to specifically target cell bodies in the prefrontal cortex and afferent fibres in the STN. Selective activation of medial prefrontal cortical neurons in mice with depressive phenotype led to an effect similar to antidepressants12. Similarly, specific activation of afferent fibres in STN, and not the local cell bodies, did lead to remediation of motor deficits in rats3. Thus, these findings indicate the targets of DBS treatment that may lead to best therapeutic effects. A brain imaging technique known as functional magnetic resonance imaging (fMRI) provides a map of neuronal activity in response to stimuli. However, it reports local changes in bloodoxygen levels in different areas of the brain, which were thought to only correlate with the activity of neurons. However, a combination a optogenetics and fMRI, referred to as ofMRI, has validated that activity of local excitatory neurons can directly cause changes in blood-oxygen-level-dependent (BOLD) signal that is detected by an fMRI scanner13. Moreover, ofMRI can be used to map working neural circuits with high accuracy. Conclusion In last few years, optogenetics has been applied in many model organisms to study networks of neurons underlying variety of behavioural systems. The precision of this method has revolutionized the way experiments are being designed and will lead to unprecedented insights into neural circuits that contribute to behaviours. Moreover, the microcircuit-level understanding of brain disorders that can be provided by optogenetics was not possible to achieve with earlier techniques. However, along with potentially humongous advantages, it also offers certain limitations that we need to overcome in next few years to make this technique more powerful. The optical fiber produces heat when high light power is used to illuminate target areas. This heat can modulate the activity of target neurons and in addition, can also cause damage to these neurons3. On the other hand, high levels of opsin expression can also cause toxicity in the target area3. Thus, it is extremely important to design proper controls to examine whether the health of neurons has been altered or not. The optogenetic toolkit has been proliferating rapidly and it is important to continually expand and refine the tools in order to widen the range of application of this technique. We need to engineer opsins with diverse ionic selectivity and spectral sensitivity. Simultaneously, the OptoXR family of proteins should be extended. Most importantly, it is necessary to device targeting strategies to express opsins selectively in subcellular compartments such as dendrites or axons. Moreover, as mentioned earlier, the light delivery methods need to be improved so that the optical fibers dissipate less heat and can also target deeper brain regions precisely. All these advances in optogenetics will result in better experimental designs to study abnormalities in neural circuits in disorders, which will enable us to formulate better treatments. 1. Yizhar O et al. (2011) Optogenetics in Neural Systems. Neuron 71:9-34. 2. Fenno L, Yizhar O, Deisseroth K (2011) The Development and Application of Optogenetics. Annu Rev Neurosci 34:389–412. 3. Tye KM, Deisseroth K (2012) Optogenetic investigation of neural circuits
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underlying brain disease in animal models. Nature Reviews 13:251-266. 4. Zhang F et al. (2010) Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nature Protocols 5(3):439-456. 5. Yizhar, O. et al. (2011) Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477:171–178 5. Yizhar, O. et al. (2011) Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature 477:171–178. 6. Zimmermann G et al. (2009) Manipulation of an innate escape response in Drosophila: photoexcitation of acj6 neurons induces the escape response. PloS One 4:e5100. 7. Tye, KM et al. (2011) Amygdala circuitry mediating reversible and bidirectional control of anxiety. Nature 471: 358–362. 8. Airan, RD et al. (2009) Temporally precise in vivo control of intracellular signalling. Nature 458:1025–1029. 9. Johansen, JP et al. (2010) Optical activation of lateral amygdala pyramidal cells instructs associative fear learning. Proc. Natl Acad. Sci. USA 107:12692–12697. 10. Adamantidis, AR et al. (2011) Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior. J. Neurosci. 31:10829–10835. 11. Stuber, GD et al. (2011) Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking. Nature 475:377–380. 12. Covington, HE et al. (2010) Antidepressant effect of optogenetic stimulation of the medial prefrontal cortex. J. Neurosci. 30:16082–16090. 13. Lee, JH et al. (2010) Global and local fMRI signals driven by neurons defined optogenetically by type and wiring. Nature 465:788–792.
