Changes in Functional Properties of Rat Hippocampal Neurons Following Pentylenetetrazole-induced Status Epilepticus

NEUROSCIENCE RESEARCH ARTICLE T. Y. Postnikova et al. / Neuroscience 399 (2019) 103–116

Changes in Functional Properties of Rat Hippocampal Neurons Following Pentylenetetrazole-induced Status Epilepticus Tatyana Y. Postnikova, a,b Dmitry V. Amakhin, a Alina M. Trofimova, a Ilya V. Smolensky a and Aleksey V. Zaitsev a,b,c,* a

Sechenov Institute of Evolutionary Physiology and Biochemistry of RAS (IEPhB), Saint Petersburg, Russia

b

Peter the Great St Petersburg Polytechnic University, Saint Petersburg, Russia

c

Institute of Experimental Medicine, Almazov National Medical Research Centre, Saint Petersburg, Russia

Abstract—Pathophysiological remodeling processes following status epilepticus (SE) play a critical role in the pathophysiology of epilepsy but have not yet been not fully investigated. In the present study, we examined changes in intrinsic properties of pyramidal neurons, basal excitatory synaptic transmission, and short-term synaptic plasticity in hippocampal slices of rats after SE. Seizures were induced in 3-week-old rats by an intraperitoneal pentylenetetrazole (PTZ) injection. Only animals with generalized seizures lasting more than 30 min were included in the experiments. We found that CA1 pyramidal neurons became more excitable and started firing at a lower excitatory input due to a significant increase in input resistance. However, basal excitatory synaptic transmission was reduced in CA3-CA1 synapses, thus preventing the propagation of excitation through neural networks. A significant increase in paired-pulse facilitation 1 d after SE pointed to a decrease in the probability of glutamate release. Increased intrinsic excitability of neurons and decreased synaptic transmission differentially affected the excitability of a neural network. In terms of changes in seizure susceptibility after SE, we observed a significant increase in the maximal electroshock threshold 1 day after SE, suggesting a decrease in seizure susceptibility. However, after 1 week, there was no difference in seizure susceptibility between control and post-SE rats. The effects of SE on functional properties of hippocampal neurons were transient in the PTZ model, and most of them had recovered 1 week after SE. However, some minor alterations, such as smaller amplitude field potentials, were observed 1 month after SE. Ó 2018 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: pentylenetetrazole model of epilepsy, electrophysiological properties, hippocampus, field EPSP, maximal electroshock threshold test.

and changes in the extracellular matrix (Beck and Yaari, 2008; Pitkanen and Lukasiuk, 2009; Amakhin et al., 2017; Plata et al., 2018; Smirnova et al., 2018). To investigate the pathophysiology of SE and its consequences animal models are frequently used. SE is usually induced in animals by electrical stimulation or chemical agents; pharmacological models include pilocarpine, organophosphates, kainic acid, pentylenetetrazole (PTZ), and other convulsants (Loscher, 2011; Nirwan et al., 2018). Different pharmacological models of SE have very diverse consequences. In kainate and pilocarpine models of SE, widespread excitotoxic death of neurons is observed after administration of the convulsants, and spontaneous recurrent seizures typically develop after a latent period lasting 2–5 wk (Magloczky and Freund, 1993; Dinocourt et al., 2003; Curia et al., 2008; Curia et al., 2014). In contrast, PTZ-induced convulsion typically does not result in substantial neuronal loss (Gallyas et al., 2008; Aniol et al., 2011; Vasil’ev et al., 2014), but only transient appearance of massively shrunken, hyperbasophilic (dark) cells in cortex and hippocampus

