Tese versao final by Joana Rodrigues

I SCHEMIA IN THE ACUTE HIPPOCAMPAL SLICE PREPARATION CONSEQUENCES FOR MEMBRANE PROPERTIES

Joana Catarina Alves Rodrigues

Dissertação para a obtenção de Grau Mestre em

Engenharia Biomédica

Júri Presidente: Orientadora: Orientador: Vogal: Vogal:

Prof. Paulo Rui Alves Fernandes Doutora Raquel Alice da Silva Baptista Dias Prof. Paulo Jorge Peixeiro de Freitas Prof. Pedro Afonso dos Santos Baltazar de Lima Prof. Ana Maria Ferreira de Sousa Sebastião

November 2012

The experimental work contained in this thesis was performed at the Institute of Pharmacology and Neuroscience, Faculty of Medicine and Unit of Neurosciences, Institute of Molecular Medicine, under the supervision of Raquel Alice da Silva Baptista Dias (PhD) and co-supervision of Prof. Paulo Jorge Peixeiro de Freitas

O trabalho experimental constante da presente tese foi realizado no Instituto de Farmacologia e Neurociências, Faculdade de Medicina e Unidade de Neurociências, Instituto de Medicina Molecular, sob orientação da Doutora Raquel Alice da Silva Baptista Dias e co-supervisão do Prof. Paulo Jorge Peixeiro de Freitas

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Success is a science; if you have the conditions, you get the result. Oscar Wilde

Acknowledgements Agradeço aos meus pais por fazerem de mim o que sou hoje. Obrigada por me terem sempre proporcionado tudo o que de melhor há na vida. Por terem sempre a palavra certa no momento certo, por me terem apoiado incondicionalmente (e por me continuarem a apoiar) em todas as grandes tomadas de decisões. Por serem, sem dúvida, exemplos de vida a seguir. Pappi, obrigada por me apoiares em tudo. Tu sempre disseste que eu daria uma excelente engenheira. Tinhas razão!! Mummy, por todos os conselhos e por toda a preciosa ajuda que sempre me deste (inclusive na formatação da tese) ! À Raquel, o meu mais sincero obrigada por estes meses, pois mais que uma orientadora, foi uma excelente amiga. Obrigada pelo entusiasmo mostrado pela biologia, pela electrofisiologia, enfim, pela ciência, que me contagiou completamente. Obrigada pelo apoio, pelos constantes elogios e, claro, pelas divertidas conversas no nosso pequeno cubículo. A opinião sobre ti é unânime, pois arranjas sempre um tempinho para nos ajudar, mesmo quando estás muito ocupada. Considero-me uma sortuda por ter conhecido e por ter tido como orientadora. OBRIGADA ! À Professora Ana Sebastião por ter aceite sem qualquer hesitação que a minha tese de mestrado fosse efectuada no laboratório e por toda a confiança que depositou em mim. Ao Professor Raul Martins, que mostrou uma enorme disponibilidade para me auxiliar na realização da tese. Obrigada por me ter esclarecido toda a parte eléctrica do Setup. À Mariana e ao Francisco pela ajuda, pelo suporte e pela companhia que me fizeram durante os dias no laboratório, principalmente nos dias de experiência em que parecia nada funcionar no setup. À Mariana pela excelente amiga que se tornou em tão pouco tempo. Nem sei como expressar todo o meu agradecimento. Por tudo. Obrigada por me teres feito sorrir, mesmo quando não tinha motivos. Pelas longas conversas, desabafos (e cusquices) na nossa salinha. Pelos passeios por Lisboa a tirar fotografias (que não foram muitos), pelas “festas do pijama” em tua casa, pelas saídas, enfim por todos os momentos. Pela tua felicidade contagiante. E ao Francisco, por me ter salvo inúmeras vezes durante as experiencias. Por me ter feito sorrir a todo o instante, pelo sua visão extremamente negativa, em oposição ao meu mundo cor-derosa. Pelas conversas à hora do almoço. Sem vocês o Patch não teria sido a mesma coisa. E a frase “Patch Clamp não é um bicho papão (só às vezes)!” irá sem dúvida ficar para sempre gravada na minha memória. Ao Diogo, também elemento do Patch-Clamp Crew, por toda a ajuda que me deu mesmo durante o pouco tempo que esteve no laboratório. Obrigada pelas explicações de neurotransmissores, que evitaram leituras exaustivas, mas também pelas discussões de ideias acerca de assuntos mais “cromos” (como quem diz, mais à engenheiro).

À Catarina Orsinha, por ser a minha estrelinha da sorte nas experiencias. Pela sua excelente companhia nas horas de almoço, que bem me sabiam quando estava num dia NÃO. E por me entender tão bem nestes últimos momentos de “stress” da entrega da tese. Ao Tiago pela sua energia contagiante, que permitiu alegrar até os dias mais cinzentos. E pela sua companhia nas manhãs e tardes de estudo na biblioteca e nos dias de experiência que pareciam não ter fim. À Sandra pela sua simpatia e pela sua ajuda preciosa nas manhãs no laboratório, e à Vânia pela sua alegria constante na sala ao lado. Ao Afonso por toda a sua ajuda, apoio e preocupação nos dias de experiencia, apesar do seu mau gosto em clube de futebol! À Rita, André, Diana, Joana, Filipa, Catarina Luís, Sara, por me terem recebido de forma tão calorosa no laboratório e por nunca me terem recusado ajuda. À Margarida, para a qual não tenho palavras para agradecer. Por toda a amizade durante estes 5 anos intensivos de curso. Por todas as nossas confidências. Por todas as nossas brincadeiras. Obrigada pelo carinho e pelo apoio que nunca me faltou. À Marta Farracho e ao João Veiga, que além de excelentes amigos, me salvaram de muito trabalho quando me ensinaram a formatar a tese. To Gulistan Kocer, for all the support and friendship during this last year. Thank you so much for all the articles, without which it would have been harder to write my thesis. E finalmente não posso também de deixar de agradecer ao Sr. João por toda a sua ajuda, e por me ter dado a conhecer uma enorme variedade de “heavy metal”.

Resumo O glutamato é o principal neurotransmissor excitatório no sistema nervoso central, tendo um papel importante ao nível da aprendizagem e memória. Contudo, em certas situações patológicas, ocorre uma libertação excessiva de glutamato para a fenda sináptica que desencadeia morte neuronal precoce por necrose ou apoptose. Estudos anteriores mostram que a eritropoietina (EPO) apresenta um efeito neuroprotector perante uma situação de isquémia, o que poderá sugerir uma acção ao nível da libertação ou acção do glutamato sobre a excitotoxicidade. Outros estudos demonstraram que a EPO também tem um efeito agudo sobre o edema celular, aspecto característico de células que sofreram um insulto de isquemia. Alterações no volume da célula influenciam a capacitância de membrana, que se considera como um parâmetro que poderia permitir avaliar variações subliminares de volume da célula. Com o objectivo de esclarecer as acções da EPO na transmissão sináptica basal, bem como alteração de parâmetros biológicos da membrana induzidos por isquemia, realizaram-se experiencias em que se avaliou a variação da actividade sináptica espontânea inibitória (mIPSC), assim como a excitatória (mEPSC) causada por período de isquemia na presença de EPO. Avaliou-se também os valores de capacitância da membrana induzida por isquemia. Relativamente à capacitância de membrana, após isquemia, o valor encontrava-se aumentado (0.49±0.005µF versus 0.35±0.003µF em células control), o que sugere que células submetidas a longos períodos de isquémia sofrem alterações significativas ao nível das suas características eléctricas. Quando comparada a frequência das mEPSCs das células expostas a isquemia à das células de controlo, as primeiras apresentavam uma depressão significativa no seu valor (0.45±0.01Hz para 0.14.00±0.01Hz), evidenciando a vulnerabilidade das células da região CA1, após isquemia. A administração de EPO (2,4 UI/ml) induziu uma redução significativa na frequência das mEPSC, when compared to mEPSC frequency of cells in the control group (0.45±0.01 Hz para 0.14±0.01 Hz, n=7, *ρ= 0.0163). Quanto à transmissão GABAérgica, a EPO conduz a um aumento significativo na frequência das mIPSCs (2±0.15Hz para 2.4±0.09Hz). Os resultados obtidos demonstram um efeito da EPO na transmissão sináptica basal, podendo estar associado ao seu papel neuroprotector.

Palavras-Chave: Eritropoietina, Hipocampo, Isquémia, Capacitância, Patch-Clamp

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Abstract Glutamate is the main excitatory neurotransmitter in the central nervous system and it has an important role in processes of learning and memory. However, increased availability of glutamate in the extracellular space may lead to cell death by necrosis or apoptosis. Several studies have provided evidence that erythropoietin (EPO) has a neuroprotective effect during cerebral ischemia, which may suggest an effect in glutamate excitotoxicity. It was also observed that EPO has an acute effect upon glial cell swelling, a characteristic feature of the penumbra region after ischemia insult. Cell swelling affects membrane capacitance, therefore this parameter might enable to evaluate small variations of cell’s volume. To clarify the effect of EPO in basal synaptic transmission, but also to observe changes in membrane capacitance induced by ischemia, a set of experiments were performed which evaluated the variation of spontaneous inhibitory synaptic currents (mIPSC) as well as excitatory synaptic currents (mEPSC) recorded after exposure to ischemia, in the absence and presence of EPO. Membrane Capacitance values induced by ischemia were also assessed. The values of membrane capacitance were increased after ischemia, when compared with control cells (0.49±0.005µF in post-ischemic slices versus 0.35±0.003µF in control ones). In addition, cells exposed to ischemia suffered a significant depression of mEPSCs frequency, when compared with control slices (from 0.45±0.01Hz to 0.14±0.01Hz), showing evidences of the vulnerability of pyramidal cells to ischemia. The administration of EPO (2.4 IU/ml) induced a significant reduction in the frequency of mEPSC (from 0.14±0.01Hz to 0.96±0.01Hz). Regarding effects upon basal GABAergic transmission, EPO application resulted in a significant increase in mIPSC frequency (from 1.99±0.15Hz to 2.4±0.09Hz). In conclusion, EPO regulates both neurotransmitters release, which might be associated with its well-established neuroprotective role from ischemia.

Keywords: Erythropoietin, Hippocampus, Ischemia, Capacitance, Patch-Clamp

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Contents 1.

Aim of the Research .............................................................................................................. 1

Introduction .......................................................................................................................... 1 2.1

Glutamate...................................................................................................................... 2

2.2

Glutamate Receptors .................................................................................................... 2

2.2.1 2.3

Erythropoietin ............................................................................................................... 7

2.3.1

Historical Background on EPO research ................................................................ 8

2.3.2

Neuroprotective Role of EPO ................................................................................ 9

2.3.3

Mechanisms and signal transduction pathways ................................................. 10

2.3.4

EPO and Glutamatergic Transmission ................................................................. 12

2.4

Glutamate Excitotoxicity ....................................................................................... 4

GABAergic Transmission ............................................................................................. 13

2.4.1

GABA receptors ................................................................................................... 14

2.4.2

GABA and EPO ..................................................................................................... 14

Technique ............................................................................................................................ 15 3.1

History ......................................................................................................................... 15

3.2

Patch Clamp Technique ............................................................................................... 18

3.2.1

Electronic components of a Patch Clamp Setup ................................................. 19

3.2.2

Four patch-clamp measurement configurations................................................. 23

3.3

Capacitance ................................................................................................................. 27

3.3.1 Measurement of Specific Membrane Capacitance in Pyramidal cells ....................... 29 4.

Hippocampus....................................................................................................................... 30

Materials and Methods ....................................................................................................... 32 5.1

Animal Model .............................................................................................................. 32

5.2

Preparation of brain slices........................................................................................... 32

5.3

Whole-Cell Recordings ................................................................................................ 33

5.3.1

mEPSC Recordings ............................................................................................... 34

5.3.2

mIPSC Recordings ................................................................................................ 34

5.3.3

Ischemia induction .............................................................................................. 34

5.4

Data analysis................................................................................................................ 34

5.5

Measurement of the Membrane Properties ............................................................... 35 ix

Results and Discussion ........................................................................................................ 35 6.1

Influence of Ischemia in membrane capacitance ....................................................... 36

6.2

Role of Ischemia in mEPSCs frequency ....................................................................... 40

6.3

Role of Erythropoietin in modulation of mEPSCs, after ischemia ............................... 41

6.4

Role of Erythropoietin in modulation of mIPSCs, in normoxic conditions .................. 44

General Discussion and Future Work .................................................................................. 47 7.1

Membrane capacitance increases after ischemia ....................................................... 47

7.2

Ischemia decreased mEPSC frequency........................................................................ 48

7.3

Erythropoietin decreases the frequency of mEPSCs after ischemia ........................... 50

7.4

Erythropoietin enhances the release of GABA, in normoxic conditions ..................... 51

Conclusion ........................................................................................................................... 53

List of Figures Figure 1. Glutamate and its receptors.. ........................................................................................ 3 Figure 2. Steps involved in the process of chemical synaptic transmission. ............................... 5 Figure 3. Excitotoxicity of glutamate. ............................................................................................ 6 Figure 4. Overview of the mechanisms due to an ischemic episode.. .................................. 7 Figure 5. Illustration of the intracellular signaling pathways resulting from Erythropoietin binding to EPOR ...................................................................................................................................... 11 Figure 6. Schematic diagram of the synthesis, transport and transmission of GABA................ 13 Figure 7. GABA receptors are characterized by different subunits. ........................................... 14 Figure 8. Galvaniâ&#x20AC;&#x2122;s experiments associated with muscle contraction. ....................................... 16 Figure 10. A two-electrode voltage-clamp circuit.. ..................................................................... 20 Figure 11. Schematic representation of the vital components of a patch clamp experimental set up. ................................................................................................................................................ 21 Figure 12. Diagram of the headstage patch clamp amplifier. ..................................................... 22 Figure 13. The different configurations of patch clamp technique and description of the process of how they are obtained. ............................................................................................................ 23 Figure 14. Equivalent circuit of whole cell configuration. ........................................................... 25 Figure 15. Transient currents due to stray (pipette) capacitance in a cell-attached record mode ..................................................................................................................................................... 26 Figure 16. Representation of voltage clamp configuration and capacitive transients. ............... 28 Figure 17. Equivalent circuit of the recording configuration ....................................................... 29 Figure 18. Current signal measured in response to a -20mv pulse step ................................... 29 Figure 19. Hippocampus. ........................................................................................................... 30 Figure 20. Pyramidal Cells dimensions. ..................................................................................... 35 Figure 21. Patch-clamp recordings............................................................................................. 36 Figure 22. Graph showing the recorded capacitive transient (blue line) and the exponential curve (green line). ....................................................................................................................... 37 Figure 23. MATLAB program output .......................................................................................... 38 Figure 24. Capacitance is increased after an ischemic episode ................................................ 39 Figure 25. Frequency of miniature excitatory postsynaptic currents is lower in cells that suffer an ischemic insult .......................................................................................................... 41

Figure 26. Protocol used to record the miniature excitatory post synaptic currents (mEPSCs) 42 Figure 27. Administration of EPO decreases the frequency, but not the amplitude, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) .......................................... 43 Figure 28. Protocol used to record the miniature post synaptic currents (mIPSCs) .................. 45 Figure 29. EPO administration increases the frequency, but not the amplitude, of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). ................................................................. 46

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List of Tables Table 1. The glutamate receptor ion channels characteristics ..................................................... 4 Table 2. Output from a Fit analysis in Graph Pad ....................................................................... 37 Table 3. Values of the Specific Membrane Capacitance.. .......................................................... 39

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List of Symbols 2+

Calcium

CaCl2

Calcium chloride

Chloride

KCl

Potassium chloride

MgCl2

Magnesium chloride

MgSO4

Magnesium sulfate

Sodium

NaCl

Sodium chloride

NaHCO3

Sodium bicarbonate

NaH2PO4

Monosodium phosphate

Nitric Oxide

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Abbreviations aCSF

Artificial Cerebrospinal Fluid

AMPA

α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate

AMPAR

AMPA receptor

Akt-1

Protein Kinase B

ATP

Adenosine triphosphate

BBB

Blood Brain Barrier

Bcl-xL

B-cell lymphoma-extra large

BDNF

Brain-derived Neurotrophic Factor

CA1-CA3

Cornu Ammonis, areas 1-3

CBF

Cerebral Blood Flow

CNQX

6-cyano-7-nitroquinoxaline-2,3-dione

CNS

Central Nervous System

CREB

Ca2+/cAMP-response element-binding protein

CPPG

(RS)-alpha-cyclopropyl-4 phosphonophenylglycine

Dentate Gyrus

DL-APV

DL-2-amino-5-phosphonovaleric acid

Entorhinal Cortex

EGTA

Ethylene Glycol Tetraacetic Acid

EPO

Erythropoietin

EPOR

EPO receptor

EPSC

Excitatory Postsynaptic Current

Field Potential

GABA

γ-Aminobutiric Acid

GABAAR

GABA-gated receptor channel (type A)

GABABR

GABA-gated receptor channel (type B)

GABACR

GABA-gated receptor channel (type C)

GAD

Glutamic Acid Decarboxylase

GluR

Glutamate Receptor

GTP

Guanosine-5'-triphosphate

GPCR

G Protein-Coupled Receptors

HIF-1

Hypoxia Inducible Factor 1

HEPES

2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

IkB

Inhibitor of Transcription Factor NFkB

JAK-2

Janus Tyrosinekinase-2

Kynuretic Acid

LTD

Long Term Depression

LTP

Long Term Potentiation

L-VCCs

L-voltage calcium channels.