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Neuroimaging Neuromarketing: The Search for the Brain’s “Buy Button” Joshua W. Villafuerte Biology Department, Action Potential College, Hillock, IL 60101; 2Psychology Department, Synaptic State University, Neuron City, CA 90101. Neuromarketing is a relatively new field that seeks to use neuroimaging tools for marketing purposes. Since its recent conception, it has received considerable attention, as many believe it will revolutionize the way we understand consumer behaviour. Marketers are particularly excited about this burgeoning field for two main reasons. First, neuroimaging is poised to offer marketers information about consumer preferences that current research techniques cannot. Second, they believe that implementing these technologies into their practices will create a more cost-effective means of doing marketing research. By using brain imaging data, marketers ultimately hope to influence the design and presentation of products in ways that will not only maximize sales, but also satisfy the consumer to the utmost extent. A Crash Course In Marketing Marketing plays an integral role in the products we consume. Through the process of coordinating its design and presentation, marketing ultimately seeks to match products with people1. Generally, marketers influence these aspects either in advance of the product’s creation, or after. In the former case, marketers inform product designers of consumer values and desires; in the latter, they strive to maximize sales through various means. These manipulations, however, are not done without first obtaining marketing input. Marketers have at their disposal an array of research techniques, each with their associated advantages and disadvantages1. Simple approaches include examples such as focus groups and surveys: these are typically easy and cheap to implement, but they can also result in biased data. Complex approaches, in contrast, comprise set-ups such as market tests. These methods offer more reliable data but can be very expensive to use. Intermediate approaches, which include simulated markets, attempt to boast and mitigate the strengths and weaknesses, respectively, of simple and complex techniques. By no means exhaustive, this list of approaches highlights the inherent problems in current market research techniques. One possible solution to these issues would be to use two or more of these methods in conjunction; however, resource and time constraints considered, this would be impractical. Ideally, marketers want to understand consumer behaviour using a single approach that would not only provide reliable, unbiased data, but also a favorable cost-to-benefit trade-off. As such, they have recently turned their attention to neuroimaging technology, with the hopes that it will improve their marketing strategies. The use of neuroimaging in marketing is a relatively new concept. As a matter of fact, one of the first studies related to neuromarketing was published in 2004. In the seminal experiment, researchers were interested in seeing how the brain responded to
different soft drinks, namely Coke and Pepsi2. What they found was that the participants’ brain responses differed not only in areas associated with sensation, but in emotion- and cognitive-processing areas as well. As the previous study underscored, people activate a myriad of mental processes, both conscious and unconscious, when engaging in consuming behaviour. Thus, an understanding of how the brain responds to a product can provide marketers with invaluable data. Such information can be obtained via a number of brain imaging techniques, including functional magnetic resonance imaging (fMRI), magnetoencephalography (MEG) and electroencephalography (EEG). Each method provides a unique way of visualizing neural activity. For instance, in fMRI, the haemodynamic response is used as a proxy for neural activity, whereas the magnetic fields generated by neuronal cells are used in MEG. Additionally, the spatial and temporal resolution, as well as the depth at which brain activity can be recorded at, differs widely among these neuroimaging techniques. By employing these tools, neuromarketers believe they can unravel the mechanisms of consumer behaviour. Decoding The Consumer Brain In gauging the viability of using neuroimaging in marketing, two important questions are posed. First, will brain activity during the decision-making process reliably predict consumption satisfaction and its associated neural response? Second, will these two neural representations be consistent regardless of the preference elicitation methods used? If the answer to both is “yes”, than the results obtained via neuroimaging may be successfully applied to marketing practices. With these basic assumptions, we can begin to make sense of the neuromarketing research. For instance, in a study investigating the neural mechanisms of the willingness-to-pay (WTP) computation, participants bid on the privilege to eat food whilst having their brains scanned using fMRI3. The researchers observed that the extent to which the participants were willing to pay to eat was positively correlated with activity in the medial orbital frontal cortex (mOFC). This was a particularly interesting finding, as activity in the mOFC has also been linked to the anticipation and consumption of rewarding stimuli4. This seemingly common neural activation suggests that brain activity associated with decision satisfaction can predict consumption satisfaction. However, when we consider that decisionmaking is a multifactorial process, involving both emotional and cognitive factors, it is not enough to conclude from this reverse inference that activity in one area of the brain will predict consumer behaviour. Perhaps, then, analyzing brain activity at the global level will beget more reliable information. Using a technique called multi-voxel pattern analysis (MVPA), obtaining such information is made possible. MVPA is a standard tool used in the statistical analysis of fMRI