INTRODUCTION Status epilepticus (SE) is characterized by abnormal electrical discharges in the brain and seizures lasting 30 or more min (Trinka et al., 2015; Seinfeld et al., 2016). SE may cause devastating damage to brain tissue and trigger epileptogenesis, which is a cascade of molecular and cellular changes that eventually lead to spontaneous seizures. SE may induce changes in intrinsic membrane characteristics and synaptic properties, neurodegeneration and neurogenesis, axonal sprouting and dendritic remodeling, neuroinflammation, proliferation of glial cells, *Correspondence to: A. V. Zaitsev, Sechenov Institute of Evolutionary Physiology and Biochemistry of RAS (IEPhB), 44, Toreza pr., St Petersburg 194223, Russia. Fax: +7-(812)-552-3138. E-mail address: [email protected] (A. V. Zaitsev). Abbreviations: ACSF, artificial cerebrospinal fluid; AHPA, amplitude of afterhyperpolarization; AHPD, duration of afterhyperpolarization; APA, action potential amplitude; APT, action potential threshold; f/I, firing rate-current; fEPSPs, field excitatory postsynaptic potentials; FVs, fiber volleys; GABA, gamma-aminobutyric acid; I/O, input–output; MEST, maximal electroshock threshold; PPR, paired-pulse ratio; Ri, input resistance. https://doi.org/10.1016/j.neuroscience.2018.12.029 0306-4522/Ó 2018 IBRO. Published by Elsevier Ltd. All rights reserved. 103

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(Zaitsev et al., 2015; Vasilev et al., 2018). In this model, almost full recovery of the animals and the absence of spontaneous recurrent seizures is usually observed (Aniol et al., 2013). As PTZ-induced SE does not trigger epileptogenesis, we suggest that some functional changes in neuronal activities following PTZ-induced SE may have compensatory effects. Acute changes in the membrane properties of neurons after seizures are not yet fully understood (Su et al., 2002; Shah et al., 2004; Jung et al., 2007; Beck and Yaari, 2008; Pilli et al., 2012; Hargus et al., 2013). In a recent study, we showed that the intrinsic membrane properties of neurons in entorhinal cortex were significantly altered soon after pilocarpineinduced SE (Smirnova et al., 2018). These changes included a decrease in input resistance (Ri) and the membrane time constant due to enhanced leak currents (Smirnova et al., 2018). However, changes in neuronal network functioning following SE in the PTZ model are mostly unexplored. In the present study, we examined the effect of PTZ-induced SE on basal excitatory synaptic transmission, short-term plasticity, and biophysical properties of pyramidal neurons in the CA1 hippocampal field in rats within the first month after seizures. We also tested the consequences of PTZ-induced SE on seizure susceptibility in a maximal electroshock threshold (MEST) model.

EXPERIMENTAL PROCEDURES Animals The study included 20- to 22-d-old Wistar rats of both sexes (35–40 g) kept under controlled lighting (12-h light/dark cycle) and temperature conditions (23 ± 1 °C), with free access to water and food. All the procedures were in accordance with the Guidelines on the Treatment of Laboratory Animals of the Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, and these comply with Russian and international standards. PTZ model of SE Seizures were induced by a single intraperitoneal injection of PTZ (70 mg/kg; Sigma, USA) dissolved in saline. Only animals with generalized tonic–clonic seizures lasting at least 30 min (i.e., exhibited SE) were included in further experiments. The control rats were given a normal saline solution. Hippocampal slice preparation Slice preparation was done as previously described (Postnikova et al., 2017a,b). On the day of the experiment, the rats were guillotined, and their brains were rapidly removed. Horizontal 400-lm-thick brain slices were cut using a vibratome HM 650 V (Microm International, Germany) in artificial cerebrospinal fluid (ACSF; t = 0 °C). ACSF composed of 126 mM NaCl, 24 mM NaHCO3, 2.5 mM KCl, 2 mM CaCl2, 1.25 mM NaH2PO4,

1 mM MgSO4, and 10 mM glucose was bubbled with carbogen (95% O2 and 5% CO2). The slices were then transferred to oxygenated ACSF and incubated for 1 h at 35 °C before electrophysiological recordings.