LY-341495

2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-D-alanine

LY 354740

(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid

MAPK

Mitogen-Activated Protein-Kinase

MCAo

Middle Cerebral Artery occlusion model

mEPSC

miniature Excitatory Postsynaptic Current

mGluR

Metabotropic Glutamate Receptors

mIPSC

miniature Inhibitory Postsynaptic Current

Mossy Fibers

mRNA

Messenger Ribonucleic acid 1,2,3,4-tetrahhydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-

NBQX sulfonamide +

Na /K+-ATPase

Sodium-Potassium Adenosine Triphosphatase

NFkB

Nuclear Factor Kappa B

NMDA

N-methyl-D-aspartate

NMDAR

NMDA receptor

OGD

Oxygen and Glucose Deprivation

PI(3)K

Phosphatidylinositol-3-Kinase

PPF

Paired-Pulse Facilitation

rhEPO

Recombinant human EPO

(RS)-APICA

(RS) 1-Amino-5-phosphonoindan-1-carboxylic acid

xvi

(S)-3,4-DCPG

(S)-3,4-Dicarboxyphenylglycine

S-4-CPG

(S)-4-carboxyphenylglycine

STAT-5

Signal Transducers and Activators of Transcription 5

Schaffer Collateral

SEM

Standard Error of the Mean

TBI

Traumatic Brain Injury

Transmembrane

TrkB

Tyrosine-related kinase B

TTX

Tetrodotoxin

xvii

1. Aim of the Research The aim of this experimental procedure was to evaluate the role of the cytokine erythropoietin in a situation of ischemia-induced neuronal swelling. In order to accomplish this aim, patch clamp technique was used to record passive and active signals from CA1 pyramidal neurons in acute hippocampal

rat

slices.

Changes

neuronal

swelling

were

assessed

through

electrophysiological recordings, by observation of changes in neuronal biophysical properties, such as membrane capacitance. Changes in synaptic transmission were also evaluated by monitoring both miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs).

2. Introduction Stroke, the third leading cause of death (Bernaudin et al., 1999), is a condition where a blood clot or a ruptured artery or blood vessel interrupts blood flow to a region of the brain, leading to a lack of oxygen and glucose supply (Lo et al, 2003). Within minutes, brain cells begin to die, and severe brain damage occurs, which can result in an inability to move one or more limbs on one side of the body, in impairment in speech and memory, as well as an incapability to see one side of the visual field. Certainly the outcome after a stroke depends on the location where the stroke occurred and how much of the brain is affected (Lo et al, 2003). There are two types of stroke: Hemorrhagic and Ischemic. Hemorrhagic stroke results from a weakened vessel that ruptures and bleeds into the surrounding brain and ischemic stroke, the most common type, is caused by a blood clot that blocks or plugs a blood vessel interrupting the blood supply (Deb et al., 2010). In both types of stroke, brain cells suffer oxygen and glucose deprivation (OGD), a condition designated as ischemia. Although the brain represents only 2.5% of the total body mass, it consumes approximately 25% of the total supply of oxygen and it has very limited capacity of energy storage. Thus it requires a continuous blood supply to provide for the constant need of oxygen and glucose, essential to execute the aerobic metabolism. If the brain is deprived of oxygen for a period of more than 60 to 90 seconds, the tissue ceases to function, and after approximately three hours, it will suffer irreversible injury, leading to necrotic death (Schaller and Graf, 2002). When analyzing a brain region after suffering an ischemic episode, it is possible to distinguish two different areas: the ischemic core and the ischemic penumbra (Lo et al, 2003). The first area corresponds to the region which suffered the most severe reduction of cerebral blood flow (CBF), experiencing within minutes permanent injury, due to the low metabolic rates of oxygen and glucose. The ischemic penumbra is the region adjacent to the ischemic core, which suffers a moderate CBF reduction (Lo et al, 2003). Neurons within this region become unable to fire action potentials. Even so, the quantity of blood that arrives allows them to remain metabolically active, and as soon as the flow improves, their ability to generate action potentials is restored

(Bretón and Rodríguez, 2011). Since there is shortage of oxygen and glucose to produce adenosine triphosphate (ATP), due to the occlusion of the brain blood vessels, the available amount of energy becomes low (Deb et al., 2010). The region of the brain affected by ischemia can still switch to anaerobic metabolism, but this procedure results in a small amount of ATP, which will not be enough to achieve all of brain energy-dependent processes essential for tissue survival. For instance, the resting membrane potential is established by the sodium potassium +

pump (Na /K+-ATPase), which in turn is operated by ATP. Thus, in a case where there is deprivation of oxygen and glucose, ATP-dependent maintenance of the resting potential will be compromised. Furthermore, the process of anaerobic respiration releases lactic acid, which can potentially destroy cells, disrupting the normal acid-base balance in the brain. The lack of ATP sets off a series of interrelated events that result in cell’s injury and eventually in their death (Bretón and Rodríguez, 2011). Excessive release of the excitatory neurotransmitter glutamate can be considered one of the early events of the death cascade.

2.1

Glutamate

Glutamate is considered the main excitatory neurotransmitter employed by pyramidal neurons in cerebral cortex and in several hippocampal tracts. It is an amino acid, and it has an essential role in cognitive functions, such as learning and memory (McEntee and Crook, 1993).

2.2

Glutamate Receptors

To develop its essential role in the brain, glutamate activates diverse receptors, which are characterized by their intrinsic characteristics and by their affinity for selective exogenous agonist and antagonists (Table 1). Receptors can be classified into two groups: ionotropic and metabotropic; and glutamate operate receptors belonging to both families. Ionotropic receptors are glutamate-gated cation channels, they can be subdivided into three sub-types, according to their selective agonist, namely: NMDA (N-methyl-D-aspartate), AMPA ( -amino-3-hydroxy-5methyl-4-isoxazolepropionate), and kainate. NMDA receptor (NMDAR) and AMPA receptor (AMPAR) are the most predominant in the hippocampus. Ionotropic receptors present a common structure, possessing four transmembrane (TM) hydrophobic regions within a central portion of the sequence, TM1-TM4. The TM2 domain forms a re-entrant loop resulting in an extracellular N-terminus and intracellular C-terminus (Mayer and Armstrong, 2004). Once the excitatory neurotransmitter binds to the receptor, it stimulates directly the ion channel, allowing ion flow and causing excitatory postsynaptic currents (EPSC). This current is depolarizing and, if enough glutamate receptors are activated, an action potential may be triggered in the postsynaptic neuron (Figure 1).

Figure 1. Glutamate and its receptors. Glutamate binds to NMDA and AMPA receptorsâ&#x20AC;&#x2122; recognition sites. The ionotropic AMPA receptors admit sodium ions when activated. This results in a moderate local depolarization that dislodges the magnesium ions, which are blocking the NMDA receptors. Calcium ions enter the cell trough NMDA receptorsâ&#x20AC;&#x2122; calcium channels. The NMDA is therefore both a ligand and a voltage gated channel. The influx of calcium into the cell results in more AMPA receptors in the postsynaptic membrane; the synapse has thus been strengthened (Adapted from Breedlove SM, 2010).

NMDA receptor has an important role in synaptic plasticity and memory function. At resting membrane potential, NMDA receptors are inactive, which is related to a voltage-dependent 2+

ions, preventing ion fluxes through it. Activation of NMDA

influx into the postsynaptic cells (see Figure 1). In order for

block of the channel pore by Mg +

receptors results into Na and Ca

the receptor to function, it is also necessary that another amino acid, Glycine, binds to the receptor together with glutamate (Mark et al, 2001). Concerning AMPAR, these receptors mediate fast synaptic transmission in the Central Nervous System (CNS) and are composed of four different subunits: GluR1-4. The AMPARs permeability to calcium and to sodium and potassium is governed by the GluR2 subunit. Therefore, if an AMPAR lacks a GluR2 subunit, it will be permeable to sodium, potassium, and calcium (Mayer and Armstrong, 2004). The principal ions gated by the majority of AMPARs are, however, sodium and potassium, which allows distinguishing AMPA receptors from NMDA receptors. AMPARs are responsible for basal excitatory synaptic transmission, but they also subserve many forms of synaptic plasticity such as Long-Term Potentiation (LTP) and Long-Term Depression (LTD), mechanisms that are thought to underlie the processes of learning and memory. Metabotropic glutamate receptors (mGluR) are G protein-coupled receptors (GPCRs) that modulate the production of intracellular second messengers, such as inositol-1,4,5-trisphosphate (IP3), calcium, and cyclic nucleotides (reviewed in McEntee and Crook, 1993; Mark et al, 2001; Rang and Dale, 2007). These receptors are subdivided in three groups, based on the pharmacology, similarity and intracellular signaling mechanisms (Table 1). Despite the several types of receptors mentioned above, AMPAR is the one which mediates most of the basal excitatory synaptic transmission in CNS.

Table 1. The glutamate receptor ion channels characteristics (Mayer and Armstrong, 2004; Rang and Dale, 2007)

Type of Receptor

Receptor Family

Subunits NR1

NMDA

NR2A-D NR3A, NR3B

Agonists

Glutamate NMDA

Antagonists

DL-APV

AMPA Ionotropic Receptors

AMPA

GluR1-4

Glutamate

CNQX

Kainate

NBQX

L-quisqualate

Kainate

Metabotropic Receptors

GluR5-7 KA1, KA2

Glutamate Kainate Domoate

CNQX Topiramate

Metabotropic group I

mGluR1

Glutamate

LY367385

mGluR5

L-quisqualate

S-4-CPG

Metabotropic group II

mGluR2

Glutamate

LY341495

mGluR3

LY 354740

(RS)-APICA

Metabotropic group III

mGluR4 mGluR6-8

Glutamate L-AP4

CPPG

(S)-3,4-DCPG

2.2.1 Glutamate Excitotoxicity Glutamate receptors play an essential role in mediating the excitatory synaptic transmission, through which brain cells communicate with each other. Glutamate is stored in vesicles in the presynaptic cell. An electrical impulse in the cell, such as an action potential, leads to an influx of calcium ions into the presynaptic terminal, and the consequent release of glutamate. The neurotransmitter diffuses across a small gap between the pre- and postsynaptic cell, the synaptic cleft, and stimulates the postsynaptic cell through the activation of ion channel-forming receptors; in the case of glutamate, the AMPA receptors mentioned above. The structure responsible for this vital function is the synapse, and it is in the synapse that the ionotropic glutamate receptors are mostly found. Once the ligand binds to the receptor, it induces a conformational change and the opening of the ion channel it comprises, enabling charged ions +

such as Na and Ca

to permeate through it. This flow of ions results in a depolarization of the

plasma membrane and the production of an electrical current, which is then propagated down through the dendrites and cell soma, reaching the axon, and eventually triggering an action potential, therefore the communication form of a neuron to another (Figure 2).

Figure 2. Steps involved in the process of chemical synaptic transmission. The release of a neurotransmitter is triggered by the arrival of a nerve impulse (or action potential) to the cell, which in turn produces an influx of calcium ions through voltage-dependent calcium-selective ion channels. Calcium ions, then, bind to membranes of the synaptic vesicles, where the neurotransmitters are localized, allowing the vesicles to fuse with the presynaptic membrane. As a result, there is the creation of a fusion pore, through which the vesicles then release their contents to the synaptic cleft. Receptors localized on the opposite side of the synaptic gap bind to the neurotransmitter molecules, generating an excitatory post-synaptic current (Adapted from Lisman et al., 2007).

However, elevated concentrations of glutamate in the extracellular space leads to excessive activation of its receptors resulting in neuronal excitotoxicity and consequently cell death (Mark et al., 2001; Purves et al, 2001). The negative effects of glutamate were initially observed in 1954 by Hayashi, a Japanese scientist, who reported that the direct application of glutamate to the CNS was responsible for the production of seizures, but his report went unnoticed for several years (reviewed in Purves et al, 2001). In 1957, Lucas and Newhouse also reported the toxicity by glutamate when they observed that the subcutaneous injection of monosodium glutamate to newborn mice destroyed the neurons in the inner layers of the retina (reviewed in Purves et al, 2001). Roughly a decade later, in 1969, Olney discovered that this phenomenon was not only restricted to the retina, but it could also be detected in the brain. Yet, the damage only occurred in the postsynaptic cells, since the dendrites of the target neurons were grossly swollen, whereas the presynaptic terminals were spared. He was, subsequently, responsible for coining the term excitotoxicity to describe this pathological process, by which excitatory amino acids elicit cell death (reviewed in Purves et al, 2001).

Figure 3. Excitotoxicity of glutamate. Glutamate is released from presynaptic terminals upon depolarization after an action potential. The glutamate release process is controlled and an excessive amount of neurotransmitter at the synapse is avoided by specific transporters. During hypoxia or hypoglycemia, however catastrophic depolarization can occur, compromising energy production and therefore the ability of the cell to maintain the membrane potential. Glutamate is released excessively at synapses, initiating a cascade of events, which results in cell death. When glutamate binds to specific receptor channels located in postsynaptic neurons originates an influx of calcium ions to the cell, which are involved in both necrosis and apoptosis of the cell (Adapted from Syntichaki and Tavernarakis, 2003).

Despite the fact that mechanisms by which the excessive release of neurotransmitter glutamate leads to neuronal injury remain still unknown in several details, there are, nowadays, two hypotheses that try to explain those neurotoxic effects. One of them proposes that glutamate 2+

excitotoxicity is dependent on extracellular Ca . In other words, excessive glutamate release 2+

induces Ca

influx into the cell, which leads to neuronal damage and eventually cell death,

through over-activation of a cascade of calcium-dependent catabolic processes (reviewed in McEntee and Crook, 1993; Mark et al., 2001) (Figure 3). Other studies showed, however, that +

glutamate neurotoxicity was also associated with the release of Na and Cl ions. Through the +

activation of an ionotropic receptor, glutamate opens membrane Na conductances, leading to a +

large influx of Na ions (see Figure 3). As a consequence, the membrane suffers depolarization -

and a secondary passive influx of Cl ions and water is observed, which results in acute neuronal swelling. Neuronal swelling is one of the main characteristic features of the penumbra region after ischemia insult, being considered that this effect is related to an alteration of the physical properties of the cell membrane (Choi, 1987; reviewed in McEntee and Crook, 1993). Understanding the mechanism underlying excitotoxicity could have important applications for protecting neurons from injury due to stroke, trauma, among others. The phenomenon of glutamate excitotoxicity is observed during an ischemic episode (Paschen, 1996). Toxic levels of extracellular glutamate accumulate in the brain due to the fact that the presence of this neurotransmitter in the synapse is regulated by ATP-dependent transporters. As it was mentioned above, in an ischemic situation, the reduced blood flow decreases the delivery of oxygen and glucose to the brain, leading to a decrease in the production of ATP and failure of energy dependent membrane receptors, ion channels and ion pumps. Therefore, the maintenance of the resting membrane potential, through the sodium-potassium pump, is

compromised and neurons suffer depolarization. Neuronal depolarization may trigger the activation of slow presynaptic inactivating voltage-dependent calcium channel, leading to excessive release of excitatory neurotransmitters, including glutamate. There is also disruption of energy-dependent glutamate reuptake from the synaptic cleft, and the subsequent overactivation of the NMDA and AMPA receptors. Water begins to enter in the cells in response to changes in ion concentrations, producing cytotoxic edema, a pathophysiological marker of 2+

ischemia (Figure 4) (Sist et al, 2011). Ca , Na and K enter the NMDA receptor channel in 2+

physiological conditions and in a controlled way, activating Ca -dependent enzymes which influence a wide variety of cellular components under physiological conditions. However, in a case of an overactivation of NMDA receptors, an excessive quantity of Ca

enters in the cell,

which due to its role as a second messenger, activates multiple signalling pathways that contribute to neuronal cell death (Sist et al, 2011) (see Figure 4). It is important to mention that the distribution of the NMDA receptor in the brain is associated with its regional vulnerability to cerebral ischemia, being higher in the hippocampus than in the cortex and higher in the hippocampal CA1 comparatively with the CA3 and dentate gyms (Cotman et al. 1987, reviewed in Paschen, 1996).

Figure 4. Overview of the mechanisms due to an ischemic episode. Energy failure leads to depolarization of neurons, followed by an excessive activation of specific glutamate receptors, which increases intracellular Ca 2+, Na+, Cllevels. Water enters into the intracellular space, via osmotic gradients, resulting in cell swelling (edema). The messenger Ca2+ activates numerous enzyme systems (proteases, lipases, endonucleases). Free radicals are also generated, damaging membranes (lipolysis), mitochondria and DNA, and consequently leading to cell death (Cartoon based on a drawing by Dirnagl et al, 1999).