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data. The advantages to using MVPA are two-fold: not only does it confer greater sensitivity in detecting neural activation, but it also provides the statistical power to predict certain behaviours. In a study that had participants perform a memory search during a free recall task, MVPA allowed the researchers to predict recall behaviour5. Based on this finding, and others like it, there is no doubt that multi-voxel pattern analysis will become a staple in neuromarketing research. Is It Worth The Cost? There is a major downside to conducting neuromarketing research: the price tag. There is no denying that neuroimaging is very expensive. Granted, some methods are more costly than others: an EEG set-up can cost thousands of dollars, whereas an MRI or MEG scanner can cost millions. Nevertheless, marketers must decide whether this cost-to-benefit trade-off is more desirable than the ones offered by other research techniques. Fortunately, some research suggests that the benefits of using neuroimaging may outweigh its high operating costs. In an fMRI study, two groups of participants were made to drink small quantities of wine6. Participants in one group were told they were given an expensive wine, whereas the others were told they were given an inexpensive wine. Unbeknownst to the individuals, the expensive and inexpensive wines were, in fact, the same. Interestingly, activity in the medial orbitofrontal cortex was greater when participants sipped the “expensive” wine in comparison to the “inexpensive” wine. Moreover, self-report ratings of how much they enjoyed the beverage correlated positively with the observed neural response. Based on this study, it is clear that expectations can have a significant effect on the way we perceive consumable goods. What implications, then, does the role of expectations play in other products? One area of focus that could benefit from these findings is in research concerned with the placebo effect. In a recent behavioural study, it was shown that the analgesic placebo effect was significantly stronger with more costly “medications”7. If such is the case, researchers may not only tailor these medications to certain individual needs, but also offer, justifiably, these products at a higher price. At the moment, it is still too early to tell how effective this strategy may be. However, as more research is carried out, marketers are sure to discover additional hidden benefits of using neuroimaging. Not Just Another Fad While some may view neuromarketing as just another fad, others see endless potential in its use and application. Indeed, the future of the field looks promising, as we are already beginning to gain insight into some of our products. One area of the brain that is of particular interest to researchers and marketers alike is the hippocampus. A fairly recent study on spatial navigation showed that the hippocampus is especially recruited when participants engage in decision-based navigation as opposed to navigation guided by external cues8. Such findings could possibly be used in architectural design, where the layout of a building (e.g., a retirement home) may be arranged so as to put as little load on the hippocampus as possible. These hippocampal results are not just applicable to architecture, however; the TV and film industry may benefit as well. In a study that asked participants to watch a TV sitcom, it was demonstrated that the ability to correctly remember certain aspects of the show’s content three
weeks later was associated with hippocampal activity at the time of viewing9. Based on this principle, a TV or film executive could hypothetically use brain imaging data to select the most memorable scenes for the final cut of a production. Neuromarketing has even shed some light on our political candidates. Some research indicates that activity in the prefrontal cortex (PFC) can provide a measure of a political candidate’s likeability10. In the study, participants were asked to watch advertisements for political campaigns. Using fMRI, the researchers were able to show that activity within the medial PFC was linked to participants maintaining their affinity towards the candidate; in contrast, activity in the lateral PFC was believed to indicate participants’ changing attitudes toward the candidate. Interestingly, another recent study showed that a political candidate had a higher chance of losing the election if a picture of that person elicited activity in participants’ insular cortex11. The Future Of Neuromarketing The field of neuromarketing is still a nascent one. Nevertheless, it is already showing many signs of potential. With the few neuromarketing studies published to date, both the scientific and general community is beginning to see the practicality in using neuroimaging for marketing purposes. Though, before the field can make further progress, certain obstacles must first be overcome. Although the cost of neuroimaging currently presents as a major setback, as technology improves, neuroimaging is expected to become cheaper and even easier to implement. Such technological improvements include better scanners and more sophisticated, rigorous analytical tools for processing brain imaging data. The act of performing neuromarketing research also raises certain ethical concerns. However, through the creation of general guidelines and expectations for transparency, the field of neuromarketing and its contributions will be viewed with the utmost regard. 1. Ariely, D. & Berns, G. S. Neuromarketing: the hope and hype of neuroimaging
in business. Nat Rev Neurosci 11, 284-292 (2010). 2. McClure, S. M. et al. Neural correlates of behavioral preference for culturally familiar drinks. Neuron 44, 379-387 (2004). 3. PlaSsmann, H., O’Doherty, J. & Rangel, A. Orbitofrontal cortex encodes willingness to pay in everyday economic transactions. J Neurosci 27, 9984-9988 (2007). 4. Kringelbach, M. L. The human orbitofrontal cortex: linking reward to hedonic experience. Nat Rev Neurosci 6, 691-702 (2005). 5. Polyn, S. M., Natu, V. S., Cohen, J. D. & Norman, K. A. Category-specific cortical activity retrieval during memory search. Science 310, 1963-1966 (2005). 6. Plassmann, H., O’Doherty, J., Shiv, B. & Rangel, A. Marketing actions can modulate representations of experienced pleasantness. P Natl Acad Sci USA. 105, 1050-1054 (2008). 7 Waber, R. L., Shiv, B., Carmon, Z. & Ariely, D. Commercial features of placebo and therapeutic efficacy. JAMA 299, 1016-1017 (2008). 8. Spiers, H. J. & Maguire, E. A. Neural substrates of driving behaviour. Neuroimage 36, 245-255 (2007). 9. Hasson, U., Furman, O., Clark, D., Dudai, Y. & Davachi, L. Enhanced intersubject correlations during movie viewing correlate with successful episodic enconding. Neuron 57, 452-462 (2008). 10 .Kato et al. Neural correlates of attitude change following positive and negative advertisements. Front Behav Neursosci 3, 1-13 (2009). 11. Spezio M. L. A neural basis for the effect of candidate appearance on election outcomes. SCAN 3, 344-352 (2008).