Field potential recordings After incubation, the slices were placed in a recording chamber and perfused with a constant flow of ACSF at a rate of 5–7 mL/min at room temperature. The electrophysiological experiment began 15–20 min after placing the slices in a chamber. Electrophysiological properties were examined 3 h and 1, 3, 7, and 30 d after SE. Field excitatory postsynaptic potentials (fEPSPs) and population spikes (PSs) were recorded from CA1 stratum radiatum and stratum pyramidale accordingly using glass microelectrodes (0.2–1.0 MX). The responses were evoked by orthodromic stimulation of afferent fibers using tungsten bipolar electrodes placed in the stratum radiatum at the CA1–CA2 border,
1 mm from the recording electrode (Fig. 1A). The slices received one paired stimulation pulse (interstimulus interval, 50 ms; duration of the pulse, 0.1 ms) every 20 s. Responses were registered using a Model 1800 amplifier (A-M Systems, USA) and were digitized and recorded to a personal computer using ADC/DAC NI USB-6211 (National Instruments, USA) and WinWCP v5.x.x software (University of Strathclyde, UK). The electrophysiological records were investigated using the Clampfit 10.2 program (Axon Instruments, USA). The amplitude and slope of the 20–80% rising phase were calculated for each fEPSP (Fig. 1B). The input/ output (I/O) relationships were measured by increasing the current intensity from 25 to 300 lA (current step = 25 lA, Fig. 1C) via an A365 stimulus isolator (WPI, USA). Fiber volleys (FVs) were measured as shown in Fig. 1B. To estimate the efficacy of synaptic excitatory neurotransmission the relationship between the fEPSP and FV amplitudes was determined for each slice. The maximum rise slope of the curve (fEPSP amplitude vs. FV amplitude) was calculated by fitting it with a sigmoidal Gompertz function (Eq. (1)): y ¼ aee

ðkðxxc ÞÞ

ð1Þ

where a is an asymptote of maximum fEPSP amplitude; e is Euler’s number (e = 2.71828. . .); k is a positive number that determines the slope of the curve; xc is the FV amplitude at which the maximum slope of the curve is observed. The maximum slope was calculated as ak=e. To measure the paired-pulse ratio (PPR) paired pulses were delivered once every 20 s at interstimulus intervals of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, and 500 ms. The PPR was calculated as

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resistance in all neurons included in the analysis was less than 20 MX and remained stable (20% increase) across the experiment. Analysis of the electrophysiological characteristics of CA1 pyramidal neurons The intrinsic properties of CA1 pyramidal cells were evaluated from the voltage responses to hyperpolarizing and depolarizing current steps (duration = 1.5 s, increment = 10–25 pA) using custom scripts (Wolfram Mathematica 10, Wolfram Research, USA) as described previously (Smirnova et al., 2018). The resting membrane potential (in mV) was determined as a mean voltage potential before the current step application. The Ri (in MX) was calculated as the voltage–current (V–I) curve slope. The membrane time constant (in ms) was assessed by the fitting of the voltage transient induced by the 25 pA current step with a single exponential function. The first spikes that were evoked by the threshold current steps were used for the estimation of the action potential Fig. 1. The positions of the electrodes in a slice and representative recordings. (A) The positions of shape. The action potential the stimulating and recording electrodes in a hippocampal slice. A stimulating electrode is placed in the stratum radiatum in afferent fibers (AF) at the CA2/CA1 border. The field recording electrode is threshold (APT; in mV) was placed in the stratum radiatum of the CA1 area. (B) The diagram is showing how the main assessed as the potential at characteristics were measured. FV – fiber volley, fEPSP – field excitatory postsynaptic potential. (C) which the interpolated rate of Representative examples of fEPSPs recorded at different strengths of extracellular stimulation in the voltage increase (dV/dt) extended control and post-SE animals. 10 mV/ms. The action potential duration (APD; in ms) was the quotient of the second and first fEPSP amplitude for estimated as its width at the level each interval. of its half-amplitude. The spike amplitude (APA; in mV) was measured from the threshold to the peak. The Whole-cell recordings in brain slices amplitude of afterhyperpolarization (AHPA; in mV) was Visualization of CA1 pyramidal neurons was done using a measured from the level of the APT to the most Zeiss Axioskop 2 microscope (Zeiss, Germany) equipped negative potential after the spike. The duration of with differential interference contrast optics and a video afterhyperpolarization (AHPD; in ms) was measured as camera (Grasshopper 3 GS3-U3-23S6M-C; FLIR the time from the peak to the most negative potential Integrated Imaging Solutions Inc., USA). Patch after the spike. electrodes (2–5 MX) were filled with an internal solution The firing rate–current (f/I) curves were used to with a composition in mM as follows: 135 K-Gluconate, describe the firing properties of neurons. The firing rate 10 NaCl, 10 HEPES, 5 EGTA, 4 ATP-Mg, and 0.3 GTP. was estimated as the number of spikes per current step. pH was adjusted to 7.25 with KOH. Whole-cell The growing part of the f/I curve was fitted with a recordings were performed at 30 °C using a Model 2400 sigmoidal Gompertz function (Eq. (1)). Several (AM-Systems, USA) patch-clamp amplifier, an NI USBparameters of the f/I-curve were estimated: a – the 6343 A/D converter (National Instruments, USA) and asymptote of the maximum action potential frequency WinWCP 5 software (SIPBS, U.K.). The data were (in Hz); a*k/e – the maximum slope of the curve (in mV/ filtered at 10 kHz and sampled at 20 kHz. Series pA); and xc – an inflection current, which was the value