2.3

Erythropoietin

Since ischemic stroke is one of the leading causes of mortality in Europe, it has become imperative nowadays to explore pharmacological agents that possess neuroprotective properties against ischemic brain injury. Despite the fact that there are already several possible therapeutic approaches to reduce the extent of brain injury, only a few of those treatment strategies effectively mitigate the harmful effects of hypoxia/excitotoxicity. Some studies reported that cytokines have a neuroprotective role against inflammatory responses related to

cerebral ischemia (Arvin et al., 1996; Bartesaghi et al., 2005). Within those studies, it was found that some hematopoietic cytokines, such as Erythropoietin (EPO), are possible candidates in processes of brain development and repair (Bernaudin et al., 1999). Erythropoietin is a 34-kDa glycoprotein hormone composed by 165 amino acids and 4 carbohydrate side chains, whose main function is to stimulate the production of erythrocytes or red blood cells, a process known as Erythropoiesis (reviewed in Bartesaghi et al., 2005). During this process, EPO stimulates erythroid precursor cells survival, as well as, their proliferation and differentiation (Brines and Cerami, 2005), in order to produce the daily requirement of about 200 billion new red blood cells to compensate their lifespan of only 120 days (Alnaeeli, 2012). The initial production of EPO, during fetal development, occurs in the liver. However, shortly after birth its main production shifts to the kidney (reviewed in Bartesaghi et al., 2005). The regulation of the EPO gene expression in most tissues is associated with Hypoxia Inducible Factor 1 (HIF-1), which is activated as a consequence of several other stress factors, such as hypoxia. Still, hypoxia is not the only factor responsible for EPO production. Metabolic disturbances, such as hypoglycemia and strong neuronal depolarization, which produce mitochondrial reactive oxygen species, may also increase EPO expression through HIF-1 activation (Bartesaghi et al., 2005; Brines and Cerami, 2005). The expression levels of EPO receptor (EPOR) are also up regulated after hypoxia (Bernaudin, M. et all, 1999). During several years it was believed by the scientific community that EPO did not possess biological functions aside from the regulation of Erythropoiesis. And although it was initially thought that EPO production occurred only in fetal liver and adult kidney, some studies reported that EPO and its receptor could be found in other tissues, which are not directly involved in the regulation of red blood cell production, such as in tissues of the CNS. In fact, several cells in the CNS, such as neurons and glial cells produce EPO and express EPOR (Genc et al., 2004). Of note, an mRNA sequence responsible for encoding EPO and EPOR was found in mouse brains (Digicaylioglu et al., 1995) raising the possibility that EPO could also be associated with other biological functions, particularly as a protective factor in the brain. A wide range of studies performed in the last ten years, allowed to demonstrate the enormous potential of EPO as a neuroprotective agent, for the treatment of ischemia, hypoxia and excitotoxic stress (Bartesaghi et al., 2005), but only a few evaluated in more detail EPO actions upon neuronal communication.

2.3.1 Historical Background on EPO research In 1882, Paul Bert, studying in Paris at that time, discovered that animals living at high altitudes, where there are lower oxygen levels, had an increased number of red blood cells (reviewed in Melli et al., 2006). A few years later, based on these studies, Friedrich Miescher proposed that the low oxygen pressure acted directly on the bone marrow, being responsible for the production of red blood cells (reviewed in Melli et al., 2006). In 1906, Paul Carnot, a professor of medicine in Paris, together with his assistant published a work, where they suggested that the

production of blood cells was regulated by an hormonal mechanism. They conducted a series of experiments on rabbits subject to bloodletting (withdrawal of small quantities of blood), and they attributed the increase in red blood cells to a humoral factor, which they called hemopoietin (reviewed in Fried, 2009). Later, Eva Bonsdorff and Eeva Jalavisto, two Finnish scientists, based on their continued work on red cell production, switched the name hemopoietin to erythropoietin (reviewed in Melli et al., 2006; Fried, 2009). In order to continue to investigate the existence of EPO, Reissman and Erslev proceeded with further studies, where they found out that a certain substance, present in the blood, was able to stimulate the production of red blood cells, increasing the hematocrit. This substance was then purified and confirmed as erythropoietin, predicting the potential therapeutic uses for EPO in certain diseases, such as anemia. In 1977, Miyake purified human EPO from the urine of anemic patients. This procedure allowed the successful cloning and transfection in mammalian cells of the EPO gene, in 1985 by Lin, in hamster ovary cells, which therefore permitted the production of industrial recombinant human erythropoietin (rhEPO) for treating patients with anemia (reviewed in Melli et al., 2006).

2.3.2 Neuroprotective Role of EPO Research during the last years has demonstrated that EPO has the potential to promote neuronal survival. As mentioned before, both EPO and its receptor are expressed in the nervous system (Digicaylioglu et al., 1995) and produced by neural cells (Masuda et al., 1993), which provided early evidence for potential activity of EPO in the brain. In fact, EPO and EPOR expression were demonstrated to change significantly during brain development, being considered that EPO/EPOR system had an essential role in regulating embryonic and adult neuronal development (Genc et al., 2004; Tsai et al., 2006). For instance, in the Tsai et al. (2006) study, it was shown that EPO and EPOR are expressed during critical periods of neuronal development, and therefore, animals with no EPO, or its receptor, in their system revealed defects in neurogenesis. It was also observed significant alterations in the expression of EPO and EPOR in regions within and around infarcts in brains of animal models of stroke and that EPO plays a neuroprotective role during the ischemic response of the brain by preventing neuronal apoptosis (Bernaudin et al., 1999). Some research groups have reported that EPO, when added to neuronal cultures, provides protection against hypoxic and glutamate excitotoxicity (Morishita et al., 1997; Bernaudin et al, 1999). EPO also provides protection to newborn mice hippocampal neurons against NMDA-receptor-mediated excitotoxicity (Keller et al., 2006). In 2002, Ehrenreich et al. reported that rhEPO administered intravenously, at high doses, to patients who suffered an acute ischemic stroke was well tolerated and safe, with an improvement in clinical outcome after 1 month. Brines et al. (2000) had previously verified that systemically administered rhEPO crossed the Blood Brain Barrier (BBB), reducing tissue damage in an ischemic stroke. According to Adamcio et al. (2008), chronic administration of EPO in healthy young animals improved hippocampus dependent memory and enhanced longterm potentiation; thus it could be potentially useful in cases of cerebral hypoxia-ischemia,

where

there

are

long-term

spatial

memory

deficits.

EPO

provides,

therefore,

microenvironment for neural plasticity during stroke recovery, besides the role in neural protection. Recent studies have shown that EPO provides substantial benefits in rodents that suffered Traumatic Brain Injury (TBI), reducing neuronal apoptosis (Yatsiv et al, 2005; Xiong et al., 2008), being those therapeutic effects independent of hematocrit (Zhang et al, 2009). Administration of soluble EPORs inhibits EPO activity, which worsens the severity of neuronal injury (Sakanaka, 1998). It was recently demonstrated that, among other actions, EPO has an acute effect upon glial cell swelling (Gunnarson et al., 2009; Krügel et al., 2010), which is thought to further restrict substrate delivery and aggravate neuronal damage in the penumbra region. In fact, experimental glial swelling induced by osmotic challenge was found to be significantly reduced by acute EPO application, in the hippocampus (Gunnarson et al., 2009), but also in the retina, through the release of Vascular Endothelial Growth Factor (VEGF) and subsequent activation of the glutamatergic-purinergic signaling cascade (Krügel et al., 2010). Finally there is evidence that treatment with EPO, after a stroke in adult rodents, resulted in a significant increase of brain levels of Brain-derived Neurotrophic Factor (BDNF) and neurogenesis, suggesting that EPO, through BDNF, may induce neurogenesis. EPO also enhances angiogenesis, through the production of VEGF, increasing neovascularization at infarct sites with a consequent improved oxygen delivery, a fact which might be related with the processes of neuroprotection and neuroregeneration (Wang et al, 2004). As summarized by Alnaeeli (2012), all the research done in this field has shown that neuroprotection associated with EPO occurs through three different mechanisms: 1) antiapoptotic response in neurons, 2) endothelial response by increased blood flow and oxygen delivery, which is triggered by an increase in vascular relaxation and angiogenesis, and 3) antiinflammatory effects.

2.3.3 Mechanisms and signal transduction pathways The binding of EPO to EPOR causes dimerization of the receptor, autosphosphorylation of Janus-tyrosinekinase-2 (JAK-2) and receptor activation (Genc et al, 2004). In turn, JAK-2 leads to the activation of several downstream signaling pathways, such as Ras-mitogen-activated protein-kinases (MAPKs), phosphatidylinositol-3- kinase [PI(3)K], and the transcription factor STAT-5 (Signal Transducers and Activators of Transcription 5) (Sirén et al., 2001; Genc et al, 2004; Bartesaghi et al., 2005). Specific inhibitors of MAPKS and PI(3)K pathways eliminate the EPO-induced increase in neuronal survival in a model of hypoxia, which can be related to the neuroprotective effect of EPO (Siren et al., 2001). It was demonstrated that there is a cross-talk between the JAK-2, STAT-5 and NFkB signal transduction system in neurons, a pathway that is used by EPO to protect cortical neurons from excitotoxic apoptosis (Genc et al, 2004; Bartesaghi et al., 2005). EPO stimulation leads to the production of a dose dependent increase in nuclear NF B and decrease in cytoplasmatic NF B, effects required for neuroprotection.

Once inside the nucleus, NF B, a protein complex that controls the transcription of DNA, bind to it, promoting the expression of neuroprotective genes. The anti-apopototic effects of NF B in neurons involve the activation of Akt-1, Bad phosphorylation and Bcl-xL upregulation (Genc et al., 2004).

Figure 5. Illustration of the intracellular signaling pathways resulting from Erythropoietin binding to EPOR. EPO binding to its receptor activates intracellular signaling pathways leading to the phosphorylation of JAK-2. Activation of JAK-2 mediates phosphorylation of STAT-5, PI(3)K, MAPK and inhibitor of the transcription factor NF B (I B). NF B dissociates from I B. STAT-5 and NF B translocate to the nucleus and bind to DNA. As a consequence, neuroprotective genes, such as and Bcl2, are expressed (Cartoon based on a drawing by Bartesaghi et al., 2005).

It is also known that Akt-1 is activated by PI(3)K (Siren et al., 2001) and that MAPK and STAT-5 are responsible for Bcl-xL upregulation. Based on these studies, it is likely that the neuroprotective role of EPO is associated with cellular pathways which are intrinsically related with each another. Activation of Akt-1 can contribute to neuronal survival by stabilizing mitochondrial membrane potential and preventing the release of cytochrome c (Kennedy et al., 1999). EPO may also exert its neuroprotective role via BDNF. As it was mentioned before, EPO induces mRNA expression and it is responsible for the production of biologically active BDNF in primary hippocampal cells. As a result there is a long-term activation of its specific receptor TrkB. The reduced neuroprotection and phosphorylation of TrkB triggered by EPO, observed when BDNF is neutralized by a specific antibody, confirm the relevance of both BDNF and TrkB in controlling EPO effect, as well as its role in rescuing neurons from damage (Bartesaghi et al., 2005). BDNF expression is triggered by the activation of voltage Ca

channels and recruitment of the

Ca -sensitive transcription factor CREB. EPO in neuronal cells induces a rapid increase in 2+

intracellular Ca , which in fact corresponds to an important step for the neuroprotective effects.

As a matter of fact, Morishita et al. (1997) showed that erythropoietin-induced Ca

influx from

outside of the cells enhances the resistance of the neurons to glutamate toxicity. The intracellular Ca

rise, through L-voltage calcium channels (L-VCCs), is responsible for

controlling the activity of NFkB, PI(3)K/Akt and MAPK and STAT-5. Thus, it is thought that to 2+

regulate neuronal survival, Ca

couples to a network of transduction cascades (Bartesaghi et

al., 2005).

2.3.4 EPO and Glutamatergic Transmission There are some studies that report the influence of EPO upon glutamatergic transmission. Weber and his colleagues, in 2002, investigated the effect of EPO preconditioning on neuronal synaptic transmission under normoxic and ischemic conditions. In these experiments hippocampal slice cultures were incubated with EPO (40 units /ml for 48 h) and subsequently exposed to oxygen and glucose deprivation for 30 min, followed by a reoxygenation period of 120 min. Extracellular Field Potential (FP) responses were recorded, and their analysis showed a significant increase in FP amplitude during and particularly following oxygen and glucose deprivation in EPO treated-slice cultures compared with the untreated control slices. These data suggested, therefore, that EPO improves synaptic transmission during and following ischemia in hippocampal slice cultures. In another study, addressing the effect of EPO on excitatory synaptic transmission, young mice were injected intra-peritoneally with EPO every other day for 3 weeks. In this study, the cellular mechanisms of EPO action on glutamatergic transmission were evaluated based on whole-cell patch-clamp recordings from CA1 pyramidal neurons in acute hippocampal slices, performed 1 week after the last injection. In this study, it was also performed a set of extracellular recordings of field excitatory postsynaptic potentials to examine the possibility of a direct effect of EPO on synaptic plasticity in hippocampus. The results obtained showed that EPO alters excitatory postsynaptic currents and improves hippocampus dependent memory (Adamcio et al., 2008). More recently, in Kamal et al. (2011) study, it was examined the direct acute effect of EPO perfusion on synaptic plasticity and transmitter release probability in hippocampal slices from one month old mice. The effect of EPO perfusion (50 U/ml) on basic synaptic transmission of hippocampal slices was evaluated, as well, as Pairedpulse facilitation, LTP and LTD that were recorded using high and low frequency stimulations. The results obtained suggested that EPO decreases the excitatory neurotransmitter release probability and enhances LTP in mice hippocampal slices, a form of synaptic plasticity which is considered as basic cellular model for learning and memory. Concerning my experiments, the effect of EPO on glutamatergic transmission was evaluated using a different protocol. First, hippocampal slices were subjected to ischemia for a period of 30minutes before the recordings. And then, after establishing whole cell configuration, CA1 pyramidal cells from hippocampal slices were exposed to aCSF containing EPO (2.4 UI/ml), for a period of 40 min. These experiments were performed to observe if EPO administration would induce alterations in frequency and/or amplitude of miniature excitatory postsynaptic currents.

2.4

GABAergic Transmission

Several experimental studies have suggested that after an ischemic stroke there is an increase in extracellular level of gamma-aminobutic acid (GABA), which may occur as a response to the excessive glutamate release, in order to inhibit its excitotoxic effect (Głodzik-Sobańska et al., 2004; Ramanathan et al., 2012). It became, therefore, imperative to understand if EPO exerted some influence on GABAergic transmission, since it seems that both have a neuroprotective role in cerebral insult. Besides that, concentration and expression of EPO and its receptor changes during development and GABAergic transmission also suffers several changes during development, which raised the possibility that again both of them could be involved in the regulation of neuronal functions in development of the brain (Wójtoicz and Mozrzymas, 2008). GABA is the main inhibitory neurotransmitter in the mammalian CNS, whose role consists of inhibiting the influx of Ca

ions into the cell, decreasing the excitability of the neuron. GABA is

released from the nerve terminal to the synaptic cleft, binding to specific receptors located in the postsynaptic membrane (Li and Xu, 2008). As a result, ion channels open, allowing the entrance of negatively charged chloride ions into the cell reducing the membrane potential, and achieving hyperpolarization (Figure 6).

Figure 6. Schematic diagram of the synthesis, transport and transmission of GABA. GABA is synthesized in inhibitory neurons from glutamate by the enzyme glutamic acid decarboxylase (GAD), and is transported into vesicles. GABA acts at inhibitory synapses in the brain, through the binding to specific transmembrane receptors in the plasma membrane of both pre- and postsynaptic neuronal processes. This binding causes the opening of ion channels to allow the flow of either negatively charged chloride ions into the cell or positively charged potassium ions out of the cell. As a result, the membrane potential suffers hyperpolarization. Reuptake of GABA by surrounding neurons and glial cells occurs through the activity of GABA transporters.