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Brain Imaging Time-lapse deep brain imaging of neurological disease progression using fluorescence microendoscopy Qiu Jing Wu Department of Human Biology, University of Toronto As many neurological diseases involve deep brain pathologies that are inaccessible to conventional light microscopy in live subjects, a time-lapse in vivo deep brain imaging using fluorescence microendoscopy is developed by Schnitzer lab to overcome this difficulty. By bilaterally implanting two hollow glass capillary guiding micro-optic probes for in vivo imaging, this technique allows repeated observations and comparison of ipsi- and contra-lateral deep brain neurons and tissue in the same animal over several weeks. Using this approach, the Schnitzer group has examined the dendritic structure of CA1 hippocampal neurons in the normal adult mice and tracked the progression of glioma angiogenesis in a mouse model, to which tumor cells were introduced into the right CA1 area while the left CA1 area served as the self-control. Contrary to their expectation, authors found a remarkable stability of the dendritic structures of CA1 hippocampal neurons in the normal adult mice, whereas progressive distortion of vascular structures and significantly slowed blood flow speed take place as glioma advances in the diseased animals. The results of the study provide the proof of principle for the applicability of the time-lapse fluorescence microendoscopy to study physiological and pathophysiological morphological plasticity of sub-cortical structures in real-time at synaptic level in vivo, and may potentially help understand the mechanisms and progression of deep-brain neurological disorders and perhaps development of focal drug treatments guided with optical-light.
and three-dimensional imaging of the tissue. In the time-lapse microendoscopy, micro-optical probes can be repeatedly inserted into surgically implanted guide tubes in the mouse brain for visualization. The capabilities of the time-lapse microendoscopy in examining dendritic structure of neurons and tracking disease progression have been illustrated by Schnitzer lab. In order to test the applicability of time-lapse microendoscopy in deep brain imaging, the dendritic structures of CA1 hippocampal neurons were examined in mice expressing fluorescent markers. Itâ€™s unknown whether CA1 neurons downstream of dentate gyrus where neurogenesis usually occurs undergo consequent dendritic remodeling (Li et al., 2009). The hypothesis is that CA1 neurons would gradually change their dendritic morphology. However, the results suggest significant stability of the dendritic structure of CA1 neurons. To demonstrate the capability of time-lapse microendoscopy to study diseases, a mouse model of glioma was examined. Glioma is one of the most common malignant brain tumors and it preferentially grows in deep brain areas (Jang et al., 2008). The local microenvironment such as angiogenesis is a major factor in tumor enlargement (Jain et al., 2007). The time-lapse microscopy enabled the scientists to track glioma angiogenesis after implanting glioma cells into the hippocampal region. The results show that local vascular morphology and flow speed are altered with glioma progression.
I. BACKGROUND Neurological diseases are usually associated with disturbances in neuronal function and changes in the local microenvironment. The mechanisms of many neurological diseases still remain largely unknown, such as the insidious progression of neurodegenerative diseases. Time-lapse in vivo imaging studies of diseased animal models yield insights into the disease mechanisms and potential therapeutic treatments (Misgeld and Kerschensteiner, 2006). Previous in vivo imaging studies were conducted in the peripheral nervous system and cortex (Grutzendler et al., 2002; Trachtenberg et al., 2002; Kerschensteiner et al., 2005; Tsai et al., 2004; Mclellan et al., 2003). However, the lack of suitable brain imaging techniques precludes the study of pathologies in deep brain areas such as hippocampus and striatum. In order to overcome this difficulty, the Schnitzer group has developed a time-lapse fluorescence microendoscopy which enables repeated observation in deep brain structures over several weeks in the same animals (Barretto et al., 2011). Microendoscopy allows imaging in deeper brain areas than the conventional light microscope and involves insertion of microlenses into brain tissue. The technique also enables high-speed
Chronic mouse preparation for time-lapse microendoscopy Figure 1 illustrates experimental paradigm for repeated deep brain imaging of mouse brain in vivo (Fig.1). Two guide tubes composed of hollow glass capillary were implanted bilaterally into the mouse brain to deliver the microoptical probes just dorsal to the CA1 hippocampal area for visualization. To initiate imaging, a micro-optical probe is inserted into the guide tube in mouseâ€™s brain and the mouse is placed under the microscope to visualize deep inside the brain. Post-mortem histology revealed glial activation near guide tubes due to the surgical implantation. They found a thin tissue layer surrounding the guide tubes had greater glial fibrillary acidic protein comparing to neighboring tissue. There was no loss of neurons around the guide tubes, and the glial activated layer did not affect the imaging of deep brain neurons.
II. Results The results and figures refer to Barretto et al., 2011
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Time-lapse imaging of glioma angiogenesis In order to demonstrate the applicability of the microendoscopy to disease studies, a mouse model of glioma was examined. The mouse model was developed by implanting glioma cells into the right CA1 region, and the left side served as a matched control. Using the time-lapse microendoscopy, the entire disease time course was tracked from glioma implantation to subject mortality over 22-24 days in total. During this period, the tumor site revealed progressive distortion of vasculatures. The vessel size was significantly increased, but the blood flow significantly dropped in the tumor vessels comparing to the normal tissue (Fig 3).
