J RODRIGUES - Master Thesis Publication

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

Joana Catarina Alves Rodrigues – IST/IMM Raquel Alice da Silva Baptista Dias – IMM/FML Paulo Jorge Peixeiro de Freitas – INESC Ana Maria Ferreira de Sousa Sebastião – IMM/FML November, 2012 Abstract Brain cells are highly vulnerable to ischemia and excessive glutamate release induced by this insult has been postulated as a principle cause of selective neuronal damage. It became therefore imperative nowadays to explore new therapeutic agents that possess neuroprotective properties against ischemic brain injury.

Erythropoietin (EPO) is considered as a potential candidate in the processes of brain development and repair. In this work the role of EPO was evaluated by monitoring both miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively). Changes in neuronal swelling were also assessed by observation of alterations in neuronal biophysical properties, such as the membrane capacitance. The values of membrane capacitance were increased after a brief period of ischemia, when compared with control cells (from 0.49±0.05µF to 0.35±0.03µF). In addition, cells expose to ischemia suffered a significant depression of mEPSCs frequency, when compared with control slices (0.45±0.11Hz versus 0.14±0.01Hz), showing evidences of the vulnerability of pyramidal cells to ischemia. EPO administration (2.4 IU/ml) induced a significant reduction in the frequency of mEPSCs (0.14±0.01Hz to 0.09±0.01Hz). Regarding the GABAergic transmission, EPO application resulted in a significant increase of mIPSCs frequency (2.00±0.15Hz to 2.4±0.09Hz). In conclusion, EPO acts on both neurotransmitters release, which might be associated with its neuroprotective role.

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

and excessive release of the excitatory neurotransmitter glutamate occurs.

Glutamate is the main excitatory neurotransmitter in the Central Nervous System (CNS), having an essential role in cognitive functions, such as learning and memory.

Nevertheless it is considered that higher concentrations of glutamate in the extracellular space and the consequent excessive activation of its receptors results in neuronal excitotoxicity and consequently cell death. The mechanisms by which the excessive release of glutamate leads to neuronal injury remain unknown. However it is thought that glutamate excitotoxicity is dependent on extracellular Ca2+. Excessive glutamate release induces Ca2+ influx into the cell, which activates multiple signalling pathways that contribute to neuronal cell death (reviewed in McEntee and Crook, 1993). Other studies showed, however, that glutamate neurotoxicity was also relate with the release of Na+ and Cl- ions. Through the activation of ionotropic receptor, glutamate opens the

1. Background 1.1 Ischemia and Synaptic Transmission 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 and glucose due to a restriction in blood supply (ischemia) for a period of few minutes, the tissue ceases to function, and after approximately three hours, it will suffer irreversible injury, leading to necrotic death (Schaller and Graf, 2002). The lack of oxygen and glucose results will affect ATP production, which in turn will not be enough to achieve all of brain energy-dependent processes essential for tissue survival. For instance, ATPdependent maintenance of the resting potential will be compromised since resting membrane potential is established by the sodium potassium pump (Na+/K+-ATPase). Cells will suffer depolarization 1

membrane Na+ conductance, leading to a large influx of Na+ ions. As a result, the membrane suffers depolarization, and a secondary passive influx of Cl- ions and water is observed, resulting in neuronal swelling (edema) (reviewed in McEntee and Crook, 1993) (Figure 1).

possibility that EPO could also be associated with other biological functions, particularly as a protective factor in the brain.

1.2.1 Neuroprotective Role Research during the last years has demonstrated that EPO has the potential to promote neuronal survival. Both EPO and its receptor are expressed in the nervous system (Digicaylioglu et al, 1995) which provided early evidence for potential activity of EPO in the brain. It was observed significant alterations in the expression of EPO and EPOR in regions within and around infarcts in brains of animal models 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), which can be related with the fact that EPO decreases excitatory neurotransmitter release probability (Kamal, 2011). Preconditioning of hippocampal slices cultures with EPO improves neuronal synaptic transmission during and following oxygen and glucose deprivation (Weber, 2002). In 2002, Ehrenreich et al. (2002) reported that recombinant human EPO (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) determined in his experiments that systemically administered rhEPO crossed the Blood Brain Barrier, 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 long-term potentiation; thus it could be potentially useful in cases of cerebral hypoxiaischemia, where there are long-term spatial memory deficits. It was recently demonstrated that, among other actions, EPO has an acute effect upon glial cell swelling (Krügel et al., 2010), a characteristic feature of the penumbra region, which is thought to further restrict substrate delivery and aggravate neuronal damage. In fact, experimental glial swelling induced by osmotic challenge was found to be significantly reduced by acute EPO application, through the release of Vascular Endothelial Growth Factor (VEGF) (Krügel et al., 2010).