2.4.1 GABA receptors The inhibitory neurotransmitter GABA activates two different types of receptors, ionotropic receptors, GABAA and GABAC receptors (GABAARs and GABAcRs, respectively), and metabotropic receptors, GABAB receptor (GABABRs). The first ones mediate fast inhibitory responses, while the latter one mediates slow inhibitory responses (Chebib and Johnston, -

1999). Ionotropic GABAARs selectively conduct Cl ions through its pore, resulting in membrane hyperpolarization. GABAA receptors are hetero-oligomeric structures, made up of a mixture of several subunits: α, β, γ, δ, ε, θ, π and ρ, which results in several possible combinations for these receptor subtypes (Farrant and Nusser, 2005). The most common combination is the triplet α1/ β2/γ2, which is detected in various cell types (McKernan and Whiting, 1996). When GABA binds to GABAARs, the receptors change conformation within the membrane, opening -

the pore, and consequently increasing the permeability of the ion pore to Cl . Once GABA is removed from the synaptic cleft, the channel returns to a closed state and can, after desensitization, be re-opened. GABAARs can however be activated by other structural analogues of GABA, such as muscimol, a natural product from the hallucinogenic mushroom Amanita muscaria (Chebib and Johnston, 1999). GABAB receptors consist of transmembrane receptors that are coupled to G-proteins and activate second messenger systems and Ca

and

K ion channels. GABAB receptors produce slow, prolonged inhibitory signals and function to modulate the release of neurotransmitters. To date, two subtypes of this receptor have been identified, GABABR-1 and GABABR-2, existing two isoforms of the first one, R1a and R1b. The receptor is only functional when both GABABR-1 and GABABR-2 are co-expressed in the same cell (Chebib and Johnston, 1999).

Figure 7. GABA receptors are characterized by different subunits. GABAAR and GABACR are both ionotropic however GABAARs are a combination of several subunit types, while GABA CRs are composed of only single or multiple subunits. GABABRs are metabotropic receptors, being associated with G proteins. The subtypes of this receptor are R1a, R1b and R2.

2.4.2 GABA and EPO The influence of EPO upon GABAergic transmission has been even less studied than the influence of EPO upon glutamatergic transmission. Wójtoicz and Mozrzymas (2008)

investigated the impact of EPO on GABA release in hippocampal neurons developing in vitro. The purpose of this study was to observe the effect of a long-term treatment (48 or 72 h) with EPO (20 U/ml), in younger rats. The analysis of miniature inhibitory postsynaptic currents revealed a major acceleration of the decaying phase of these currents after EPO administration, suggesting that EPO has a modulatory action on GABAergic transmission in developing neural networks. In another study addressing the effect of EPO on GABA release, young mice were injected intra-peritoneally with EPO every other day for 3 weeks. Whole-cell patch-clamp recordings were performed on CA1 pyramidal neurons in acute hippocampal slices 1 week after the last injection, in order to study the cellular mechanisms of EPO action on inhibitory transmission (Adamcio et al., 2008). Regarding my experiments, the effect of EPO on GABAergic transmission was evaluated using a different protocol. During patch clamp recordings, after establishing whole cell configuration, CA1 pyramidal cells from hippocampal slices were exposed to aCSF containing EPO (2.4 UI/ml), for a period of 40 min. These experiments were performed in order to observe if EPO administration would induce alterations in frequency and/or amplitude of miniature inhibitory postsynaptic currents.

3. Technique 3.1

History

Electrophysiology can be described as the study of the electrical properties of biological cells and tissues. In the case of neurosciences, it is associated with the measurement of voltage change

and

electric

current of

neurons

and,

particularly,

action

potential

activity.

Electrophysiology studies date from 1660’s, when Jan Swammerdam, a Dutch microscopist and natural scientist, was responsible for the development of a neuromuscular preparation. In his experiments, Swammerdam used a frog leg and he proceeded to the stimulation of the nerve (process that he called “irritation”), which triggered muscle contraction (reviewed in Verkhratsky et al, 2006). Later, this scientist improved his previous preparation, placing the muscle into a glass and attaching needles to each one of the muscle ends. The movement of the needles due the nerve stimulation could be used to monitor the contraction phenomenon and to record it. In one of his several experiments, the nerve was fixed by a brass ring, and the “irritation” was done by a silver wire. In fact, Swammerdam was really close to understand the mechanism behind the propagation of the electrical signal between nerves and muscles (reviewed in Verkhratsky et al, 2006). It was, however, Isaac Newton, who first observed the nature of nerve signals (reviewed in Verkhratsky et al, 2006). Nevertheless, support experiments that proved the electric nature of the impulse were only found eighty years later, in 1791, with Luigi Galvani, when he published his fundamental work, De Viribus Electricitatis in Motu Musculari Commentarius, related with animal electricity. This work described 10 years of observations of contractions of frog-nerve muscle preparations, which consisted of the inferior limbs with the crural nerves, connected with the spinal cord and a metal wire that was inserted across the

vertebral canal. With these preparations, Galvani was able to identify the electrical excitation of the nerve-muscle unit to find the relationship between muscle contraction and stimulus intensity as well as to describe the refractory phenomenon by showing that nerve stimulation, if repeated several times, leads to the disappearance of contractions that can be restored after a period of rest. Yet, it was between the years 1794-1797 that fundamental experiments took place (reviewed in Piccolino, 1998). During this period, Galvani used two frog legs, attached with sciatic nerves and he noticed that when the nerve of the first preparation was in contact with the nerve of the second one, contraction was observed in both preparations (Figure 8), corresponding to the propagation of the action potential (reviewed in Folgering, 2008; reviewed in Verkhratsky et al., 2006). These experimental achievements allowed Galvani to develop the theory of electrical excitation. According to him, biologic tissues existed in a state of “disequilibrium” (the tissue at rest is capable of reacting to external stimuli, by generating electrical signals) and that the “electricity” presented in animals was a result from accumulation of positive and negative charges on external and internal surfaces of muscle or nerve fiber. Furthermore Galvani considered the existence of water-filled channels, as the pathway needed for the electrical current to flow, which penetrated the surface of the fibers, allowing electric stability (reviewed in Piccolino, 1998; reviewed in Verkhratsky et al., 2006).

Figure 8. Galvani’s experiments associated with muscle contraction. a) The surface section of the nerve touched the muscle and as a result the leg contracted (1974); b) the surface of the section of the right sciatic nerve touched the muscle, which resulted in both legs getting contracted (1797) (adapted from Piccolino, 1998).

Galvani’s discovers ended up inspiring several other scientists that followed his ideas, contributing for the development of electrophysiology. Within those scientists, it is important to mention the roles of Emile du Bois-Reymond and Hermann von Helmholtz. The first one measured electrical events accompanying the excitation of nerve and muscle, concluding that excitation leads to a decrease in the potential difference between the cut surface and the intact tissue. He termed the excitatory electrical response the “negative Schwankung”, which means negative fluctuation (reviewed in Verkhratsky et al., 2006). Concerning Helmholtz, in 1850-1852, using the nerve-muscle preparation, he estimated the speed of nerve impulse propagation, which was in the range of 25-40m/s (reviewed in Verkhratsky et al., 2006). In 1868, Julius Bernstein introduced the “differential rheotome”, scientific machinery that made possible to record the first values of resting and action potentials (Seyfarth, 2006). The value of the resting potential was about 60 mV, which Bernstein associated to the fact that the membrane is

selectively permeable to K . Regarding the action potential (still called “negative Schwankung” at the time), Bernstein determined that it had a rise time of about 0.3ms and a duration of ~0.80.9ms; he also confirmed that it was possible to observe the “sign reversal” (when the potential crossed the “zero potential”), moment correspondent to the action potential overshoot. Furthermore, Bernstein estimated the speed of nerve impulse propagation and he obtained values very similar (∼25–30 m/s) to the ones acquired by Helmholtz (reviewed in Verkhratsky et al, 2006). Later Charles Ernst Overton showed that the “negative Schwankung” was actually +

related with the exchange of Na and K ions between the extracellular and intracellular mediums (reviewed in Verkhratsky et al., 2006). Simultaneously new discoveries about the structure of the cell were made, which also helped in electrophysiology studies. Within those discoveries, Danielli and Dawson, found that the cell presents a bilayer lipid membrane associated with numerous proteins and penetrated by narrow water-filled pores (Danielli and Dawson, 1935). The Voltage Clamp technique was developed in 1949, by Cole and Marmont, which helped Hodgkin and Huxley clarifying the mechanisms underlying the generation of the action potential in nerve fibers. These scientists demonstrated that membrane excitability is determined by passive ion fluxes according to their electro-chemical gradients. The studies carried out after Hodgkin and Huxley elucidated the mechanism of ion permeation involved in membrane electrical excitability, which was probably the most important achievement of contemporary membrane electrophysiology, leading to the development of electrophysiology techniques (reviewed in Piccolino, 1998; reviewed in Verkhratsky et al., 2006). In 1949, Gilbert Ling and Ralf Gerard developed microelectrodes pulled from glass pipettes, which were appropriate for low-traumatizing penetrations of individual cells. These microelectrodes started to be used for electrophysiology recordings from all types of cells (reviewed in Verkhratsky et al., 2006). It was Alfred Strickholm who performed the first extracellular recordings from cellular membranes, placing a pipette against a muscle in a way that the cell surface under it was electrically isolated. This procedure allowed him to measure the impedance of frog muscles and record currents, flowing through the small membrane patch under the tip of the extracellular pipette (Strickholm, 1962). A few years later, based on previous studies, Neher and Sakmann pressed an electrode tip, gently, on the surface of a denervated frog muscle fiber, allowing the recording of single ion channel currents from a cell membrane. The First Patch Clamp experiment was achieved. The first recordings of single ion channel currents were, however, associated with some background noise due to the relatively low seal resistance (MΩ) established between the cell membrane and the recording pipette (Neher and Sakmann, 1979). The discovery, that application of negative suction enables the establishment of high resistance seal (10-100 GΩ), termed as the gigaseal, in 1980, allowed to obtain better electrical recordings, since the interaction between the pipette and the membrane became significantly more stable and tight (reviewed in Verkhratsky et al. , 2006). The creation of the Patch Clamp technique was considered an important science development and for that reason the Nobel Prize for Physiology and Medicine was attributed to the scientists Neher and Sackmann, in 1991 (reviewed in Folgering, 2008).

3.2

Patch Clamp Technique

Patch Clamp Technique can be defined as an electrophysiology technique, which allows the study of ion currents mediated by single or multiple ion channels of a cell, under voltage-clamp. It can be applied to a wide variety of cells, but it is especially used in excitable cells, such as neurons and muscle fibers (Kornreich, 2007). Patch clamp recordings make use of an electrode, a glass micropipette with an open small tip diameter of roughly 1 Âľm, used to seal onto the surface of the cell membrane. The pipette interior is filled with an electrolyte solution associated with the type of the patch-clamp configuration to be established. The solution matches the ionic composition of the bath solution in cell-attached and inside-out recording configuration, also known as external solution, and matches the cytoplasm composition in the case of the whole cell and outside-out recording configuration, known as internal solution (Safronov and Vogel, 1999). In other words, it matches the ionic composition of the fluids in contact with the side of the membrane with which the electrode is going to be in continuity. It is necessary to place a filter between the fill capillary tube and the syringe for backfilling the pipette. The electrolyte solution is positioned in contact with a chloride silver wire, which conducts the electric current to the amplifier. The pipette is then placed into the bath solution, being important to lower the pipette as quickly as possible. But first, it is essential to apply positive pressure to the back of the electrode, in order to keep the tip of the pipette free of contamination with impurities present on the surface of the bath (Kornreich, 2007). Inside the bath, the electrode is advanced until the tip is in contact with the cell surface (Figure 9A). The micropipette is then pressed against the cell membrane, increasing the resistance of the electrode (Figure 9B). Simultaneously, sometimes it is possible to observe a dimple on the surface of the cell produced by the stream of the solution inside the electrode, after which the positive pressure should be released and a small amount of suction (negative pressure) should be applied to the electrode. This process allows the beginning of the sealing process (Figure 9C).

Figure 9. Currents that results from a test pulse (a square pulse of 5mV). A. The electrode is lowered into the external recording solution. The current resulted from the application of a 5mV test pulse of 25 ms duration is approximately 2.5 nA. B. Contact with the cell surface partially occludes the pipette tip, increasing the value of the resistance and reducing the value of the test pulse current. C. Application of negative pressure results In the formation of a giga-seal, with a further decrease in the current to approximately 3 pA (current flat) and an increase in the value of the resistance (adapted from Kornreich, 2007).

When the value of the total resistance is about 30 MΩ, the experimenter should change to the voltage-clamp mode, using a holding potential of -20 mV, and as the cell seals, the potential has to be adjusted to -60/70 mV. The formation of the “gigaseal” (achievement of the cell-attached mode) can occur immediately on contact with the cell surface or it can take a little while. In general, the gigaohm seal is slower in hippocampus slices than in cultures of neuron cells (Gibb and Edwards, 1994). The high resistance of the seal is imperative since the higher the resistance the more complete is the electrical isolation of the membrane patch and more stable is the recording, decreasing the electric current noise (Odgen and Stanfield, 1994). Most of the patch clamp measurements are performed in a voltage-clamp mode, which allows ion flow across a cell membrane to be measured as electric current, whilst the membrane voltage is held at a constant value (Halliwell et al., 1994). Changes in the current values are associated with changes in resistance (opening and closing of ion channels). Ohm’s Law (Equation 1) provides the basis for the voltage clamp technique, defining the relationship between the current I, measured in amperes A, the voltage V, measured in volts V, and the resistance R measured in Ohms Ω. Equation 1 shows that an increase of the voltage leads to an increase in the current; and a decrease in the resistance leads to an increase in the current. Therefore, according to Ohm’s law, when the potential across the membrane is kept constant, changes in the current will account for changes in the resistance. (1)

During the patch clamp technique, when the pipette is in contact with the cell membrane, the occlusion of the pipette tip increases the resistance, resulting in a decrease in the current. The negative pressure applied after to create the seal will increase again the resistance, reducing the value of the current across the pipette-membrane interface. Figure 9 illustrates the changes in the ion currents during the gigaseal formation in a path clamp recording, for the case of a 5mV test pulse, value that is commonly used in the beginning of any patch clamp recording to monitor the establishment of a gigaseal. The resistance of the electrode before touching the cell (Figure 9A) is easily calculated by the Ohm’s Law. For this case, a 5mV test pulse, with duration of 25 ms, results in a 3 nA current. The value of the resistance is therefore: (2)

3.2.1 Electronic components of a Patch Clamp Setup The first voltage-clamp experiments used a circuit with two electrodes both interconnected through a feedback amplifier. One of the electrodes served as a voltage sensor, while the other acted as a current source, injecting the current needed to maintain the membrane potential. The voltage electrode was connected to pre-amplifier, which in turn, fed a signal to the clamping amplifier that also received command voltage as input. The clamping amplifier compared the

command potential with the recorded membrane potential, and sent an output through the current electrode. In other words, changes detected by the voltage electrode in the holding potential, resulted into current injection, which was equal and of opposite polarity to the current flowing through the cell membrane (Figure 10). The monitoring of the injected current corresponded to an accurate reproduction of the currents flowing across the membrane. The application of this technique was, however, restricted to cells that are large enough (> 20 Âľm) to allow inserting two electrodes (Sontheimer and Ransom, 2002).

Figure 10. A two-electrode voltage-clamp circuit. Two microelectrodes are inserted in large cells, one to record voltage and the other to pass current. The first one is connected to a first amplifier and records membrane potential. The second one is connected to the clamping amplifier (feedback amplifier), which passes current to the cell to maintain the membrane potential (Sontheimer and Ransom, 2002).

In order to solve this problem, the single electrode switching amplifier was developed, which made use of only one microelectrode to serve double duty as voltage and current electrode. During a short period of time, the amplifier connects its voltage-sensing input to the electrode, takes a reading, and subsequently switches rapidly (at 3-20kHz) to the current source output, in order for the same electrode to deliver current to the cell (Halliwell et al., 1994). Due to its characteristics, this approach is limited in its time-resolution by the switching frequency between the two modes, which must be set based on the cellâ&#x20AC;&#x2122;s RC time constant (Sontheimer and Ransom, 2002). The whole-cell patch clamp amplifier uses similarly only one electrode. Nevertheless, in contrast with the previous techniques, it uses one electrode continuously for the voltage recording and passage of current (Sontheimer and Ransom, 2002). Figure 11 represents a general scheme of a standard patch-clamp setup. The patch pipette with the internal recording electrode is connected to the headstage, which in turn is connected to a micromanipulator. The micromanipulator function is to allow positioning the electrode in the right position in order to touch the surface of the cell. The hippocampus cells are visualized with an upright light microscope. The microscope and headstage are placed on a table, used to isolate these components from vibrations which would interfere with the gigaseal formation. The table is inside a Faraday cage, which protects the setup from ambient electrical noise. The acquired

analog signal from the headstage is then passed to an analog to digital converter (AD-DA), where the signal is digitized and sent to a computer for data analysis (Safronov and Vogel, 1999; Kornreich, 2007).

Figure 11. Schematic representation of the vital components of a patch clamp experimental set up. The patch pipette with internal recording electrode and reference electrode are connected to the headstage, which in turn is mounted on a micromanipulator. The hippocampal cells are visualized with an inverted light microscope. The microscope, micromanipulator, and headstage are placed on a table inside of a Faraday cage. The acquired analog signal from the headstage is passed through an AD converter, where the signal is digitized and then sent to a computer for analysis (Adapted from Safronov and Vogel, 1999; Kornreich, 2007).