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of the current (in pA) at which the maximum slope of the curve was observed. MEST test The effect of PTZ-induced SE on susceptibility to seizures was tested using the MEST model 1 and 7 d after PTZ administration. Each of the four groups (two controls and two PTZ) contained 10 rats. The rats were exposed to alternative current (current intensity from 12 to 100 mA, pulse duration: 0.7 s, frequency: 100 Hz, and pulse width: 0.7 ms) using a pulse generator ECT Unit 57800 (Ugo Basile). Electrodes (ear clips) were moistened with saline before attaching them to the ear of the rat to improve electrical contact. To find thresholds, we used the ‘‘up-and-down” staircase method (Racusen et al., 1990). The first rat received a mid-strength current (40 mA). If tonic hindlimb extension occurred, the second rat received a lower current (32 mA). Otherwise, it received a higher current (50 mA). Each next rat received lower or higher currents, depending on the presence or absence of convulsions in the previous rat. The steps of the current were administered at intervals in a log scale equal to 0.1. In determining the probability of convulsions, we assumed that if convulsions occurred after one current, they would also occur in the same rat after higher currents. Equally, if convulsions did not occur after one current, they would not occur in the same rat after lower currents. The probability of convulsions was calculated as the ratio between the number of rats with convulsions after a specific current and the total number of rats that received the current. Then, a probability sigmoid curve was fitted with Boltzmann’s equations, and the EC50 was calculated as the current with 50% probability of convulsions. Statistical analysis The statistical analysis of the results was done using Sigmaplot 12.5 (Systat Software Inc., USA) and OriginPro 8 (OriginLab Corp., USA). To identify and exclude the outliers Dixon’s Q test (at a 90% confidence level) was used. The Kolmogorov–Smirnov test was used for evaluation of the normality of the sample data. For normally distributed data, paired or unpaired Student’s t-test and a one-way analysis of variance (ANOVA) were used, where appropriate. The effect of the stimulation strength on field responses was assessed with repeated measures ANOVA. For data that did not pass the normality test, the Kruskal–Wallis one-way ANOVA on ranks was employed. The results were considered significant when P < 0.05. All data are given as the mean and the standard error of the mean.

RESULTS The amplitude and slope of fEPSPs were significantly reduced after PTZ-induced SE We evaluated the synaptic neurotransmission at CA3CA1 pyramidal neuron synapses in hippocampal slices from rats in which SE was induced by PTZ on the 21st day of life (post-SE rats), as well as from control