Figure 1. 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 Ca2+, Na+, Cl- levels.

Water enters into the intracellular space, via osmotic gradients, resulting in cell swelling (edema). The messenger Ca2+ activates numerous enzyme systems. Free radicals are also generated, damaging the membrane and the DNA, and consequently leading to cell death.

Ischemia also affects the GABARergic transmission. GABA is the main inhibitory neurotransmitter in the mammalian CNS, whose role consists of inhibiting the influx of Ca2+ ions into the cell, decreasing the excitability of the neuron. 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).

1.2 Erythropoietin Recently, some studies have reported that some cytokines have a neuroprotective role against inflammatory responses related to cerebral ischemia (Arvin et al., 1996; Bartesaghi et al., 2005), such as Erythropoietin (EPO). EPO is responsible for the survival, proliferation and differentiation of erythroid precursor cells, a process known as Erythropoiesis (reviewed in Bartesaghi et al., 2005). During several years it was believed that EPO did not possess biological functions aside from the regulation of the erythropoiesis. However, some studies reported that EPO and its receptor (EPOR) could be found in other tissues, which are not directly involved in the regulation of red blood cell production (Genc et al., 2004), raising the 2

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 (Wang et al., 2004).

The membrane potential of the cell, Vp, is measured and compared to the command potential, Vref. 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. The injected current measured during a patchclamp resembles the current flowing through the cell membrane, although with opposite polarity (Odgen and Stanfield, 1994).

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. There are several possible configurations, however the most widely used one is the whole-cell configuration, since it enables the measurement of macroscopic currents flowing the membrane of the entire cell. Briefly, a glass pipette (1Âľm diameter) is approached to the cell membrane, after which it is applied mild suction in order to obtain a tight seal between the pipette and the cell membrane.

Subsequently, more strong pulse of suction is applied in order to pull the patched membrane away, providing total access of the intracellular space of the cell.

The most important component of a patchclamp setup is the current-to-voltage amplifier, placed in the headstage (Figure 2). This amplifier also acts as a differential amplifier, operating to make the output equal to the difference between the two inputs. In short, 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 Ohm’s Law.

3. Capacitance The cells membrane is composed of a lipid bilayer (8–10 nm thick), which acts as a leaky capacitor. The capacitance per unit area of membrane is referred to as specific capacitance (Cm). This parameter allows to understand cells electrical behavior (Gentet et al., 2000) and it is an indirect measure of cells volume (Chi and Xu, 2000). Membrane capacitance can be calculated using capacitive transients, obtained as a response from the application of a voltage step to the cell.

The amplitude, decay time constant, and steady state level of this transient can be analyzed to estimate the series resistance of the recording pipette (Ra), the patch (or cell) membrane conductance (Gm), and membrane capacitance (Cm).

(1)

(2) (

),

(3)

where Vstep is the amplitude of the voltage-clamp step, I0 is the peak amplitude of the current transient immediately after the step is applied, is the decay time constant of the current, and Iss is the steadystate current at longer times following the step. The parameters, , I0, and Iss were determined by fitting a single exponential function to the current transient recorded during the voltage step.

Figure 2. Diagram of the headstage patch clamp amplifier.

The amplifier gain is set by the value Rf and is given by . The membrane potential of the cell, , is measured and compared to the command potential, Vref.

The voltage output is adjusted by the amplifier, in order to maintain a constant pipet potential at the desire Vref (Cartoon based on a drawing by Odgen and Stanfield, 1994).

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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), with an osmotic interval 280–290 mOsm. For mIPSCs, the internal solution is composed by (in mM): Kgluconate 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 voltageclamp mode (Vh=-70 mV) set up by an Axopatch 200B (Axon Instruments) amplifier. The offset potentials were nulled before the giga-seal formation. Firing pattern was usually determined at the beginning of each experiment, through a 500 ms depolarization step 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.