Figure 1 Chronic mouse preparation for time-lapse microendoscopy. (a) Two imaging guide tubes were implanted bilaterally into the mouseâ€™s brain. (b) Three microendoscope probes can be inserted into the guide tubes repeatedly to visualize deep brain neurons after mouse recovered from surgery. (c) The microscope objective lens focus illumination onto the probes and allow visualization deep in the brain.
Time lapse imaging of CA1 hippocampal neuronsâ€™ dendritic structures In order to test the basic abilities of the microendoscopy, the dendritic structures of CA1 neurons are monitored over 7 weeks in mice expressing yellow or green fluorescent protein (YFP or GFP) in a subset of CA1 neurons (Fig.2). The results revealed only 16 cases of dendritic turnover among 33596 observations of dendrites over the entire tracking period, which yield a probability of <0.00013+ 0.00003 for a single dendrite to undergo a remodeling. Thus the dendritic structures of CA1 neurons are extremely stable.
Figure 3 time-lapse microendoscopy of glioma angiogenesis in CA1 region of the mice. (a) A colored image of glioma cells (green) and microvasculature (red) on day3 after glioma cell implantation. (b,c) time-lapse images of tumor vessels revealed progressive deformation of the microvasculature. (d) Comparison of average erythrocyte speed in left (control) and right (tumor) CA1 area on day 20 after glioma implantation.
Quantitative analysis of angiogenesis The changes in vessel morphology and blood flow speed were analyzed and quantified. Individual mice showed variations between subjects and also on a day to day basis, but the average vessel diameter was maintained at a constant level in normal tissue. Similarly, there was no significant change in the erythrocyte flow speed in normal tissue (Fig 4). In contrast, changes in the vessel diameter and flow speed were significant at tumor sites comparing to the normal tissue. The vessel diameter was significantly increased and flow speed was significantly reduced. The glioma also had effect on the vasculature because the vessel branching ratios, calculated as vesselâ€™s total length over number of branches, were significantly decreased in tumor tissue.
Figure 2 time-lapse microendoscopy of CA1 hippocampal neurons. (a,b,c) Two-dimensional projections of three-dimensional stacks. b is the enlarged image of the framed area in a. (d) Enlargement of a single dendrite showing dendritic spines
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Figure 4 Quantitative analysis of glioma angiogenesis in CA1 region. (a) Graph of flow speed versus vessel diameter in normal (left) and tumor (right) tissue. The plots for the tumor sites showed a significant increase in vessel diameter and decrease in flow speed as the disease progressed. (b) Average erythrocyte flow speed (left) and vessel diameter (right) were ploted against time. Vessel diameter and flow speed were significantly changed in tumor tissue compaing to control.
Discussion The newly developed time-lapse fluorescence microendoscopy enables cellular level deep brain imaging in vivo over weeks. In this study, the ability of the time-lapse microendoscopy to visualize dendritic structures of deep brain neurons and to track disease progression was well demonstrated. The results revealed that the CA1 hippocampal neurons are extremely stable in terms of dendritic structures. The time-lapse microendoscopy in a mouse model of glioma also showed progressive distortion of microvasculature at the tumor sites. The mean blood vessel diameter was significantly increased, and the average blood flow was significantly decreased in tumor tissue. CA1 hippocampal neurons are known to be essential for spatial and episodic memory, but the stability of their dendrites had never been investigated. The current study is the first to assess the longterm stability of the hippocampal dendrites. The authors expected that the dendritic structures of CA1 neurons would be constantly changing in order to accommodate the upstream neurogenesis occurring in dentate gyrus. However, the marked stability of CA1 neurons shown in the results disputes against the idea that CA1 neurons would undergo gradual dendritic remodeling. The probability of dendritic turnover was estimated as <0.00013, indicating CA1 neurons are extremely stable under normal conditions. However, it is surprising that plastic changes in spine number, size and shape were not quantified, because these parameters (other than dendritic branches) have been implicated in activity-dependent synaptic remodeling in vitro and learning and memory in vivo. The findings in this study nevertheless help establish a basal stability level which may serve as a reference for comparisons with diseased or aged animals in future studies. It would be interesting to test if manipulating the local microenvironment such as growth factors would have an impact on the stability of CA1 hippocampal neurons in future experiments. Furthermore,