animals, which were injected with saline at the same age. Afferent fibers were electrically stimulated at a range of current intensities (25–300 mA). In each experiment, the following was recorded: (1) presynaptic FVs, which reflected the number of CA3 axons that fired action potentials, and (2) the amplitude and slope of fEPSPs, which reflected the sum of excitatory postsynaptic responses occurring in CA1 pyramidal neuron dendrites evoked by stimulation of afferent fibers. All measurements were done at different time points after PTZ-induced SE: 3 h (N = 8 rats; n = 29 slices), 1 d (N = 18; n = 36), 3 d (N = 7; n = 26), 7 d (N = 11; n = 24) and 30 d (N = 7; n = 21). As developmental changes occur at this age, we initially used three control groups: 1 (N = 22; n = 44), 7 (N = 5; n = 17) and 30 (N = 5; n = 11) d after saline injection (22, 28, and 51 d old, respectively). No difference in properties of fEPSPs was found between the control animals of 22 and 28 d old. Therefore, we compared the synaptic neurotransmission properties of the PTZ-exposed rats during the first week after SE with those of the combined control group (n = 61). The results obtained in rats 30 d after SE were compared with those of an age-matched control group (N = 5; n = 11). As shown by a repeated measures ANOVA, the amplitude of the fEPSPs was significantly altered during the first week after SE (F44,1617 = 2.47; p < 0.001, Fig. 2A). According to the LSD post hoc test, the amplitude of fEPSPs did not differ from control values 3 h after SE. However, there was a significant decrease in fEPSP amplitudes in a wide range of stimulation currents 1 and 3 d after SE. The fEPSP amplitude did not return to normal even 30 d after SE (F11,341 = 4.10; p < 0.001, Fig. 3A). The slope of the fEPSPs was also significantly reduced in post-SE rats 3 h and 1, 3, and 7 d after PTZ treatment (F44,1529 = 3.44; p < 0.001, Fig. 2B). However, there was no statistically significant difference in the slope of the fEPSPs 30 d after SE (F11,330 = 0.07; p = 0.10; Fig. 3B). In contrast, the amplitudes of the presynaptic FVs did not change during the first week following SE (F44,1617 = 1.11; p = 0.29, Fig. 2C) or 30 d after SE (F11,319 = 0.22; p = 0.10; Fig. 3C). The slope of the I/O curve was significantly reduced during the first week after SE To determine whether the smaller fEPSP amplitude in the post-SE rat hippocampus was due to reduced efficacy of synaptic transmission, the I/O relationship between the fEPSP and FV amplitudes was determined for each slice (Fig. 2E). The I/O relations constructed from individual experiments were well fitted with a sigmoidal Gompertz function (Eq. (1)). The maximum slope of this fit reflected the composite cellular transfer function between presynaptic action potentials and postsynaptic membrane response. Therefore, the maximal I/O slope may be considered as a measure of synaptic strength. According to the ANOVA, the slope of the I/O curves was significantly reduced in post-SE rats during the first week (F4,143 = 4.87; p < 0.001). In the post hoc

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Fig. 2. Stimulation response relationships for fEPSP amplitudes (A) and slopes (B) and presynaptic fiber volley (FV) amplitude (C) recorded from the hippocampal CA1 area. Each point represents mean ± SEM. (D) Representative examples of I/O relationships between the fEPSP and FV amplitudes in hippocampal slices in rats (ctrl – control rat, 1 d and 7 d – post-SE rats 1 d and 7 d after PTZ-induced SE). (E) The maximal I/O slope is significantly reduced during the first week after SE. *p < 0.05, **p < 0.01, ***p < 0.001 – the difference between control and post-SE groups.

analysis, no changes in the efficacy of synaptic transmission were observed until 3 h after SE (Fig. 2D). According to our data, the recovery of synaptic transmission to the control level occurred 30 d after SE (F1,29 = 0.22; p = 0.64; Fig. 3D).

Paired-pulse facilitation was altered in hippocampal slices following SE A decrease in the I/O slope can arise from alterations in presynaptic neurotransmitter release. To determine whether PTZ-induced SE contributed to a decrease in

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Fig. 3. Stimulation/response relationships for fEPSP amplitudes (A) and slopes (B) and presynaptic FV amplitude (C) recorded from the hippocampal CA1 area in control (ctrl) and post-SE rats (30 d after SE). Each point represents mean ± SEM. *p < 0.05, **p < 0.01 – the difference between control and post-SE groups. (D) The maximal I/O slopes are similar in control rats and rats 30 d after SE.