4. Methods 4.1 Animals 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.

4.2 Brain slices Preparation The rats were sacrificed by decapitation under deep isofluorane anesthesia. Their brains were quickly removed and placed in an ice-cold solution.

The two hippocampi were then dissected and cut on a Vibratome (VT1000 S; Leica, Germany) to obtain transverse slices (300μm). 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. First, slices were incubated at 35°C for a period of 30 minutes, in artificial cerebrospinal fluid (aCSF), containing 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, followed by a recovering period for at least 1 hour, at room temperature.

4.4 mEPSC Recordings 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), to block voltage-gated sodium channels, preventing the propagation of action potentials in nerves, and Gabazine (Gabazine, 2 µM), an antagonist of GABA receptors. After approximately 15-20 min, Erythropoietin (EPO, 2.4 IU/ml) was added to the solution containing already TTX and Gabazine.

4.3 Whole-cell patch clamp-recordings All recordings were performed in pyramidal cells located at CA1 stratum pyramidale. The recordings were made at room temperature and 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. EPO was then added to the superfusion solution, reaching the recording chamber within approximately 1 min. 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) and characterized by a resistance of 4–7 MΩ, when filled with an internal solution, which depended on the type of currents that were being recorded.

4.5 mIPSC Recordings mIPSCs, responses resulted from the spontaneous release of GABA by interneurons, were recorded in aCSF solution, to which was added Tetrodoxin (TTX, 0.5 µM) and Kynurenic Acid (KA, 1 mM), an antagonist of ionotropic glutamate receptors. Erythropoietin (EPO, 2.4 IU/ml) was added to the solution after approximately 15-20 min of stable baseline.

4.6 Ischemia induction Pyramidal cells were subjected to 30 min of ischemia, which was induced, by replacing 10mM glucose-containing aCSF with one containing 7mM sucrose (3mM glucose), gassed with 95%N2/5%CO2 (Rossi et al., 1999).

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4.7 Data analysis

5. Results

Analysis of mEPSCs and mIPSCs were performed with Mini analysis software. The data was sampled at 5 kHz and filtered using a low-pass Gaussian Filter (600Hz with a -3dB cut-off).

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.

5.1 Specific Capacitance increases due to ischemia Neuronal 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. The purpose of these experiments was to evaluate the influence of ischemia in neuronal electrical behavior, by measuring membrane capacitance.

The average transient from each membrane cell was fit with a single exponential function, and the time constant and amplitude parameters were used to estimate Ra, Rm, and Cm (see Materials and Methods). The value of Cm was divided by the measured surface area of the nucleated patch to calculate C. 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).

The values obtained for the specific membrane capacitance C were analyzed and compared, so that it would be possible to detect differences in ischemic and control cells. Figure 3 shows evidences of a significant increase in the specific membrane capacitance of cells previously exposed to ischemia (0. 49±0.05 µF, n=8, ρ*=0.0461), when compared to the control baseline level (0.36±0.03 µF, n=8, ρ*=0.0461).

4.8 Measurement of Capacitances 500 ms hyperpolarizing square pulses of 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 Graph Pad Prism, the average transients, in response to the stimulation, were fit with an exponential (one phase) decay with the use of an iterative sum-of-squares minimization algorithm according to ( )

(

)

(

)

(4)

The parameters obtained by the fitting curve expression were used to estimate Ra, Gm, and Cm, using equations (1), (2) and (3). 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 cell, 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 (expression (5)). 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). (

) ( )

Figure 3. Capacitance is increased after 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 glucosecontaining 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).

(5)

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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; Weber et al., 2002). In my experiments, I address the EPO effect on basal excitatory synaptic transmission after CA1 pyramidal cells had suffered an ischemic insult, in order to mimic what happens in a clinical situation.

The effect of EPO perfusion on hippocampal slice, after 30 minutes of ischemia, is shown in Figure 5. The averaged mEPSC frequency after EPO application was significantly lower than the averaged frequencies in baseline (0.14±0.01Hz to 0.09±0.01Hz, measured 30-40 min after EPO application, n=7, *ρ=0.0163). The mEPSC amplitude suffered only slight decrease (13.9±1.3 pA to 12.9±1.1 pA, measured 30-40 min after EPO application n=7, ρ=0.5600).