the ability of the microendoscopy to track changes in dendritic morphology suggests that it could also aid in the studies of neurological disorders involve alterations of dendritic structures. Glioma is the most common deep brain tumor in humans. Lack of appropriate in vivo imaging techniques limits the study of disease mechanism and progression. Given that angiogenesis is a major factor causing tumor enlargement, the scientists were able to track the glioma angiogenesis over several weeks using the developed time-lapse microendoscopy. As expected, progressive deformation of vessels was shown as disease advanced but again CA1 neuronal dendrite/spine remodeling in the vicinity of glioma was not characterized in this study. The tumor growth also significantly affected the vessel diameter and flow speed. The data not only provided important information on the disease progression of glioma, but also illustrated that the time-lapse microendoscopy could facilitate the future studies of deep brain diseases. Further insights into whether and how glioma impacts neuronal morphology and functions (or vice versa) awaits simultaneous imaging of vessels, neurons and glia. A key advantage of the time-lapse microendoscopy is that it enables longitudinal studies in individual live animals rather than tissue specimens taken from different animals used in histology. The longitudinal imaging study not only greatly reduces the number of animals required, but also provides dynamic data from individual animals, which is essential in studying disease progression. One limitation would be the invasiveness of surgical implanted guide tubes which may damage brain tissues and limits its use in a clinical setting. Alternatively one could only implant the micro-optical probes into the brain, but the guide tubes allow the same micro-optical probes in a number of animals, and enable observing one site with different probes used in different optical resolutions and dimensions. In conclusion, time-lapse microendoscopy allows studies of deep brain disease such as epileptic, neurodegenerative, cerebrovascular and trauma-induced conditions that were not possible by conventional light microscopy. One can envisage that the time-lapse microendoscopy in combination with portable optical fibers and minimicroscopes may enable deep brain imaging in freely moving mice in near future, presenting unparalleled opportunities to unravel dynamic plasticity of brain cells in health and diseases. 1. Barretto, RPJ. et al. (2011) Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy. Nature Medicine 17, 223-228. 2. Grutzendler, J et al. (2002) Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816. 3. Jain, R.K. et al. (2007) Angiogenesis in brain tumours. Nat. Rev. Neurosci. 8, 610–622. 4. Jang, T. et al. (2008) A distinct phenotypic change in gliomas at the time of magnetic resonance imaging detection. J. Neurosurg. 108, 782–790. 5. Kerschensteiner, M. et al. (2005) In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat. Med. 11, 572–577 6. Li, Y., Mu, Y. & Gage, F.H. (2009) Development of neural circuits in the adult hippocampus. Curr. Top. Dev. Biol. 87, 149–174. 7. McLellan, M.E. et al. (2003) In vivo imaging of reactive oxygen species specifically associated with thioflavine S–positive amyloid plaques by multiphoton microscopy. J. Neurosci. 23, 2212–2217. 8. Misgeld, T. & Kerschensteiner, M. (2006) In vivo imaging of the diseased nervous system. Nat. Rev. Neurosci. 7, 449–463. 9. Trachtenberg, J.T. et al. (2002) Long-term in vivo imaging of experiencedependent synaptic plasticity in adult cortex. Nature 420, 788–794. 10. Tsai, J. et al. (2004) Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. Nat. Neurosci. 7, 1181–1183.
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Molecular Biology Gene editing nucleases Alan M. Xi Department of Human Biology, University of Toronto, Toronto, Ontario, Canada M5T 1K5 Creation of engineered gene editing nucleases (GENs) offered a new approach to modify genomes at desired locations. Three classes of GENs are the most common: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases. ZFN, the most studied of the three, will be examined in detail in this paper. A ZFN is made by joining two components, a nuclease domain and a DNA binding domain, which consists of 3 to 5 zinc finger modules juxtaposed in an array. When expressed in the target cell, ZFNs incur double-stranded breaks at a locus dictated by the configuration of zinc fingers. The error-prone DNA repair mechanism introduces mutation into the genomic sequence, whose function is thereby disrupted. A multi-tude of studies can be conducted with this new technique. This review investigates two of such studies. In one, ZNF considerably reduced CAG repeat expansion in cultured human cells, which posed a potential gene therapy for Hungtinton’s disease. In the other, ZFN enabled resear-chers to study the talpid 3 gene by reverse genetics, which shed light on its role in Hedgehog signaling and neural tube generation. These findings captured the epitome of an exciting field of future advances made possible by the novel method. However, ZFN technology does face a number of problems. These limitations will be discussed at the end of this paper, along with suggestions for the future course of improvement. I. BACKGROUND As more and more genomes from different species were being sequenced, biologists were eager to decipher, and subsequently make use of, the function conferred by every minute stretch of the double helix. To achieve this, a tool must be available that allows researchers to manipulate specific parts of the genome and study the consequences. Techniques like this are of such contemporary significance that their inventers are sometimes awarded on the caliber of the Nobel Prize. RNA interference, for instance, relies on targeting and destroying the RNA product of transcription, thus indirectly controlling expression of a gene or other genomic elements. RNAi is noted for its ease of use and applicability in many different species. This technique is limited, however, by the extent to which knock-down occurs; it often results in incomplete elimination of a target and leaves plenty of room for false negatives. Also widely used are two related techniques, transgenics and gene targeting, which are used to insert exogenous genes and substitute for endogenous genes, respectively. While both techniques have matured substantially over the past decades, their application is confined in mice and yeast. This requires that the homologue for a gene of interest be found in mice or yeast, which is sometimes problematic and even impossible. GENs evolved as a product of several branches of research, taking advantage of our foremost understanding of DNA