presynaptic neurotransmitter release, we compared presynaptic facilitation in hippocampal slices in rats 3 h (N = 5; n = 16) and 1 (N = 7; n = 32), 3 (N = 6; n = 7), and 7 d (N = 6; n = 9) after SE with that in the controls (N = 8; n = 12). We used a paired-pulse protocol to measure the PPR of the fEPSP amplitude (Fig. 4A, B). The PPR was determined using interpulse intervals of 10–500 ms. The possible changes in the PPR point to differences in the probability of neurotransmitter release (Dobrunz and Stevens, 1997; Zucker and Regehr, 2002; Zaitsev and Anwyl, 2012). We built facilitation curves for each group of animals (Fig. 4C, D). Although the curves did not coincide, their maxima were observed at the same time intervals of 30–100 ms. To estimate the effects of the interstimulus intervals (Factor 1) and SE (Factor 2) on the magnitude of short-term facilitation, we employed a two-way ANOVA. Each of the factors had a significant effect on the PPR (Factor 1: F14,952 = 88.0; p < 0.001; Factor F2: F4,68 = 2.537; p < 0.05). However, the effect of the factor interaction was not significant (F56,882 = 0.64, p = 0.98), which supported the observation of no changes in the shape of the facilitation curve after SE (Fig. 4C). Applying Fisher’s LSD post hoc test, we found that the PPR significantly increased in rats 1 d after SE

(at interpulse intervals of 20–300 ms). Three hours after SE, there was a significant increase in facilitation only for the shortest interstimulus intervals of 10–20 ms. Thirty days after SE, there was no difference in the PPR(50 ms) in the control (1.52 ± 0.08, N = 5, n = 11) and post-SE rats (1.38 ± 0.03, N = 7, n = 14, p = 0.11). These results suggested a decrease in presynaptic glutamate release probability at hippocampal neuron synapses during the first week after PTZ-induced SE. Excitatory postsynaptic potential-spike coupling was disturbed after SE Next, we investigated whether the excitatory postsynaptic potential-spike coupling was disturbed after SE. For this purpose, we made several simultaneous recordings of evoked responses in stratum radiatum (Fig. 5A, top) and stratum pyramidale (Fig. 5A, bottom) to orthodromic stimulation of increasing intensity. The appearance of population spikes on the records from the stratum pyramidale corresponded to an obvious notch in the descending phase of fEPSPs recorded in the stratum radiatum. Therefore, in most experiments, we detected a population spike threshold by the appearance of the

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F1,31 = 9.57; p < 0.01, Fig. 5C). However, the threshold current inducing fEPSPs in slices from the post-SE rats did not differ from that in slices from age-matched controls (first week: F4,157 = 1.95; p > 0.05; 30 d after SE: F1,31 = 0.33; p = 0.57, Fig. 5B). It should be noted that the difference between these two thresholds has been significantly reduced (Fig. 5D). These changes indicated that the excitability of CA1 neurons increased after seizures. Biophysical properties of CA1 hippocampal pyramidal neurons were altered after SE

Fig. 4. Paired-pulse facilitation is altered in hippocampal slices following SE. Representative examples of pair-pulse responses from the hippocampal CA1 area in control (ctrl) and post-SE rats (1 d after SE), using interpulse intervals of 30 ms (A) and 80 ms (B). (C) and (D) Diagrams showing the paired-pulse facilitation in hippocampal slices across different inter-stimulus intervals (C, 10–80 ms; D, 10–500 ms). Each point represents mean ± SEM. *p < 0.05 – the difference between control and post-SE groups.

notch in the fEPSPs. In slices obtained from the post-SE rats, population spikes appeared in response to a lower current intensity than in slices from the control animals (first week: F4,157 = 3.96; p < 0.01, 30 d after SE:

Input–output properties of neurons depend on the efficacy of excitatory/inhibitory synaptic connections and intrinsic excitability. To evaluate if changes in the excitability of CA1 neurons depend on intrinsic membrane properties we compared them in CA1 pyramidal neurons from the control and post-SE rats using the whole-cell patchclamp recording in hippocampal slices. First, we compared the subthreshold membrane properties of neurons, including resting membrane potential, Ri, and the membrane time constant (Fig. 6A–C). Only Ri significantly increased from 111 ± 7 MX in the control group (n = 18) to 154 ± 15 MX 1 d after PTZ-induced SE (n = 16), and it returned to 125 ± 7 MX 7 d after SE (n = 18) (one-way ANOVA, F2,51 = 5.2, p < 0.01, followed by Dunnett’s post hoc test). No significant changes in other subthreshold properties were found (Fig. 6C). Next, we investigated changes in the properties of action potentials, evoked by the threshold current. Analysis of changes in the properties of action potentials revealed that none of the tested parameters (APT, APA, APD, AHPA, and AHPD) had been altered after SE (Fig. 6D). This finding suggested that the properties of most voltage-dependent ionic channels are not be affected by seizures in the PTZ model. Changes in Ri significantly disturb the transduction of synaptic input in neuronal firing (Beck and Yaari, 2008). Therefore, we compared how neurons of the control and post-SE rats transformed a constant depolarizing current into firing (Fig. 7A). We fitted the rising parts of the f/I curves with Eq. (1) (Fig. 7B) and investigated how the parameters of this equation changed after SE. The results revealed that the inflection current decreased by 27% one day after SE, but it did not differ from control value 7 d after SE (F2,51 = 5.3, p < 0.01, one-way ANOVA followed by Dunnett’s post hoc test, Fig. 7C). No significant changes were detected in the maximal firing frequency or the maximal slope of f/I. These results indicated that f/I curve was shifted to the left as compared to the control curve on the first day after SE (Fig. 7B) suggesting that a smaller current was required to induce the same firing frequency in post-SE neurons. To investigate whether the shift of the f/I curve was dependent on changes in Ri, the values of the injected currents were normalized to those of Ri for each cell. No significant changes were detected in the maximal normalized slope and normalized inflection current (Fig. 7E). This finding indicated that the shift in f/I curves after SE was mostly attributed to the changes in Ri.

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Our results suggest that increased excitability of CA1 neurons at short intervals after SE (one day or a few days) is due acute changes in intrinsic membrane properties but a week after SE this factor does not play a significant role. MEST increased in rats 1 d after SE To investigate whether PTZinduced SE affected seizure susceptibility, the MEST test was conducted 1 and 7 d after PTZ administration. One day after PTZ administration, the threshold for electroshock-induced convulsions (tonic hindlimb extension) significantly increased (Fig. 8A). The probability curve was shifted toward larger currents: EC50 increased from 54.4 ± 0.9 mA in the control group (N = 11) to 106 ± 3 mA in the post-SE group (N = 11, p < 0.001). Seven days after PTZ administration, we detected no between-group difference in the threshold for electroshock-induced convulsions (Fig. 8B). The EC50 was 40.8 ± 0.6 mA in the control group (N = 10) and 40.3 ± 1.0 mA in the post-SE group (N = 11).

DISCUSSION In the present study, we investigated the effects of PTZinduced SE on the functional properties of neurons in the area of the brain most vulnerable to seizures: the hippocampus. A peculiarity of the PTZ model is that the acute seizures it induces are followed by almost complete recovery of the animals, and spontaneous recurrent seizures do not usually develop (Aniol et al., 2011, 2013). We found that 1 d after SE the hippocampal neurons became more excitable and started firing at a less excitatory input due to a significant increase in Ri. The inflection current was smaller for neurons in post-SE slices than in control slices indicating the higher firing rate of neurons

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Fig. 6. Changes in membrane properties of CA1 neurons after PTZ-induced SE. (A) A representative example of subthreshold responses to current steps from 75 to +25 mV. Gray bar indicates the time interval (50 ms) which was used to obtain averaged values of membrane potential for V-I relationships. (B) The averaged V-I characteristics of hippocampal neurons in control group and groups after PTZ-induced SE. (C) Bar graphs showing the average values of subthreshold parameters of CA1-neurons in control rats (ctrl) and post-SE rats 1 and 7 d after PTZ-induced SE. Ri, input resistance; VRest, resting membrane potential; sm, membrane time constant. *p < 0.05 – the difference between control and post-SE groups. (D) The average values of the action potential shape parameters. APT, action potential threshold; APA, action potential amplitude; APD, action potential duration; AHPA, amplitude of afterhyperpolarization; AHPD, duration of afterhyperpolarization.