5.2 mEPSCs frequency was suppressed by ischemia 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 neuronal condition becomes irreversible (Xu and Pulsinelli, 1994; Tanaka et al., 1997). Several studies have been done to evaluate the effect of an ischemic insult upon glutamatergic transmission, which had yielded to conflicting results. Using extracellular techniques in vivo, some studies showed that the spontaneous firing rate in CA1 hippocampus was increased following ischemia (Chang et al., 1989) whereas others reported that spontaneous neuronal activity and evoked responses were suppressed after ischemia (Furukawa et al., 1990).

A set of experiments was performed to compare the average mEPSCs frequency registered from ischemic cells with control cells. The averaged frequency of cells expose to ischemia was significantly lower (0.14±0.01Hz, n=7, *ρ=0.0163, Figure 4), when compared to the frequency of cells in the control group (0.45±0.11Hz, n=7).

a)

b)

Figure 4. 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. 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).

Figure 5. Administration of EPO decreases the frequency, but not the amplitude, of 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) Column graphs representing the averaged mEPSC frequency and amplitude 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. EPO administration caused a significant decrease in the frequency towards the control baseline level (0.14±0.01Hz to

5.3 EPO decreases mEPSC frequency, but not their amplitude Several studies demonstrate that EPO has the ability of protecting nerve cells from glutamate toxicity, decreasing the excitatory synaptic 6

0.09±0.01Hz, 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).

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.

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).

5.4 EPO enhances mIPSCs frequency This set of experiments performed in mIPSCs allowed the study of the effect of EPO in basal GABAergic transmission. The results show an increase in the mIPSCs frequency of 2.4±0.09Hz (n=6, *ρ=0.0461, Figure 6a), compared with the baseline (2.0±0.15Hz, n=6), but not in the amplitude, which remained roughly constant (36.00±3.7pA, n=6) upon EPO administration (36.02±3.0pA, n=6, ρ=0.9962, Figure 6a). Figure 6b illustrates the changes in mIPSCs frequency and amplitude, as a result of EPO application, in a representative experiment.

6. Discussion and Future Work 6.1 Specific Capacitance increases after ischemia The study of 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 were estimated in two distinct conditions: in ischemia and in control situation, in order to see if ischemia was responsible for producing changes in neuronal electrical behavior. During a stroke episode, due to the decrease of ATP, the membrane potential value is compromised. Therefore, neuronal membrane depolarizes, triggering a cascade of events that can lead to cell swelling (reviewed in McEntee and Crook, 1993), which varies the cell body volume. And changes in the cell volume, may lead to alterations in the value of capacitance. In fact, it is usually said that when determining the membrane capacitance, one is indirectly measuring the cell volume (Chi and Xu, 2000). The cells exposed to 30 min ischemia present a significant increase in the value of the capacitance when compared to control cells (Figure 3). In 1995, Graf and his colleagues used whole-cell patch-clamp technique to study changes in membrane conductance and capacitance, after inducing osmotic swelling in rat hepatocytes. After osmotic swelling the value of specific capacitance of liver cells substantially exceeded the value for biological membranes, which suggested that changes in cell volume due to edema affect the capacitance membrane of rat liver cells.

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 alteration in neuronal function.

In some studies, it was demonstrated that EPO application after brain injury resulted in a

a)

b)

Figure 6. EPO administration increases the frequency, but not the amplitude, of mIPSCs. a) Column graphs representing

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significant reduced infarct cell volume (Krügel et al., 2010), which might be related with EPO neuroprotective role. Therefore it would be interesting to measure the capacitance values after EPO administration, in order to elucidate a bit more about the ability of EPO in protecting brain cells.

spontaneous activity), and consequently the ability of inducing plasticity.

6.3 EPO decreases mEPSCs frequency, but not their amplitude 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 is possible to observe that EPO decreases the frequency of mEPSCs, supporting a presynaptic effect of EPO in glutamatergic currents.

My study is based on basal synaptic transmission; therefore it only possible to speculate about a potential neuroprotective role of EPO 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 (excitotoxicity). In my experimental conditions, EPO induced only a slight decrease in mEPSC amplitude (no significant effect was verified), visible in Figure 5b. 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 field potentials (FP) amplitudes, after EPO application. These discrepancies may be related with the differences between the experimental protocols.