Patch-clamp technique makes use of a feedback circuit to set the membrane potential of a cell to a desired command value. The membrane potential is, therefore, maintained constant and the membrane current is measured for that potential value. The patch-clamp amplifier, placed in the headstage is considered one of the most important features of the setup. This amplifier functions as a current-to-voltage converter, allowing the current that is measured, to be displayed on the computer screen. It is considered that the amplifier behaves as an ideal amplifier, which means that it is characterized by a large openloop voltage gain and it does not draw any input current. Input impedance is therefore considered to be infinite and the output impedance is considered to be zero (Odgen and Stanfield, 1994). The patch-clamp amplifier is also a differential amplifier, since it operates to make the output equal to the difference between the two inputs (Figure 12). Briefly, the current flows through the electrode (ip) across a resistor of high impedance (Rf), generating a voltage drop, Vp, proportional to the measured pipet current ip, according to Ohmâ&#x20AC;&#x2122;s Law (Equation 3). The feedback resistance Rf, is the component in the patch-clamp amplifier circuit, which turns it into a current-to-voltage converter.

(3)

â&#x20AC;&#x201C;

The membrane potential of the cell, Vp, is measured and compared to the command potential, Vp. The role of the amplifier is to adjust the voltage output to maintain a constant pipet potential at the desire reference potential (Vref). When current flows across the membrane through ion

channels, Vp is instantaneously displaced from Vref. To prevent changes in the membrane potential, the amplifier changes Vout in order to generate an ip that will exactly oppose the displacement of Vp from Vref. As it was mentioned before, the current measured during a patchclamp by the experiment resembles the current flowing through the cell membrane, although with opposite polarity. The injected current is recorded, being possible to take conclusions about the membrane conductance. Figure 12 illustrates the components of the current-to-voltage converter amplifier, characterized by a high gain, set by the large feedback resistor, Rf.

Figure 12. Diagram of the headstage patch clamp amplifier. The amplifier gain is set by the value and is given by . The membrane potential of the cell, , is measured and compared to the command potential, . The voltage output is adjusted by the amplifier, in order to maintain a constant pipet potential at the desire (Cartoons based on a drawing by Odgen and Stanfield, 1994).

At the beginning of the experiment Vref is set for zero current by offsetting electrode potentials, procedure that can be done manually or by using the ‘tracking mode’ of the patch clamp amplifier, which uses an integrator to keep the current at zero, adjusting Vref accordingly. After the formation of a high resistance seal, and the ‘tracking mode’ of the patch clamp amplifier being switched to voltage clamp mode, Vref may be changed without causing large currents between pipette and bath. Nevertheless, fast changing commands (the leading edges of rectangular pulses for instance) leads to large currents, due to charging stray capacitance associated with pipette and cell. In these situations, the amplifier may saturate and compensation circuits have to be applied in order to offset ‘fast’ (pipette) and ‘slow’ (cell) capacitive transients (Odgen and Stanfield, 1994). To measure the capacitance changes due to alterations in biophysical properties of the cell (one of my goals) only the fast capacitive transients had been compensated. Indeed these are the ones that may saturate the amplifier.

3.2.2 Four patch-clamp measurement configurations Within the basic technique there are several configurations that can be applied, according to what the researcher wants to study (Figure 13). The difference between the configurations is related with alterations in membrane integrity, membrane orientation, or continuity between the intracellular space and the pipette solutions (Kornreich, 2007) and each one of the configurations has its own advantages and disadvantages. All recordings configurations involve the initial formation of a high resistance gigaseal between the cell membrane and the tip of the pipette (Kornreich, 2007).

Figure 13. The different configurations of patch clamp technique and description of the process of how they are obtained. When the pipette sealed to the membrane cell, in the cell attached mode, it is possible to record single channel currents. The seal (gigaseal) is very stable, and therefore the patch can be pulled off the cell and dipped in a wide variety of tested solutions. On the other hand, the cell-attached patch can be ruptured by applying suction, resulting the whole-cell configuration. This last configuration has the advantage of allowing the study of the allowing the study of an ensemble response of all ion channels present in a cellâ&#x20AC;&#x2122;s membrane. Pulling the pipette away from the cell in the whole-cell configuration results in the formation of an outside-out patch (Adapted from Kornreich, 2007).

The cell attached configuration is the first step required to establish the other configurations. In order to form the cell attached mode the tip of the pipette is lowered onto the cell of interest using the micromanipulator. When the pipette is placed on the surface of the cell, and after the formation of a low resistance (seal) there is an application of a slight suction to the inside of the recording pipette to form the gigaseal. This configuration allows the recording of currents through single ion channels in the patch of the membrane, without disrupting the interior of the cell. After the formation of the gigaseal, by quickly pulling the pipette away from the cell the inside-out configuration is obtained. The patch of the membrane is ripped off the cell and stays

attached to the micropipette, exposing the intracellular surface of the membrane to the bath solution. This technique is applied to study the effects of manipulating the environment at the intracellular surface of ion channels (Safronov and Vogel, 1999; Kornreich, 2007). The whole cell configuration is achieved by applying negative pressure after attaining the cell attached configuration, which will rupture the patch of membrane inside the tip of the electrode. The pipette solution diffuses into the cell, and due to the fact that the internal volume of the recording pipette is larger than the internal volume of the cell, the pipette interior solution will replace completely the intracellular solution. This technique is applied to study the ensemble response of all ion channels within a cellâ&#x20AC;&#x2122;s membrane (Safronov and Vogel, 1999; Kornreich, 2007). More details about the whole-cell configuration will be given further, since this was the configuration used in the present work. After reaching the whole cell configuration, another configuration can be achieved by slowly pulling away the pipette from the cell, resulting in the separation of a bleb of the membrane of the cell and formation of a patch on the tip of the pipette. The internal surface of the cell membrane is exposed to the pipette solution and the external surface stays in contact with the batch solution. This configuration is known as outside-out patch, and single channel recordings are possible if the bleb of the membrane presents small dimensions. This configuration is used to study the properties of an ion channel when isolated from the cell, with the possibility of changing external solutions (Safronov and Vogel, 1999; Kornreich, 2007). Within all the configurations, the whole cell is the one most frequently used. This configuration allows to establish electrical continuity between the pipette solution and the interior of the cell, and compared to other configurations, where only current passing through a single channel is recorded, in whole cell configuration, it is possible to record macroscopic currents flowing through the membrane, allowing the study of an ensemble response of all ion channels present in a cellâ&#x20AC;&#x2122;s membrane. The whole cell configuration can be represented by an electric circuit demonstrated in Figure 14. The current ip, the current in the pipette, is considered positive flowing from the pipette to the cell. The resistance Rs in series with the cell membrane pretends to illustrate the pipette tip. The cell membrane is represented as a Capacitor Cm in parallel with a Resistance Rc.

Figure 14. Equivalent circuit of whole cell configuration. Pipette current, ip, corresponds to the sum of (current that ďŹ&#x201A;ows in the cell resistance ) and (current in the capacitance) and ďŹ&#x201A;ows in the series resistance between pipette and cell. The pipette current produces a voltage error . (b) Time course of changes of in the value of and ip following a step of (Adapted from Odgen and Stanfield, 1994).

According to Kirchoff's law, in an electrical circuit the sum of currents flowing into a node (junction) is equal to the sum of currents of flowing out that node. This implies that the pipette current its divided in two currents, one ionic whose value is given by capacitive, given by

and the other

. Equation 4 can be used to determine the time course of the

change in the potential of the cell. ( ) where

( ( )

( )) (

)

( ) corresponds to the value of the membrane potential for a time ,

value and

(4) ( ) the initial

the time constant (that characterizes the response of the voltage clamped cell to

applied voltage steps and currents), which is given by (5)

The values for

and

are given using equation 6 and 7, respectively.

(

(6)

)

(7) (

)

(

)

During the establishment of a whole cell voltage clamp recording different current responses are observed in the computer screen, that are similar to the ones represented in Figure 15, where a 5mV square pulse was used to monitor the setting of a gigaseal (Figure 15A). The size of the

test pulse current gives the experimenter a measure of the electrodeâ&#x20AC;&#x2122;s trip resistance. Therefore, changes in the size of the test pulse current will be associated with changes in the electrodeâ&#x20AC;&#x2122;s resistance. As it was pointed out before, the electrode tip is lowered until it touches the cell membrane, moment where the value of its resistance increases. The change in resistance together with the appearance of a dimple (observed in a video monitor) in the site of contact with the cell membrane can be used as a sign to release the positive pressure and apply gentle suction; resulting in the formation of a small seal. Together with gentle suction to continue to pull the small patch of the cell membrane into the pipette, the experimenter should deliver negative command potential since it aids the formation of the seal. The test pulse current continues to decrease until no current flows between the electrode tip and the cell surface. At this moment, the cell attached configuration is achieved (gigaseal is formed) (Figure 15B). Meanwhile the fast capacitive transients are nulled (Figure 15C), and the application of strong suction results in the rupture of the membrane patch, shown by the appearance of large capacity transients at the leading and trailing edges of the pulse (Figure 15D).

Figure 15. Transient currents due to stray (pipette) capacitance in a cell-attached record mode. a) Illustration of a 5 mV rectangular test pulse; b) Formation of a giga-seal as a result of gentle suction. The resistance is high, so except for the capacitance transients the test pulse current is virtually flat; c) The electrode capacitance transient is nulled; d) Whole-cell configuration is achieved by applying strong suction that removes that patch of the membrane in the electrode tip, but leaves the seal and cell intact. The value of the resistance decreases and large capacitance transients are observed (Adapted from, Odgen D, Stanfield P, 1994)

The whole-cell recording configuration is associated with errors set by series resistance and cell capacitance. The series resistance (also termed as access resistance) becomes in series with the cell membrane (which in turn acts as a capacitor). From this combination results a low-pass RC filter, which introduces artifacts. As a consequence there will be a slow exponential charging of the cell membrane potential, characterized by =RsCm, in case of a response to a square

voltage step. The time constant of the error voltage due to series resistance can be minimized, within other solutions, simply by keeping Rs as small as possible by using low resistance pipettes (Odgen and Stanfield, 1994). Another error comes from the dilution of cytosol components. In the whole cell configuration, the internal volume of the recording pipette is larger, when compared to the volume of the cell, which means that the interior solution will replace the contents of the interior of the cell. The loss of the cytosolic components into the pipette leads to a cascade of events, such as alterations in the properties of ionic currents, and cell responses involving second messengers, such as calcium release (Horn and Marty, 1988; Odgen and Stanfield, 1994). Performing perforated patch-clamp technique, which is a variation of the whole-cell configuration, can avoid the rundown of cell responses. This technique does not use suction to rupture the patch membrane, but, instead, an electrode solution containing an antifungal or antibiotic agent. The antibiotic molecules diffuse into the membrane cell and perforate it, providing electrical access to the cell interior. Still, there is a higher access resistance, comparatively to the whole-cell configuration, due to the partial membrane occupying the tip of the electrode. As a consequence, electrical access decreases, as well as, the current resolution; the recording noise increases and the series resistance error is magnified. Not to mention that the membrane under the electrode tip, due to the antibiotics perforations becomes weak and can rupture, which means that the one is then in the presence of whole-cell recording, but with antibiotic contaminating the inside of the cell (Horn and Marty, 1988; Safronov and Vogel, 1999). Nevertheless and in spite of its limitations, the whole cell configuration is widely used and proven a reliable way to evaluate the flow of ions across the cell membrane.

3.3

Capacitance

As it was previously mentioned, in stroke, due to the incapacity of cells to maintain the ionic and fluid homeostasis, there is excessive release of glutamate to the synaptic cleft. Through the +

activation of ionotropic receptor, this neurotransmitter opens the membrane Na conductance, +

leading to a large influx of Na . The membrane suffers strong depolarization and an influx of Cl

ions and water is observed (Choi, 1987; reviewed in McEntee and Crook, 1993). This phenomenon results in neuronal swelling (edema), which is associated with an increase in the cellsâ&#x20AC;&#x2122; volume. One way to measure this effect on cells is through the determination of the membrane capacitance. In fact, it is considered that the membrane capacitance is an indirect representation of cell volume (Chi and Xu, 2000). Capacitance is the quantity charge, measured in Faraday, required to create a given voltage difference between two conductors. A typical capacitor in an electronic circuit is comprised of two surfaces (plates) separated by a substance called dielectric (Kornreich, 2007). The membrane of the cell (composed of a lipid bilayer with thickness of 8â&#x20AC;&#x201C;10 nm (Gentet et al., 2000) can be considered as an excellent insulator between the two conducting surfaces (in this

case the cytoplasm and the extracellular medium), acting the membrane as a capacitor (Barbour B, 2011). Both inside and outside regions of the cell are composed by different ions and their movement across the membrane creates the voltage difference, which corresponds to the basic form of electrical communication in nerve cells. The main characteristic of a capacitor is that it was the ability to store charge, which is proportional to the applied potential or voltage. In most of the articles, in which membrane capacitances are measured, the authors present the values of this parameter per unit area, known as specific membrane capacitance, C (Odgen and Stanfield, 1994; Gentet et al., 2000), which is an important parameter to model the electrical properties of neurons. In fact, since membrane capacitance is directly proportional to the membrane surface, the estimation of this parameter allows understanding if changes in the cell surface are accompanied by changes in cellular electric properties (Gentet et al., 2000; Golowasch et al., 2009). Membrane capacitance can be calculated using capacitive transients, which in turn are obtained by the application of a voltage step to the cell. The capacitive transient obtained in response to a command voltage step Vp is represented in Figure 16, which shows evidences that the interaction of the electrode resistance and cell capacitance results in a low-pass filtering effect (Barbour B, 2011). The current relaxes to the final value according to an exponential decay with a time constant

ReCm, which is responsible for all the interactions

between cell and amplifier.

Figure 16. Representation of voltage clamp configuration and capacitive transients. The inverting input is clamped to the command voltage Vc by the negative feedback. Since the pipette current must pass through the feedback resistor to ensure this clamp, the output voltage is proportional to the current. The traces shown correspond to the capacitive transients, which are obtained in response to a command voltage step. The current transient resembles the RC filter response, which means that the interaction between the electrode resistance and cell capacitance cause a strong low pass-filter effect (adapted from Barbour B, 2011 ).

Most of the studies that use capacitive transients to determine the specific membrane capacitance, have achieved capacitance values very similar among different types of cells, which means that the membrane capacitance is independent of the type of cell. The value of capacitance in hippocampal pyramidal neurons was estimated to be between 0.5-1 ÂľF/cm (Solsona et al., 1998; Gentet et al, 2000; Golowasch et al., 2009).

3.3.1 Measurement of Specific Membrane Capacitance in Pyramidal cells There is a simple and direct approach for measuring the specific membrane capacitance, which requires only electrophysiology equipment and a computer. A simplified circuit can be used to describe the whole cell recording configuration (Figure 17). This simplification requires that the membrane is isopotential, and that no voltage-dependent conductances are active. Besides, this model circuit predicts that the current transient following a step in the voltage-clamp command potential will have an exponential time course (Figure 16). A time domain analysis of capacitive transients obtained by square wave stimulation allow to obtain the values for the recording pipette (Ra), cell membrane resistance (Rs) and membrane capacitance (Cm), which equations are expressed in (7), (8) and (9), respectively.

(7)

(8)

(

)

(9)

Figure 17. Equivalent circuit of the recording configuration. It contains only 3 fundamental components: the access resistance,

, the membrane resistance

and the membrane capacitance,

. It is used a simple version to

facilitate the estimation of the membrane capacitance, although a full theoretical analysis of it is actually far from straightforward.

VStep corresponds to the amplitude of the voltage-clamp step, I0 to the peak amplitude of the current transient immediately after the step is applied,

the decay time constant of the current,

and Iss the steady-state current at longer times following the step. The parameters , I0, and Iss were determined by fitting a single exponential function to the current transient between the dot red lines (see section Materials and Methods: 5.5 Measurement of Capacitance).

Figure 18. Current signal measured in response to a -20mv pulse step. This current transient was recorded from a pyramidal cell located in CA1, in whole cell configuration and characterized by a holding potential of around -70mV.

There is another technique that can be used to measure the membrane capacitance, which involves an analysis of the frequency domain. However, this method is not as flexible as the

time domain one, due to the need of implementation of special hardware, such as lock-in amplifier. This was the main reason for implementing the first method to calculate the membrane capacitance.

4. Hippocampus The hippocampus is an important component of humans and other vertebratesâ&#x20AC;&#x2122; brains and its study has attracted the interest of scientists since a long time ago. It can be easily found and removed intact from the brain, after which, it is usually cut into transverse slices (perpendicular to the septal-temporal axis). Hippocampal slices have been widely used since they offer several technical advantages for both biochemical and electrophysiological studies. For instance, with slices, it is possible to control directly the environment of the slice and the drugs can be applied in known concentrations to the entire slice or only to specific areas, according to the goal of each study (being also possible to remove the drug easily from the tissue when desired).