binding proteins, molecular engineering, DNA self-repair, and so on. Three classes of GENs are used most widely in today’s research laboratories: zinc-finger nuc-leases (ZFNs), transcription activator-like effector nucle-ases (TALENs), and meganucleases1. ZFNs, the most commonly used of the three, are made by joining the Fok1 nuclease domain to an array of 3 to 5 zinc fingers, each recognizing one specific trinucleotide unit2. Each ZFN therefore binds to a unique strand of DNA 9 to 15 nucleo-tides in length. In general, two ZFNs are designed to bind either strand of the DNA, with a spacing of 5 to 7 nucleotides in between. Upon dimerization the two proteins cleave the straddled DNA by creating double-stranded breaks (DSBs). TALENs bind and cleave in a similar fashion, the main difference being that the DNA binding domain consists of tandem TALEN repeats, each of which targets one of the four nucleotides. As a result, a string of TALEN repeats pinpoints a single genomic locus with extremely high specificity. Meganucleases, on the other hand, cleave DNA at long target sites which must be integrated beforehand into the genome. The cell conducts self-repair upon discovery of DSBs. It does so via two principal pathways, nonhomologous endjoining (NHEJ) and homology-directed repair (HDR). However, both repair mechanisms are prone to error. NHEJ results in various kinds of mutation, ranging from frame-shifts to largescaled deletions or even chromosomal rearrangements. These artificial mutations can serve as the basis for reverse genetics studies. On the other hand, HDR is carried out in the presence of homologous DNA. If an exogenous sequence tailored to a specific purpose is added at this time, it can be incorporated into the host genome and subsequently exert its function. The power of GEN technology lies in their universality, which allows it to overcome some of the crucial limitations of other genome editing methods. Since they directly recognize target DNA, GENs can be used to modify not only coding sequences, but also non-coding regions such as introns, promoters, repressor and activator binding domains, and theoretically anywhere else in the entire genome. This makes it superior to RNA interference, with which one is unable to work with untranscribed genomic elements. Furthermore, GENs is in theory applicable to many difference species that depend on the same error-prone DNA repair mechanisms. Experiments can be conducted both in vivo and in vitro, potentially bypassing the expensive and time-consuming procedure associated with gene targeting and transgenics in mice. Studies far more diverse than before can be carried out to characterize certain genes in human cells and in species closely related to humans. II. APPLICATION- Treatments for Neurological Disorders As shown by Mittelman et al, ZFNs can be used as a potential treatment for neurological disorders that involve CAG tandem repeat expansions, such as Hungtinton’s disease3. In this study, two
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ZFNs were designed to target either GCA or GCT. The core amino acid series of the zinc finger DNA binding domain was QSGDLTR for zfGCA and QSSDLTR for zfGCT. The binding domains were fused to their respective Fok1 endonuclease domain using the L6 glycine-lysine linker. Plasmids containing these ZFNs were transfected into human and rodent cell lines that carried extensive CAG expansion. The APRT and HPRT systems were used as reporters of cleavage efficiency, marking rodent cells and human cells, respectively. The intronic CAG repeats in AP-(CAG)95-RT and HP(CAG)95-RT interfered with proper splicing; APRT and HPRT were only expressed if the repeat number was reduced to â‰¤38. Results showed that zfGCT alone was sufficient to increase APRT+ colony frequency by 10-fold (p=0.0004) and HPRT+ colony frequency by 7-fold (p=0.0009). (Figure 1) However, zfGCA treatment yielded no significant difference from control. The combination of zfGCT and zfGCA was the most efficient, resulting in a 15-fold increase in APRT+ colony frequency, which also surpassed the effect of zfGCT alone (p=0.03). On the other hand, shortening the CAG repeat tracts compromised the efficiency of ZFN-induced cleavage. APRT+ and HPRT+ colony frequencies were significantly lowered if AP-(CAG)61-RT (p=0.006) or HP-(CAG)68-RT (p=0.004) were used instead of the 95-repeat constructs. As revealed by sequencing analysis, 56% of these interferences resulted from contractions, or uninterrupted shortening of pure CAG repeats.
genetic disorder that involves simple changes in the genome. Sickle cell anemia, for instance, is caused by a single nucleotide substitution in the gene that codes for hemoglobin. In the presence of GEN, microhomology-mediated end joining can be harnessed to exchange the mutated nucleotide with a corrected one carried by exogenous DNA. This opens the door for numerous novel treatments that were unthinkable in the past.