6.2 mEPSCs frequency was suppressed by ischemia Another important characteristic of this present study is related with the differences in frequency of glutamatergic transmission between cells that were exposed to ischemia and cells in normoxic conditions (control cells). The present data shows that the frequency of the mEPSCs frequency recorded after 30 minutes of ischemia was greatly reduced, comparatively to the control cells. Studies investigating the spontaneous firing rate of CA1 neurons after transient ischemia have yielded conflicting results. Some studies show that spontaneous firing rate in CA1 neurons increased following ischemia (Chang et al., 1989) while others showed evidences that neuronal activity was significantly suppressed after this insult (Furukawa et al., 1990). Figure 4 illustrates the significant decrease in mEPSCs frequency of pyramidal cells that suffered the insult, which reflects the vulnerability of CA1 pyramidal neurons.

The reduced frequency observed in my experiments brings out another important point, the synaptic plasticity in ischemia. 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 plasticity are similar to those activated during excitotoxicity. The function of this pathologic type of plasticity is still not completely addressed and this phenomenon is only observed 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

6.4 EPO enhances mIPSCs frequency EPO is known to have a modulatory action on GABAergic transmission, in young rats. In Wójtowicz and Mozrzymas (2008) study, long-term EPO treatment of hippocampal neurons developing in vitro resulted into significant changes in mIPSCs time course. Besides, data from patch clamp recordings from previous studies showed evidences that EPO facilitates GABA release, increasing the inhibitory postsynaptic currents frequency in acute brain slices (Adamcio et al, 2008). 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.

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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 GABA in order to inhibit the excitotoxic effect (Głodzik-Sobańska et al., 2004). Therefore, 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 (Figure 6).

Although GABA has received until now relatively little attention in the area of cerebral ischemia-induced neuronal death, the truth is GABAergic system may be of particular importance, since it functions in opposition to that of glutamate.

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). In conclusion, it would have been of great interest to study GABA release in pyramidal cells subjected to a 30 min ischemic insult, in order to see if GABA release frequency would be as affected as the frequency of pyramidal cells was, after a 30 min period of oxygen and glucose deprivation. 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.

Prof. Raul Martins from Instituto Superior Técnico, for his help during this project.

7. Conclusion

Calabresi P, Centonze D, Pisani A, Cupini LM, Bernardi G (2003) Sinaptic Plasticity in the ischaemic brain. The Lancet Neurology 2: 622-629

References Adamcio B, Sargin D, Stradomska A, Medrihan L, Gertler C, Theis F, Zhang M, Müller M, Hassouna I, Hannke K, Sperling S, Radyushkin K, El-Kordi A, Schulze L, Ronnenberg A, Wolf F, Brose N, Rhee JS, Zhang W, Ehrenreich H (2008) Erythropoietin enhances hippocampal longterm potentiation and memory.

BMC Biology 6: 1-16 Bartesaghi S, Marinovich M, Corsini E, Galli CL, Viviani B (2005) Erythropoietin: A Novel Neuroprotective Cytokine. NeuroToxicology 26: 923-928 Bernaudin M, Marti, HHM, Roussel S, Divoux D, Nouvelot A, MacKenzie ET, Petit E (1999) A Potential Role for Erythropoietin in Focal Permanent Cerebral Ischemia Mice. Journal of Cerebral Blood Flow and Metabolism.

19: 643-651 Brines M, Ghezii P, Keen S, Agnello D, Lanerolle, NC, Cerami C, Itri L, Cerami, A (2000) Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proceedings of the National Academy of Sciences of USA 97: 1526-1531

My results show that the ischemia changes the electrical properties of CA1 pyramidal cells, namely the value of membrane capacitance, which shows evidence of the extreme vulnerability of cells to oxygen and glucose deprivation. Concerning, EPO, although not providing a complete insight about the mechanisms of EPO in the brain, the present data suggests that this cytokine has an effect on neurotransmitter release, which might be related with its neuroprotective role.

Chang

HS, Sasaki T, Kassel NF (1989) Hippocampal unit activity after transient cerebral ischemia in rats. Stroke 20: 10511058

Chi XX, Xu ZC (2000) Pyramidal Neurons After Transient Forebrain Ischemia Differential Changes of Potassium Currents in CA1.

Journal of Neurophysiology 84: 2834-2843

8. Acknowledgments

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The author would like to thank to Prof. Ana Sebastião for allowing the realization of this master thesis in Instituto de Medicina Molecular (Faculdade de Medicina de Lisboa) and to Drª.

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