Figure 19. Hippocampus. a) Structure of left rat hippocampus. All forebrain structures except those at the mid-line were removed (O'Keefe and Nadel, 1978); b) Different regions, layers, and fiber pathways present in the hippocampus (Amaral and Lavenex, 2007).

The hippocampus belongs to the limbic system and it is known for its role in memory and spatial navigation. There are two hippocampi in the brain, one in each side, being both characterized by a C-shaped structure (Figure 19a). In rodents, the hippocampus is positioned so that one end is close to the top of the head (the dorsal or septal end), and the other end is near the bottom of the head (the ventral or temporal end). The hippocampus can be further divided in several subfields. Although the terminology varies among authors, the terms most frequently used are the dentate gyrus (DG) and the Cornu Ammonis (CA) (O'Keefe and Nadel, 1978; Amaral and Lavenex, 2007). The neuroanatomist Rafael Lorente de NĂł differentiated the last subfield in three different regions: CA1, CA2 and CA3, in which CA1 and CA3 are the largest ones and the easiest to identify (Figure 19b). The CA regions can be differentiated in eight defined strata or layers: the alveus, stratum oriens, stratum pyramidale, stratum lucidum, stratum

radiatum,

stratum

lacunosum,

stratum

moleculare

and

the

hippocampal

sulcus or fissure. The alveus corresponds to the deepest layer and it is composed by pyramidal

neurons axons, passing on toward the fimbria. Stratum oriens, the next layer superficial to the alveus, contains the cell bodies of inhibitory basket cells and horizontal trilaminar cells, as well as, the basal dendrites of pyramidal neurons. Of note, in rodents the two hippocampi are highly connected, but in primates this commissural connection is much sparser. In the stratum pyramidale, cell bodies of the pyramidal neurons, which are the principal excitatory neurons of the hippocampus, can be found. In this stratum it is also possible to find the cell bodies of many interneurons, including axo-axonic cells, bistratified cells, and radial trilaminar cells. Stratum lucidum is one of the thinnest strata in the hippocampus only found in the CA3 region. Fibers from the dentate gyrus granule cells course through this stratum in CA3. Stratum radiatum contains septal and commissural fibers, but also Schaffer collateral fibers, which project forward from CA3 to CA1 pyramidal neurons. It is also possible to find in this stratum some interneurons such as basket cells, bistratified cells, and radial trilaminar cells. Regarding the stratum lacunosum, this thin stratum contains Schaffer collateral fibers and perforant path fibers from the superficial layers of entorhinal cortex. The stratum molecular is the most superficial stratum, where the perforant path fibers form synapses onto the distal, apical dendrites of pyramidal cells. At last there is the hippocampal sulcus or fissure, which is a cellfree region that separates the CA1 field from the DG (O'Keefe and Nadel, 1978, Amaral and Lavenex, 2007). The dentate gyrus is composed of three layers: The polymorphic layer, the stratum granulosum and the stratum moleculare. The polymorphic layer is the most superficial layer of the dentate gyrus, being composed mainly by interneurons and the axons of the dentate granule cells that pass through this stratum on the way to CA3. In rats, the thickness of this layer is approximately m. The stratum granulosum contains cell bodies of the dentate granule cells. Concerning the stratum moleculare, this layer is the closest to the hippocampal fissure. The inner third of the stratum moleculare is the layer where commissural fibers from the contralateral dentate gyrus form synapses, but also where inputs from the medial septum terminate. In the external two thirds of the stratum moleculare, the perforant path fibers make excitatory synapses onto the distal apical dendrites of granule cells (O'Keefe and Nadel, 1978, Amaral and Lavenex, 2007). Sometimes it is common to use the term fascia dentate to refer to these two last mentioned stratum together. In the transverse hippocampal slices, it is possible to recognize almost all the subfields mentioned above, but also the intrinsic hippocampal circuitry. The hippocampal circuitry, the main glutamatergic, excitatory, unidirectional circuit of hippocampus, involves three connected pathways. The Entorhinal Cortex (EC) can be considered the first step in the circuit. Cells in the superficial layers of the EC project, among other destinations, to the dentate gyrus via the perforant path. Second, the granule cells of the dentate gyrus project to the pyramidal cells of the CA3 field of the hippocampus, via Mossy Fiber (MF) projections. In the last step, pyramidal cells from CA3 field project to the pyramidal cells in CA1 via Schaffer Collaterals (SC). The CA1 field of the hippocampus then projects unidirectionally to the subiculum neurons, providing its major excitatory input, which, in turn send, the main hippocampal output back to the EC,

resulting in a loop. (Amaral and Lavenex, 2007). The hippocampus circuitry, also known as the “trisynaptic” loop, can be represented by ECDG (synapse 1), DGCA3 (synapse 2), CA3CA1 (synapse 3) The principal type of neural cells present in the hippocampus is the pyramidal neurons, first discovered and studied by Santiago Ramón y Cajal. Pyramidal neurons are characterized by a triangular shaped cell body (just like their name suggests), a single axon, a large apical dendrite, and several smaller dendrites at the base. In the hippocampus it is also possible to find another type of important cells: the interneurons (Amaral and Lavenex, 2007). One of the main electrophysiological differences between these two types of cells is related to their firing pattern. Pyramidal neurons are characterized by long action potentials, several degrees of spike frequency adaptation and slow firing frequencies (Madison and Nicoll, 1984). Interneurons mediate GABAergic transmission, while pyramidal neurons send excitatory outputs. Excitatory pyramidal neurons receive both inhibitory and excitatory inputs, which were both recorded in this work.

5. Materials and Methods 5.1

Animal Model

Experiments were performed using acute hippocampal slices from 3 to 4 weeks old Wistar rats (Harlan Iberia, Spain). The rats were housed in standard plastic cages and kept under standardized temperature and lighting conditions, being provided water and food ad libitum. All experiments with animals were approved by and conducted in accordance with the European Community Guidelines and Portuguese Law on animal care.

5.2

Preparation of brain slices

The rats were decapitated (after being subjected to deep isofluorane anesthesia), and their brains quickly removed (<1 min) (Gibb and Edwards, 1994) and placed in an ice-cold solution. The low temperature helps decreasing the cell metabolism, and simultaneously, keeps the consistency of cerebral tissue. It is important to refer that, in general, it is easier to make healthy slices from younger animals, with around 3 weeks, since the skull is soft and the brain can be removed more rapidly. Besides that, younger rats have smaller brains that cool more rapidly than a large brain from an adult. Not to mention that the younger brain may be more resistant to anoxia (Gibb and Edwards, 1994). The two hippocampi were then dissected. The dissecting solution contained: Sucrose 110 mM, KCl 2.5 mM, CaCl2 0.5 nM, MgCl2 7 mM, NaHCO3 1.25 mM, glucose 7 mM, oxygenated with 95%O2 and 5% CO2, pH 7.4. In order to obtain the slices, the hippocampus was cut on a vibratome (VT 1000 S; Leica, Nussloch, Germany). The 300 mthick transverse slices were transferred to a beaker, incubated at 35°C, containing a solution

corresponding to the artificial cerebrospinal fluid (aCSF), composed by NaCl 124 mM, KCl 3 mM, NaH2PO4 1.25 mM, NaHCO3 26 mM, MgSO4 1 mM, CaCl2 2 mM and glucose 10 mM, pH 7.4, gassed with 95%O2 and 5% CO2. The slices were maintained at 35°C during a period of 30 minutes, to facilitate the functional recovery of the tissue, period after which the beaker is placed at room temperature. The slices recovered for at least 1 hour at room temperature before beginning recordings.

5.3

Whole-Cell Recordings

The whole-cell recordings were made, as mentioned before, from pyramidal cells located at CA1 stratum pyramidale. The recordings were made at room temperature and were performed using a microscope (Zeiss Axioskop 2FS) equipped with infrared video microscopy and differential interference contrast optics. The hippocampal slices were placed in the recording chamber, fixed with a grid, being continuously superfused by a gravitational superfusion system, with aCSF at room temperature. Erythropoietin was then added to the superfusion solution, reaching the recording chamber minutes after. The patch pipettes, used in the setup, were made from borosilicate glass capillaries (1.5 mm and 0.86 mm, outer and inner diameters, respectively, from Harvard Apparatus) in two stages on a pipette puller (PC-10 Puller, Narishige Group). They were characterized by a resistance of approximately 4–7 MΩ (if microelectrodes have a high resistance value, it is usually more difficult to break through the whole-cell configuration), when filled with an internal solution, which depended on the type of currents that were being recorded. For mEPSCs the internal solution is composed by (in mM): K-gluconate 125, KCl 11, CaCl2 0.1, MgCl2 2; EGTA 1, HEPES 10, MgATP 2, NaGTP 0.3 and phosphocreatine 10. The pH value was around 7.3, adjusted with NaOH (1 M), within an osmotic interval 280–290 mOsm. For mIPSCs, the internal solution is composed by (in mM): K-gluconate 125, NaCl 8, CaCl2 1, EGTA 10, HEPES 10, MgATP 5, NaGTP 0.4 and glucose 10. The pH value was around 7.2, adjusted with CsOH (1 M), with an osmotic interval 280–290 mOsm. The recordings currents were performed in the voltage-clamp mode (Vh=-70 mV) set up by an Axopatch 200B (Axon Instruments) amplifier.

The offset potentials were nulled before the

gigaseal formation. After establishing whole-cell configuration, it was possible to determine the membrane potential of the neurons in the current-clamp mode. Firing pattern was usually determined at the beginning of each experiment, through 500 ms depolarization steps up to 40mV. Small voltage steps (5 and 20mV, 500 ms) were delivered before starting the recording miniature currents, to monitor the access resistance and membrane capacitance.

5.3.1 mEPSC Recordings Miniature excitatory postsynaptic currents (mEPSCs), which represent postsynaptic responses that result from the spontaneous release of glutamate by Schaffer collateral afferents into pyramidal cells, were recorded in aCSF solution, to which was added tetrodoxin (TTX, 0.5 µM) and gabazine (2 µM). TTX is a potent neurotoxin, which selectively blocks voltage-gated sodium channels, preventing the propagation of action potentials in nerves (Chau et al, 2011). The application of TTX allowed, therefore, the observation of currents resulting from a spontaneous transmitter release. Gabazine acts as an antagonist at GABA A receptors. It binds to the GABA recognition site of the receptor-channel complex, reducing GABA-mediated synaptic inhibition by inhibiting chloride flux across the cell membrane, and thus

inhibiting neuronal

hyperpolarization, as a consequence of GABA release. After approximately 15-20 min, Erythropoietin (EPO, 2.4 IU/ml) was added to the solution containing already TTX and gabazine.

5.3.2 mIPSC Recordings Some of my experiments involved the recording of miniature inhibitory postsynaptic currents, mIPSCs, which resulted from the spontaneous release of GABA by interneurons. In those cases, instead of gabazine, it was added Kynurenic Acid (KA, 1 mM) to the aCSF solution, which is an antagonist of ionotropic glutamate receptors, blocking the glutamergic currents. After approximately 15-20 min, Erythropoietin (EPO, 2.4 IU/ml) was added to the solution containing already TTX and Kynurenic Acid.

5.3.3 Ischemia induction In some of my experiments, pyramidal cells were subjected to a 30 min of ischemia, which was induced, substituting 10mM glucose-containing aCSF with one containing 7mM sucrose (3mM glucose), gassed with 95%N2/5%CO2.

5.4

Data analysis

Analysis of mEPSCs and mIPSCs were performed with Mini analysis software. The data was sampled at 5 or 10 kHz and filtered using a low-pass Gaussian Filter (600 Hz with a -3dB cutoff). Statistical analyses were carried out using the Prism Version 5.01 for Windows (GraphPad Software). Results were expressed as the mean±SEM of n experiments. The statistical significance was assessed by a two-tailed Student’s t test, and statistical significance was assumed if ρ value was 0.05 or less.

5.5

Measurement of the Membrane Properties

500 ms hyperpolarizing square pulses of 5 or 20 mV were applied to cells with a holding potential between -50 to -70 mV, and around 50 capacitive transients were recorded and averaged. Making used of GraphPad Prism, the average transients, in response to the stimulation, were fit with an exponential (one phase) decay with the use of an iterative sum-ofsquares minimization algorithm according to

( )

(

)

(

)

(10)

The parameters obtained by the fitting curve expression were used to estimate Ra, Gm and Cm, using equation (7), (8) and (9). The signal was sampled at 5 kHz and the currents I, I0 and Iss were measured with respect to the baseline. The value of I0 was determined by extrapolating the fitted exponential curve back to the start of the current response. Regarding the value of the capacitance, Cm was then divided by the surface area of the patch, which allowed to obtain the value of the specific capacitance C (Gentet et al., 2000). Pyramidal cells were considered to be approximately ellipsoids, to simplify the determination of the surface area. The area was calculated using expression 11, where the major and minor axes are represented in the Figure 20. The dimensions of the cell were measured in a television screen from images captured at high magnification (40x objective coupled with a 4x magnification lens). (

) ( )

(11)

Figure 20. Pyramidal Cells dimensions. Hippocampal cells were considered to be ellipsoids. The two major axes were measured from images in a screen television captured by 40x objective coupled with a 4x magnification lens.

6. Results and Discussion The role of erythropoietin was assessed through recordings of mEPSCs and mIPSCs using whole-cell patch-clamp recordings, which were obtained from pyramidal cells located in stratum pyramidale of CA1 hippocampal area. CA1 pyramidal cells were visually identified by their characteristic morphological features (Figure 21a), and functionally by their firing patterns,

obtained in response to current injection through the recording electrode. Therefore, to ensure that the recorded cells were indeed CA1 pyramidal cells, firing patterns were routinely determined at the beginning of each experiment (Figure 21b). Pyramidal cells are characterized by slow firing frequencies (â&#x2030;¤5Hz), longer action potentials (â&#x2030;Ľ0,8ms) and for featuring spikefrequency adaptation (Madison and Nicoll, 1984).

b) a)

Figure 21. Patch-clamp recordings. a) Recordings were performed in pyramidal cells located in stratum pyramidale of CA1 area of rat hippocampal slices. b) The identification of a pyramidal cell was also confirmed by its firing pattern, which was obtained in response to a current injection through the recording electrode.

6.1

Influence of Ischemia in membrane capacitance

Biological membranes are lipid bilayers interspersed in proteins, which constitute approximately 50% of the membrane content. Biological membranes are characterized by two constants, G m (membrane conductance) and Cm (membrane conductance), values seem to be fairly constant among different types of cells, and even within different species (Sontheimer and Ransom, 2002). Cellsâ&#x20AC;&#x2122; swelling, a characteristic feature of the penumbra region after stroke, which is thought to further restrict substrate delivery and aggravate neuronal damage, is believed to change the value of Cm. After beginning an experiment and before recording miniature excitatory postsynaptic currents, hyperpolarizing square pulses were applied to the cells (see Methods and Materials 5.5). The measure of the membrane capacitance was estimated by the analysis of the cell response to that pulse. The purpose of these experiments was to evaluate the influence of ischemia in neuronal biophysical properties such as membrane capacitance. Therefore, capacitive transients were acquired from both cells in ischemic and in normoxic conditions (control cells). Taking into account the methods used in other studies to measure membrane capacitance (Lindau and Neher, 1988; Gentet et al., 2000; Zhou et al., 2006), this parameter was calculated by performing an analysis of recorded capacitive transients obtained by application of a 20mV hyperpolarizing pulse (Figure 18). I also did some transients recordings using 5mV pulses, but the results obtained were roughly similar and since most articles used a 20mV pulse, in order to get a more trustworthy comparison, I chose not to include the quantifications using a 5mV pulse. After obtaining the current transients, they were averaged, so that the noise would be

decreased and the resolving power increased. The transient was then fit with a single exponential function (using Graph Pad software) and the time constant and amplitude parameters (I0, Iss and ) (Table 2) were used to estimate Ra, Gm and Cm . Since all my results 2

were dependent on how well this curve fitted my data, besides checking the value of R , which quantifies the goodness of the fit, after each analysis, I also inserted the expressions of the exponential fit curves in MATLAB and compared them with the recorded capacitive transients (Figure 22). Table 2. Output from a Fit analysis in Graph Pad. From this analysis, the parameters I0, Iss and regression were estimated to generate an exponential curve that could fit the capacitive transient.

Iss

-432.2 pA

-120.4 pA

for a nonlinear

Exponential Fi Curve Expression 6.136 ms

-432.2e-12+120.4e-12)*exp(I(:,1)/(0.006136))-120.4e-12))

Figure 22. Graph showing the recorded capacitive transient (blue line) and the exponential curve (green line). Both curves were drawn in MATLAB, in order to see if the exponential curve provided a good fit to the series of data points.

The parameters obtained from Graph Pad were then used as an input for a MATLAB function, which was built to facilitate the calculation of Ra, Gm and Cm (Figure 23).