ZFN Used In Reverse Genetics Ben et al. examined the effect of eliminating the talpid 3 gene on neurodevelopment in zebrafish4. To generate mutant alleles, two types of ZFN mRNA were microinjected into one-cell stage zebrafish embryos. One ZFN carried zfGCC, zfGGC, and zfGTG as its DNA binding domain; the other carried zfGGA, zfTGA, and zfGTT. In situ hybridization (ISH) was performed with anti-nkx2.2 RNA probe, which stained for ventral neural tube. Immuno-fluorescence was performed using anti-acetylated alpha-tubulin (marker for cilia), and images were captured through Olympus confocal microscope. In the mutant, expression of nkx2.2 was significantly reduced as shown by ISH, suggesting developmental defect of the ventral neural tube. The same result had been observed in ta3 mutant chicks. It was also found in im-munofluorescence imaging that ta3 mutant exhibited a reduction of acetylated tubulin production in the neural tube, another morphological defect associated with ta3 knockout. Although previous genomic searches failed to find a homologue of the talpid 3 gene in zebrafish, neuro-developmental problems here observed in the zebrafish knockouts mirrored the mutant phenotype in chicks, indicating that a functional orthologue very likely existed in zebrafish. A causal link was also established between the generation of neural tube and Hedgehog signaling pathway, in which ta3 plays a critical role. This study showed that GENs can be used in reverse genetics to supplement or replace traditional methods, including mouse transgenics, homologous recombination, yeast two-hybrid screening, RNA interefence, morpholino oligonucleotide, etc. In the prospect Figure 1 APRT and HPRT colony frequencies in response to ZFN mutagenesis. By itself, zfGCT increased APRT colony frequency 10-fold (A) Figure 1 APRT+ and HPRT+ colony frequencies in response to ZFN mutagenesis. By itself, and HPRT colony frequency 7-fold (B). The effects were mitigated by reducing the number of CAG repeat. Combined treatment with zfGCA that automated genomic sequencing continues zfGCT increased APRT+ colony and zfGCT increased APRT frequency 15-fold. frequency 10-fold (A) and HPRT+ colony frequency 7-fold (B). to generate more readouts, phenotypic analyses The effects were mitigated by reducing the number of CAG repeat. Combined treatment with must be actively carried out to understand the zfGCA and zfGCT increased APRT+ frequency 15-fold. function of the new genes. It is likely that GEN will serve a critical role in facilitating this process, carving into do Taken together, these results indicated that CAG repeat mains which other techniques cannot reach, potentially uncovertracts were vulnerable to ZFN attack. It was interesting that zf- ing previously unknown idiosyncrasies of the human genome, and GCT alone was able to do so quite efficiently, probably through contributing to the development of novel treatment for diseases. homodimerization. The authors indicated that this could be attributed to the very similar configurations of the two zinc finger Limitations and Future Directions arrays, which differed by only one amino acid residue. Also, the Despite its great potential, GEN technology does have cleavage was length dependent, with shorter repeat tracts cleaved several limitations. One of the most encumbering issues is the less frequently than longer ones. These characteristics make ZFN a difficulty to synthesize these nucleases. Take ZFN for example. good candidate for future therapeutic use in treating CAG expan- The natural preference of zinc fingers is for G-rich consensus sesion diseases, since it not only reduces tract length by contraction, quences, because molecular interaction is maximized if the argibut it does so rather selectively at the most pathological sites (i.e. nine residue is juxtaposed with a guanine base5. Some researchlongest expansions) while leaving the shorter, less tracts intact. ers attempted to bypass this constraint by using overlapping zinc Gene therapy like this is, of course, not limited to neu- fingers, which has nonetheless proved unsuccessful in many cases. rological conditions alone. GENs show great promise for any The second limitation comes from the 5- to 7-bp spac +
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ing between the two Fok1 nuclease domains which must dimerize before making the cut. One way of getting around this problem is by using long linkers, which coalesce Fok1 with its partner zinc fingers. This solution has its own problem: increasing the length of the linker also increases the rate of promiscuous binding, which compromises specificity. An alternative approach suggested by the author is to develop an altered form of Fok1 that dimerize at more flexible spacings. This may not be impossible as novel Fok1 mutants have been engineered, including the â€œSharkeyâ€? mutant that showcases an improved cleaving ability. In addition to overcoming these limitations, future work should be directed at enhancing the effect of GENs. Any step in the binding-cleavage-repair process could be targeted to achieve this purpose. For instance, a certain molecular modulator could be developed to reduce repair accuracy at the site of cleave (but nowhere else). An increased rate of error would increase the occurrence of mutagenesis. 1. Baker, M. Gene-editing nucleases. Nat Methods 9, 23-27 (2012). 2. De Souza, N. Primer: genome editing with engineered nucleases. Nat Methods 9, 27 (2012). 3. Mittelman, D. et al. Zinc-finger directed double-stranded breaks within CAG repeat tracts promote repeat instability in human cells. PNAS 106, 9607-9612 (2009). 4. Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates. Development 138, 4969-4978 (2011). 5. McMahon, M. A., Rahdar, M. & Proteus, M. Gene editing: not just for translation anymore. Nat Methods 9, 28-31 (2012).
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The undergraduate neuroscience review journal - Human Biology, University of Toronto