Figure 23. MATLAB program output. This MATLAB program has as input the value of the step, the I0, Iss and as output the values of Ra, Gm and Cm. This image is an example of one of the capacitance analysis.

and

These experiments were carried out in 16 pyramidal cells, from which 8 corresponded to cells that suffer 30 minutes of ischemia (aCSF containing sucrose 7nM / glucose 3nM, gassed with 95%N2/5%CO2), while the other 8 corresponded to control cells (aCSF containing 10 mM, gassed with 95%O2/5%CO2). As mentioned before, most of these experiments were performed before recording mEPSC, but it was only possible to study the capacitances if the whole-cell configuration was completely achieved. The values obtained for the Ra, Gm and Cm can be found in Table 3. The value of Cm was then divided by the measured surface area to achieve the value of the specific capacitance C, values which are also reported in Table 3.

Table 3. Values of the Specific Membrane Capacitance. The data presented in this table correspond to the values of

Normal Conditions

Ischemia Conditions

Ra, Gm and Cm , from both Ischemia and normal conditions, as well as the dimensions from the pyramidal cell, which allowed the calculation of the area and consequently the specific capacitance membrane.

Exp

Ra (Ω)

Gm (S)

Cm (F)

Major axis (µm)

Minor axis (µm)

Area (cm2)

C (F/cm2)

3.57E+07

6.49E-09

1.46E-10

1.30E+01

5.55E+00

0.000268803

5.44E-07

2.24E+07

8.62E-09

1.70E-10

1.02E+01

7.40E+00

0.000242594

7.01E-07

2.52E+07

7.23E-09

1.52E-10

1.48E+01

4.63E+00

0.000296355

5.12E-07

1.80E+07

1.05E-08

2.18E-10

1.57E+01

6.48E+00

0.000387076

5.64E-07

2.94E+07

1.38E-08

1.26E-10

1.39E+01

5.18E+00

0.000285173

4.44E-07

3.52E+07

6.45E-09

1.03E-10

1.24E+01

9.25E+00

0.000367964

2.79E-07

3.33E+07

4.12E-09

1.38E-10

1.11E+01

5.55E+00

0.00021773

6.33E-07

2.54E+07

6.57E-09

1.25E-10

1.57E+01

8.33E+00

0.000454276

2.76E-07

4.03E+07

6.67E-09

1.17E-10

1.30E+01

6.48E+00

0.000296355

3.94E-07

3.44E+07

1.07E-08

1.06E-10

1.67E+01

5.55E+00

0.000387076

2.75E-07

3.34E+07

6.07E-09

1.54E-10

1.48E+01

7.40E+00

0.000387076

3.98E-07

3.35E+07

6.73E-09

1.61E-10

1.30E+01

7.40E+00

0.000325251

4.95E-07

3.22E+07

1.03E-08

1.09E-10

1.11E+01

7.40E+00

0.000268803

4.06E-07

3.68E+07

6.87E-09

1.12E-10

1.85E+01

8.33E+00

0.000565157

1.99E-07

4.63E+07

8.34E-09

8.14E-11

1.48E+01

3.70E+00

0.000268803

3.03E-07

4.27E+07

7.34E-09

1.10E-10

1.57E+01

3.70E+00

0.000296355

3.72E-07

The values obtained for the specific membrane capacitance C were analyzed and compared, so that it would be possible to detect differences between ischemic and control cells. The comparison of C values obtained in both conditions is shown in Figure 24, in which there is evidence of a significant increase in the specific membrane capacitance of cells that previously suffered a 30 minutes period of ischemia (0.49±0.05 µF, n=8, *ρ=0.0461, Figure 24), when compared to the control baseline level (0.36±0.03 µF, n=8, *ρ=0.0461).

Figure 24. Capacitance is increased after an ischemic episode. The value of membrane capacitance was significantly higher (0.49±0.05 µF, n=8, *ρ=0.0461) in cells that had been exposed to 30 min of ischemia (induced by replacing 10mM glucose-containing with aCSF containing 7mM sucrose/ 3mM glucose, gassed with 95%N2/5%CO2.), comparatively with control cells (0.36±0.03 µF, n=8, *ρ=0.0461). Values are mean±SEM. * p<0.05 (two tailed unpaired Student’s t-test, compared with control situation, using absolute membrane capacitance values).

6.2

Effect of ischemia in mEPSCs frequency

The mechanisms of ischemic cell damage triggered by the occurrence of an ischemic episode are still not completely understood. For instance, it has not been established if it is the disturbances occurring during ischemia or those that follow the insult that play the prominent role in the manifestation of cell damage. Hippocampal pyramidal neurons from CA1 region are known to be quite vulnerable to brief episodes of ischemia or hypoxia (Cotman et al. 1987, reviewed in Paschen, 1996). In response to both oxygen and glucose deprivation CA1 neurons suffer a massive depolarization and neurons’ condition becomes irreversible (Xu and Pulsinelli, 1994; Tanaka et al., 1997). Several studies have been done in order to evaluate the effect of an ischemic insult upon glutamatergic transmission. However, investigation of the firing rate of neurons in CA1 region, following an ischemic episode, had led to conflicting results. Relevant for the present work are the studies by Furukawa et al. (1990) and Xu and Pulsinelli (1994), which show that spontaneous synaptic activity from CA1 neurons was significantly depressed after brief periods of ischemia. In opposition, in Chang et al. (1989) study, it was reported that neurons in the CA1 region suffered an increase in their firing rate 2 and 3 days after 10 minutes of transient cerebral ischemia. This hyperexcitability could be related with the ability of cells to induce synaptic plasticity as a response to acute energy deprivation, a maladaptive synaptic plasticity (Calabresi et al., 2003). Synaptic plasticity can be defined as the ability of neurons to create new neuronal connections or strengthen existing ones in response to either use or disuse of transmission over synaptic pathways.

This

process

usually

associated

with

changes

the

quantity

of neurotransmitters released into a synapse and in the number of postsynaptic receptors. Therefore, I thought it would be interesting to observe if there were significant differences between the mEPSC frequency in cells that did and cells that did not suffer an ischemic insult. In order to accomplish this task, a set of experiments was performed, in which mEPSCs were recorded, in whole-cell configuration, from CA1 pyramidal cells in normoxic conditions (considered as control cells) and from cells after suffering an ischemic insult. In short, these latter cells were placed, for 30 min, in an aCSF solution containing sucrose 7mM/ glucose 3mM and gassed with 95%N2/5%CO2, before the recordings, in order to mimic an insult of ischemia in brain cells. The average mEPSC frequency of cells from both conditions was then compared. Figure 25 illustrates the changes observed in mEPSC frequency between control with ischemic cells. After ischemia, there is in fact a depression of glutamatergic transmission, since mEPSC frequency in cells which suffered deprivation of oxygen and glucose is significantly lower (0.14±0.01 Hz, n=7, *ρ= 0.0163), when compared to mEPSC frequency of cells in the control group (0.45±0.01 Hz, n=7).

Figure 25. Frequency of miniature excitatory postsynaptic currents is lower in cells that suffer an ischemic insult. The column graph shows a comparison between values of mEPSC frequency in control experiments (aCSF containing glucose 10mM, oxygenated with 95%O2 and 5%CO2) with the mEPSC frequency of cells, which were incubated for 30 minutes in an ischemia solution (aCSF containing sucrose 7mM/ glucose 3mM, gassed with 95%N2/5%CO2), before the recordings. These data values correspond to average frequency observed before initiating EPO perfusion (baseline values). Each value corresponds to the average of individual macroscopic responses to spontaneous glutamate release recorded from different cells, after adding TTX (0.5 µM) and gabazine (2 µM) and achieving a stable baseline. The experimental conditions to which each cell was subjected are indicated below each column. Values are mean±SEM. * p<0.05 (two tailed unpaired Student’s t-test, compared with control situation, using absolute frequency current values).

6.3

Role of Erythropoietin in modulation of mEPSCs, after ischemia

Several reports demonstrate that EPO has the ability of protecting nerve cells from glutamate toxicity, role that has been investigated during the last decade (Morishita et al., 1997; Ehrenreich et al., 2002; Wang et al., 2004, Bartesaghi et al., 2005; Xiong et al., 2008). There are already some findings that show that EPO decreases the excitatory synaptic transmission in normoxic conditions (Adamcio et al., 2008; Kamal et al., 2011). However, most of the studies have focused their attention on the role of EPO in glutamate release under hypoxia/ischemic situations (Morishita et al., 1997; Sakanaka et al. 1998; Weber et al., 2002). Therefore I thought it would be interesting to address the EPO effect on basal excitatory synaptic transmission after CA1 pyramidal cells had suffered an ischemic insult, in order to mimic what would happen in a clinical situation. Using the same data set from the previous study and after recording mEPSC baseline frequency, EPO was added to system. In these experiments, before starting recording miniature excitatory postsynaptic currents, pyramidal cells were subjected to a period of 30 minutes of ischemia, which was induced by replacing artificial cerebrospinal fluid containing glucose 10mM by sucrose 7mM/ glucose 3mM (not a complete deprivation of glucose). After this period, cells were placed in aCSF (glucose 10mM) in the recording chamber of the patch clamp setup (beginning of the recovery process). A cell from the CA1 region was chosen and after establishing the whole cell configuration, mEPSCs started to be recorded. The sodium channel blocker, tetrodotoxin (TTX, 0.5 µM), was applied to prevent action potential generation, and thus, neuronal communication. Gabazine (2 M), a selective GABA receptors antagonist, was added as well, in order to isolate the excitatory component of synaptic transmission, and usually after a period of around 15/20 minutes, stable baseline recording was achieved. After this interval, slices were perfused with Erythropoietin (2.4 IU/ml) contained in aCSF solution, for

a period of 40 minutes. In the end of EPO incubation, it was initiated a 15 minutes period of washout (Figure 26).

Figure 26. Protocol used to record the miniature excitatory post synaptic currents (mEPSCs). Pyramidal Cells were exposed to ischemia (induced in vitro by replacing 10mM glucose containing aCSF with that containing 7 mM sucrose and 3 mM glucose, gassed with 95%N2/5%CO2 for 30 min). After the insult, cells were placed in the recording chamber in the patch clamp setup. The experiments were conducted in the presence of aCSF supplemented with TTX (0.5 µM) to block action potential generation, together with gabazine (2µM), a selective GABA receptors antagonist, which acted during 15 min, upon achieving a stable baseline. Afterwards, slices were incubated with EPO (2.4 IU/ml) for 40 minutes, period after which the period of washout was initiated.

Under these experimental conditions, EPO administration caused a significant decrease in the frequency towards the control baseline level (0.14±0.01 Hz to 0.09±0.01 Hz, measured 30-40 min after EPO application, n=7, *ρ= 0.0163, Figure 27), which shows evidences of an inhibitory effect on glutamate release. EPO application did not affect significantly the value of mEPSC amplitude, which suffered only a slight decrease (n=7, ρ=0.5600). In absolute values, the mEPSCs amplitude was 13.9±1.3 pA in the baseline period and 12.9±1.1 pA, after the presence of EPO in the superfusion solution.

Figure 27. Administration of EPO decreases the frequency, but not the amplitude, of spontaneous miniature excitatory postsynaptic currents (mEPSCs). (a) Representation of mEPSCs tracings from a CA1 pyramidal cell, that has been previously exposed to ischemia, in the absence (top trace) and presence (bottom trace) of the cytokine EPO (2.4 UI/ml). b) Representative average tracings of mEPSCs of two superimposed events in the absence (1) and presence (2) of the erythropoietin (2.4 IU/ml), for a representative cell. In all experiments, it was used a sodium channel blocker, tetrodotoxin (TTX, 0.5 mM) and gabazine (2 mM), so that only the mEPSCs resulting from a spontaneous transmitter release would be recorded. These average tracings were obtained, analyzing two 10 min periods, one immediately before addition of EPO and the other corresponding to the final 10 min period recorded in the presence of this cytokine. (c) Column graphs representing the averaged mEPSC frequency (left) and amplitude (right) recorded from each cell 30-40 min after EPO administration (black column) or during a period of 10-15 min of baseline (absence of EPO, white column), indicated below each data set. This data set is the same as in Figure 25. EPO administration caused a significant decrease in the frequency towards the control baseline level (0.14±0.01 Hz to 0.09±0.01 Hz, measured 30-40 min after EPO application, n=7, *ρ= 0.0163). Values are mean±SEM. * p<0.05 and n.s. p>0.05 (two tailed unpaired Student’s t-test, compared with control experiments, using absolute frequency current values).

Role of Erythropoietin in modulation of mIPSCs, in normoxic conditions 6.4

A proper neuronal transmission requires a regulated balance between excitatory and inhibitory signals. Therefore when studying the effect of a drug in the CNS, it is always important to analyze both components of synaptic transmission. Not to mention, that there are only a few studies addressing the effect of EPO upon GABAergic transmission. For instance, Wojtowicz and Mozrzymas (2008) investigated if long-term EPO treatment of hippocampal neurons developing in vitro affected GABAergic transmission. Therefore, they analyzed GABAergic miniature postsynaptic currents and they reported that, within a restricted window of time, EPO could induce significant changes in mIPSC time course. Ramanathan and his colleagues, in 2012, induced transient focal ischemia in rats, by using a middle cerebral artery occlusion model (MCAo), in order to investigate the role of endogenous GABA against excitatory neurotransmitters in different regions during ischemia. The results showed that during ischemia there is an extracellular accumulation of GABA, which might be considered as a defense mechanism in the neuronal cells to antagonize accumulation of the excitatory neurotransmitters in the ischemic status, even though this phenomenon did not give full protection to the neuronal cells. Głodzik-Sobańska et al., in 2004, reached similar results. Their experimental studies have suggested that in response to the excessive accumulation of excitatory neurotransmitter in the extracellular space due to ischemic stroke, results in a compensatory concentration of gammaaminobutyric acid (GABA) in order to inhibit its excitotoxic effect. In fact, in a situation of excessive glutamate release it seems that EPO and GABA have similar goals. Therefore, I decided to perform also a set of experiments, in which I would evaluate changes in the frequency and amplitude of miniature inhibitory postsynaptic currents caused by EPO perfusion. These experiments were performed in aCSF supplemented with TTX (0.5 µM) and Kynuretic Acid (KA, 1mM) to prevent action potential-driven synaptic transmission and contribution of glutamatergic transmission, respectively. This way, only the mIPSCs resulting from a spontaneous transmitter release were recorded. In this set of experiments, EPO effect on hippocampal pyramidal cells was tested directly by perfusing the slices with aCSF-containing EPO (2.4 UI/ml). In other words, after 15/20 min of superfusion incubation with TTX and KA, it was usually achieved a stable baseline, a period after which Erythropoietin was added to the slices, for 40 minutes. A 15 minutes period of washout was initiated, after the end of EPO administration (Figure 28).

Figure 28. Protocol used to record the miniature post synaptic currents (mIPSCs). The experiments were conducted in the presence of aCSF supplemented with TTX (0.5 µM) to prevent action potentials and Kynuretic Acid (1mM), an antagonist of glutamate receptors (AMPA, NMDA and kainite receptors), which acted during 15 min until achieving a stable baseline. Subsequently, slices were incubated with with EPO (2.4 IU/ml) for 40 minutes, period after which the period of washout was initiated.

The results show an increase in the mIPSCs frequency of 2.4±0.09Hz (n=6, *ρ=0.0480, Figure 29a), compared with the baseline (1.99±0.15Hz, n=6), but not in the amplitude, which remains roughly constant (36.00±3.7pA, n=6) upon EPO administration (36.02±3.0pA, n=6, ρ=0.2367, Figure 29b). Figure 29c and d illustrates the enhancement of mIPSCs frequency, as a result of EPO application, of a representative experiment, as well as, the mIPSCs amplitude, which was not affected.

Figure 29. EPO administration increases the frequency, but not the amplitude, of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). a) Column graphs representing the averaged mIPSC frequency (left) and amplitude (right) recorded from each cell 30-40 min after EPO administration (2.4 IU/ml, black column) or during a period of 10-15 min of the baseline period (absence of EPO, white column); b) Representative time course changes in mIPSC frequency (left ) and amplitude (right), in which each point corresponds to the average of individual macroscopic responses to spontaneous glutamate release, every 2 minutes, in a representative cell; c) Representative tracings of mIPSCs of two superimposed events in the absence (1) and presence (2) of the erythropoietin (2.4 IU/ml), for a representative cell. It is important to mention that the sodium channel blocker, tetrodotoxin (TTX, 0.5mM) and the kynuretic acid (KA, 1mM) were present throughout recordings of these spontaneous events; d) Average time-course of normalized current frequency changes caused by 2.4 UI/ml EPO. Values are meanÂąSEM. * p<0.05 and n.s. p>0.05 (two tailed unpaired Studentâ&#x20AC;&#x2122;s t-test, compared with control experiments, using absolute frequency current values).

7. General Discussion and Future Work Investigation of the modulatory effect of erythropoietin upon both inhibitory and excitatory synaptic transmission is of great importance, in order to fully understand if EPO can be used as a therapeutic treatment in brain damage. Therefore, to date, most of studies have focused their attention on EPO neuroprotective role in hypoxia and cerebral ischemia. The results obtained in my experiments might contribute to explain the mechanism of action of erythropoietin, when delivered after exposure to ischemia, and therefore in conditions that mimic clinical trials.

7.1

Membrane capacitance increases after ischemia

The study of the membrane capacitance has proven to be a powerful approach to understand the electrical properties of neurons. The values obtained for Cm for pyramidal neurons membrane (Table 3) were estimated in two distinct conditions: in ischemia and in a control situation, in order to see if this insult was responsible for producing changes in the electrical properties of the neurons. During a stroke episode, due to the decrease of the intracellular +

concentration of ATP, the activity of Na /K pump is affected and membrane potential value is compromised. The neuronal membrane depolarizes, triggering a cascade of events that can lead to cell swelling (Choi, 1987; reviewed in McEntee and Crook, 1993), which varies the cell volume. And changes in the cell volume, may lead to alterations in the value of capacitance. In fact, it is usually accepted that when determining the membrane capacitance, one is indirectly measuring the cell volume (Chi and Xu, 2000). The comparison of the capacitances in the two distinct conditions (Figure 24) demonstrates that cells, that suffered ischemia for 30 minutes, presented a significant increase in the value of the capacitance. There are few articles mentioning the effects of ischemia in the value of membrane capacitance. In 1995, Graf and his colleagues used whole-cell patch-clamp technique to study changes in membrane conductance and membrane capacitance, after having induced osmotic swelling in rat hepatocytes. The purpose of their study was to see if the rise in the values of membrane conductance was accompanying with changes in membrane capacitance, which they observe not to be the case. However, after osmotic swelling they estimated the value of specific capacitance of liver cells (assuming they were spheres) and they obtained a value of ~3 ÂľF/cm

(cell radius, 9-12 Âľm; total membrane capacitance, ~50 pF), which substantially 2

exceeded the value for biological membranes (1 ÂľF/cm ). Therefore, they suggested that changes in cell volume due to edema affect the capacitance membrane of rat liver cells. It is important to mention that in the work herein reported the surface area determined for each cell to achieve the specific capacitance was not different in cells in the ischemia group or in the control group. In fact, they were similar, which means that the increase in the specific capacitance was not influenced by area values. Therefore, based on my results, I can state that I could successfully evaluate changes in membrane capacitance in cells that suffered an ischemic insult, even in conditions where cell volume was not markedly changed. Subtle

changes in passive biophysical properties of cell could therefore be used to quantify early signs of alterations in neuronal function. Some of the values of capacitance in hippocampal pyramidal neurons, although with the same 2

order of magnitude, do not fit in that range 0.5-1 ÂľF/cm (Solsona et al., 1998; Gentet et al, 2000; Golowasch et al., 2009). These small discrepancies may be related with some inaccuracies in the determination of cell dimensions to calculate the area. The dimensions were measured directly during the electrophysiological recordings, using a television screen, which is not completely accurate. Another possible explanation for the reported differences may be associated with the different protein content of cells. Neuronal membrane has a low density of voltage-gated channels, but it might contain other proteins that may alter the value of C m, including a significant density of ligand-gated receptors (Thurbon et al., 1998). In some studies, it was demonstrated that EPO application after brain injury resulted in a significantly reduced infarct cell volume (Gunnarson et al., 2009; KrĂźgel et al., 2010), which might be related with EPO neuroprotective role. Cellular edema is associated with excessive glutamate release, due to an increase in Na+ entrance in the cells. Thus, since EPO acts on glutamatergic synaptic transmission, it is possible that when decreasing glutamate release (which decreases cell depolarization), this hormone acts on neuronal swelling, reducing it. Based on the results of the studies previously mentioned, and on data obtained by me, it would be interesting to measure in the future the capacitance values after EPO administration, and compared those with the ones obtained in the absence of EPO, either under normoxic or hypoxic conditions. Those results could elucidate a bit more about the ability of EPO in protecting brain cells.

7.2

Ischemia decreased mEPSC frequency

Another important aspect of this present study is related with the differences in frequency of glutamate release between cells that were subject to ischemia, and cells that did not suffer the insult. It was possible to observe that the frequency of the mEPSCs recorded after 30 minutes of ischemia was greatly reduced, comparatively to the frequency of cells that were not subjected to energy deprivation. Studies investigating the spontaneous firing rate of CA1 neurons after transient ischemia have yielded conflicting results. Thus, Furukawa and his colleagues, in 1990, exposed male Wistar rats to ischemia of 5 or 20 min duration, after bilaterally occluding their vertebral arteries. Spontaneous action potentials were recorded, indicating that the function of CA1 neurons was not affected by 5 min of ischemia, but it was significantly depressed after 20 min. Similar conclusions were reached by Xu and Pulsinelli (1994). They analyzed electrophysiological responses of CA1 pyramidal neurons to 5 min forebrain ischemia (an occluding device was placed around each rat carotid artery), and they reported that spontaneous synaptic activity was greatly reduced within tens of seconds after the onset of ischemia. Nevertheless, in Chang et al. (1989), they reported that neurons in the CA1 region

showed marked increases in firing rate 2 and 3 days after 10 minutes of transient cerebral ischemia, induced by clamping of both carotid arties combined with hypotension. It was considered that the excessive release of glutamate could be associated with some metabolic or other intrinsic disorder that affected neurons in a way that they could no longer maintain their normal resting potential. The CA1 region is known to be particularly vulnerable to oxygen and glucose deprivation (Cotman et al. 1987; Furukawa et al., 1990; reviewed in Paschen, 1996), and thus after a brief period of cerebral ischemia neurons are selectively damaged, which eventually will lead to their death. Figure 25 illustrates the significant decrease in the frequency of pyramidal cells that suffered the insult, which means that CA1 neurons were, in fact, quite affected by 30 min of ischemia. The reduced frequency observed in my experiments brings out another important point, the synaptic plasticity in ischemia, more specifically Long Term Potentiation (LTP). LTP is an increase in synaptic strength induced by high-frequency stimulation of a chemical synapse and it has been regarded as cellular substrate for memory and learning processes (Malenka and Nicoll, 1999). Recently, several studies have reported that hippocampal CA1 pyramidal neurons may respond to acute energy deprivation by triggering pathological forms of synaptic plasticity, which is probably associated with the fact that most of the molecular processes involved in the induction or maintenance of physiological LTP are similar to those activated during excitotoxicity. Among these processes, the increase in intracellular calcium levels during the induction phase is a basic requirement for both forms of synaptic plasticity (Calabresi et al., 2002; Calabresi et al., 2003). In short, physiologic LTP is mainly triggered by activation of postsynaptic NMDA receptors, which is accomplished through repetitive tetanic stimulation of 2+

synapses. As a consequence, cells suffer strong depolarization, which results in Mg

dissociation from its binding site within NMDA receptor, allowing calcium ions influx ions to the cell. The rise of Ca

is the critical trigger of LTP (Malenka and Nicoll, 1999).

Excessive increase in intracellular levels of calcium is also observed in glutamate excitotoxicity and ischemia-induced neuronal death (reviewed in McEntee and Crook, 1993; Mark et al., 2001). Therefore, it makes sense that when exposed to ischemia cells tend to induce synaptic plasticity, a pathologic type of LTP, whose function is still not completely addressed. This type of LTP is only induced if the period of energy deprivation is short. For a case, in which the period of energy failure is longer than a few minutes, such as it happens in my experiments, disruption of ionic homeostasis occurs, resulting in irreversible membrane depolarization and neuronal swelling (Calabresi et al., 2003). In my study, the hippocampal cells were deprived from oxygen and glucose for a period of 30 minutes, which probably strongly disable several cells (reflected by the reduced frequency of spontaneous activity), and consequently the ability of inducing plasticity.

7.3

Erythropoietin decreases the frequency of mEPSCs after ischemia

Since it was discovered that EPO and its receptor were produced in the CNS (Genc et al., 2004), several studies have been undertaken to clarify the impact of the expression of EPO and its receptor, EPOR, in the brain (Buemi et al., 2002; Noguchi et al., 2007). Most of the studies have investigated the role of EPO, using animal models of nervous system injury, after focal and global cerebral ischemia episode (Bernaudin et al., 1999; Brines et al. 2000), which makes sense, because one might be dealing with a possible therapeutic drug to reduce the harmful effects of a stroke. EPO is known for reducing the frequency of glutamatergic transmission in normoxic conditions (Adamcio et al., 2008; Kamal et al., 2011). In my studies, cells were exposed to an OGD environment before evaluating excitatory synaptic transmission, despite the fact that it is known that ischemia reduces significantly, by itself, glutamatergic transmission frequency (Figure 25). The study of basal synaptic transmission in hippocampal cells after ischemia, allows to mimic the effect of EPO in the penumbra region, in vivo. Regarding the results obtained in this set of experiments, it was possible to observe that EPO has an effect on the excitatory neurotransmitter release, decreasing the frequency of mEPSCs, which supports a presynaptic effect of EPO in glutamatergic transmission. In Kamal et al. (2011) study, in which it was not induced ischemia, they investigated the role of EPO in hippocampal slices and they reported that erythropoietin had a direct effect on the excitatory neurotransmitter release probability. Although, using a different protocol, their study suggests that perfusion of hippocampal slices with EPO (50 U/ml) affects the excitatory transmission by reducing glutamate release. The average responses of recorded evoked field excitatory postsynaptic potential from the dendritic layer, in the last minutes of the experiments, were significantly lower than the average responses before the perfusion with EPO. Paired pulse facilitation (PPF) was also measured before and 60 minutes after the perfusion of the slices with EPO, reflecting a reduction in the transmission release probability, after EPO administration. By comparing these data with my results is related to the fact that it appears that the action of EPO on glutamate transmission is independent of ischemia induction. Indeed, EPO seems to affect presynaptic regulation of glutamate release both in basal conditions and after ischemia. Since my study is based on spontaneous synaptic transmission, it is not possible to assure that EPO has indeed a neuroprotective effect in the brain. Yet, it is possible to speculate about a potential neuroprotective role of erythropoietin in the brain, in which reducing the glutamatergic transmission may be one of the mechanisms by which this cytokine protects the synapses from toxic levels of glutamate (the phenomenon of excitotoxicity). In my experimental conditions, EPO induced only a slight decrease in mEPSC amplitude (no significant effect was verified), visible in Figure 27c (left). This small variation might result from an indirect effect due to frequency changes, instead of being a direct effect of EPO on the postsynaptic glutamate receptors. In Adamcio et al. (2008), it was reported that EPO lead to a

significant decrease of both amplitude and frequency of the recorded excitatory postsynaptic currents. Also, Weber et al. (2002), observed alterations in evoked extracellular ďŹ eld potentials (FP) amplitudes, after EPO application. These discrepancies may be related with the used protocol. In Adamcio et al. (2008), the experiments were performed with 28 days old mice, which were injected intra-peritoneal with EPO or placebo (diluent for EPO), every other day for three weeks. And the whole-cell patch clamp recordings were performed only one week after the last injection. Besides, once again, in this study, cells did not suffer an ischemic insult. Concerning Weber et al. (2002) study, it was shown that EPO treated cultured slices (40units/ml for 48 h) signiďŹ cantly decreased FP amplitude, during and following oxygen and glucose deprivation, when compared with untreated control slices. The hippocampal slice cultures were pre-incubated with EPO for 48h, before electrophysiology recordings. In my experiment, in contrast, EPO was contained in an aCSF solution and it was directly administered to the hippocampus slice for a period of 40 minutes (acute perfusion), instead of directly administrated in the animal. The findings here reported that EPO decreases excitatory synaptic transmission in slices that were exposed to a long period of ischemia. This fact might suggest that EPO could be useful to prevent the excessive plasticity pointed out by Calabresi et al. (2003), when cells suffer a brief ischemic episode. It would be interesting to do a set of experiments to observe the effect of EPO after a small period of ischemia, when glutamate release in the extracellular space presents toxic levels.

7.4

Erythropoietin enhances the release of GABA, in normoxic conditions

GABA is an inhibitory neurotransmitter in the CNS, and therefore it reduces neuronal firing and consequently their excitability. It is known that suitable neuronal transmission requires a regulated balance between excitatory and inhibitory signals. Therefore it is important, when studying the effect of a drug in the CNS, to observe changes in both excitatory and inhibitory neurotransmitter release. Wojtowicz and Mozrzymas (2008) demonstrated an effect of EPO on GABAergic transmission in developing hippocampal neurons in culture. The analysis of mIPSCs revealed that 48 or 72 h treatment with Epo (40 U/ml) results in a major acceleration of the decaying phase of those currents. This study was made in postnatal day 1-3 Wistar rats and the results indicated that EPO may exert a modulatory action on GABAergic transmission in developing neural networks. Since there are only a few articles studying the interaction between EPO and GABA, I proposed to test if EPO would change the frequency and/or amplitude of miniature inhibitory postsynaptic currents. The results obtained in my experiments, concerning GABAergic transmission, demonstrate that mIPSCs frequency in CA1 pyramidal neurons increased after acute EPO administration, whereas the amplitude remained unchanged. Data from patch clamp recordings from previous studies showed evidences that prolonged treatment with EPO facilitates GABA

release, increasing the frequency of spontaneous inhibitory postsynaptic currents (IPSC) in CA1 in pyramidal neurons, but with no change in the amplitude (Adamcio et al, 2008). In that study, young (28 days old mice) were injected intra-peritoneal every other day for 3 weeks with EPO (5 IU/g in 0.01 ml) or placebo (diluent for EPO, 0.01 ml/g) and a direct effect of EPO in synaptic transmission was tested by performing patch clamp recordings at CA1 hippocampal pyramidal neurons, at 1 week after the cessation of treatment. Although Adamcio et al. (2008) used a protocol different from mine (they used prolonged-EPO treatment and I tested the effect of acute EPO application), both sets of data are in accordance, reinforcing the conclusion that EPO has a presynaptic and a facilitatory effect on GABAergic transmission. As I previously mentioned, it is thought that in response to the excessive accumulation of excitatory neurotransmitter in the extracellular space, there is a compensatory increase in the release of gamma-aminobutyric acid (GABA) in order to inhibit the excitotoxic effect (GĹ&#x201A;odzikSobaĹ&#x201E;ska et al.,2004). It appears that EPO and GABA have both a neuroprotective role against excitotoxicity, and therefore it makes sense that through EPO administration, GABA release increases in the brain. For an appropriate neuronal transmission, a regulated balance between inhibitory and excitatory signals is indispensable. Besides it is thought that an increase in glutamate release is followed by an increase in GABA transmission, in order to establish brain synaptic homeostasis (Turrigiano and Nelson, 2004). Therefore, in order to guarantee that the effect of EPO on GABAergic currents was independent of excitatory inputs arriving to pyramidal cells, as well as, from glutamatergic transmission mediated by AMPA or NMDA receptors, these experiments were made in the presence of TTX, which prevented the firing action potentials, as well as with kynurenic acid, an antagonist of AMPA, Kainate and NMDA receptors. Therefore, the changes observed in the frequency of mIPSC, under these conditions, demonstrate a presynaptic effect of EPO upon spontaneous GABAergic currents, free from any excitatory contamination. Although GABA has received until now relatively little attention in the area of cerebral ischemiainduced neuronal death, the GABAergic system may be of particular importance, since it functions in opposition to that of glutamate (Schwartz-Bloom and Sah, 2001). Ramanathan and his colleagues (2012) observed that depolarization caused by ischemia leads to an extracellular accumulation of GABA in different brain regions. Thus, it could be relevant to test if the facilitation action of EPO upon GABA release still holds in ischemia injured neurons. Another study addressing the effect of ischemia in GABAergic transmission, reported that the GABAergic interneurons are more resistant to ischemia than pyramidal cells (Furukawa et al., 1990). Therefore, it would be interesting to study GABA release from interneurons subjected to a period of 30 min of ischemia, in order to see if the frequency of spontaneous events would be as affected as the frequency of pyramidal cells was. Not to mention that it could be also important to investigate the effect of acute EPO perfusion in interneurons in basal transmission, as well as, after OGD. These experiments

8. Conclusion The present results provide evidences that EPO has a direct modulatory role on both excitatory and inhibitory neurotransmitter release. In glutamatergic transmission, EPO has an inhibitory effect, decreasing spontaneous glutamate release, even in cells that had been exposed to ischemia for a 30 minutes period. My experiments also demonstrated that hippocampal CA1 pyramidal cells are easily damaged by long periods of ischemia, and in fact OGD may induce changes in membrane electrical properties, which is observed by the differences in membrane capacitance values between cells that did and did not suffer ischemia. EPO role in GABAergic transmission was also evaluated, only in basal transmission, and the results obtained showed an increase in mIPSC frequency 30-40 minutes after EPO application. All the results seem to indicate that EPO might have a crucial neuroprotective role in the CNS. In conclusion, further experiments have to be performed to fully understand the mechanisms underlying behind the action of Erythropoietin. Still, this cytokine has a relevant impact in the pathophysiology of insulted neuronal cells, which definitely opens new frontiers in the treatment of diseases of the CNS.

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