at a less excitatory input. The observed changes in intrinsic membrane properties were transient and disappeared during a week after SE. At the same time, we observed a decrease in basal excitatory transmission in CA3-CA1 synapses at least

for a week. As these two factors act on the excitability of the neural network in opposite directions, we investigated whether seizure susceptibility changed after PTZ-induced SE. We observed a significant increase in the MEST 1 d after SE, suggesting a decrease in

3 Fig. 5. Excitatory postsynaptic potential-spike coupling is disturbed after SE. (A) A representative example of simultaneous recordings of evoked responses in stratum radiatum (top) and stratum pyramidale (bottom) to orthodromic stimulation of increasing intensity. On the right, the same recordings are shown with the shift. Note the notch (arrows) in the fEPSP recordings corresponding to the population spikes (arrowhead). (B) The threshold current inducing fEPSPs in slices from the control and post-SE rats. (C) The threshold current inducing population spikes in slices from the control and post-SE rats. (D) The difference between threshold current inducing population spikes and current inducing fEPSPs in slices from the control and post-SE rats. *p < 0.05, **p < 0.01, ***p < 0.001 – the difference between control and post-SE groups.

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Fig. 7. Changes in firing properties of CA1 neurons after PTZ-induced SE. (A) The voltage responses to 125 and 200 pA current steps in three representative neurons from control rats (ctrl) and post-SE rats 1 and 7 d after PTZ-induced SE. (B) Three representative examples of f/I curves for control and post-SE neurons. (C) The bar graphs showing average values of the parameters of f/I curves. **p < 0.01 – the difference between control and post-SE groups. (D) The bar graphs showing average parameters of the normalized f/I curves. No significant differences between groups are observed.

seizure susceptibility. However, after 1 wk there was no difference in seizure susceptibility between the control and post-SE rats. Although most functional properties of hippocampal neurons recovered 1 wk after PTZ-induced SE, some minor alterations, such as smaller amplitude fEPSPs, were observed 1 mo after SE.

Seizure-related intrinsic plasticity in hippocampal pyramidal neurons Neurons respond to diverse types of perturbation by a set of homeostatic mechanisms that include reciprocal synaptic scaling of excitatory and inhibitory synapses

and changes in intrinsic neuronal excitability (Davis and Bezprozvanny, 2001; Turrigiano and Nelson, 2004; Beck and Yaari, 2008; Joseph and Turrigiano, 2017). Activity-dependent intrinsic plasticity in neurons is a well-known phenomenon (Desai et al., 1999; Sourdet et al., 2003; Cudmore and Turrigiano, 2004). Abnormal, excessive neuronal activity during seizures has been shown to induce intrinsic plasticity in hippocampal and cortical neurons, including plasticity of different types of conductances, such as decreases in A-type potassium currents (Bernard et al., 2004) and hyperpolarizationactivated currents (Shah et al., 2004; Jung et al., 2007; Marcelin et al., 2009), increases in T-type calcium

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Fig. 8. MEST increased in rats 1 d after SE. Diagrams showing the probability of tonic hindlimb extension in control rats and rats 1 d after SE (A) or 7 d after SE (B) in response to stimulation current. Probability sigmoid curves were fitted with Boltzmann’s equations, and the EC50 was calculated as the current with 50% probability of tonic hindlimb extension.

currents (Sanabria et al., 2001; Su et al., 2002; Yaari et al., 2007) and persistent sodium currents (Royeck et al., 2015). The aforementioned alterations lead to substantial changes in passive and active neuronal membrane properties. In our study, the most robust change in membrane properties was the transient increase in Ri of hippocampal CA1 pyramidal neurons 1 d post-SE. Previous reports on changes in neuronal Ri after seizures are contradictory. In a lithium-pilocarpine model, 1 d post-SE, we observed reduced Ri in pyramidal neurons in the entorhinal cortex but not in the prefrontal cortex (Smirnova et al., 2018). In contrast, previous research found no significant changes in Ri of CA1 pyramidal neurons (Royeck et al., 2015; Tamir et al., 2017) or entorhinal cortex principal cells (Kobayashi et al., 2003; de Guzman et al., 2008) in pilocarpinetreated chronically epileptic rats. However, research reported an increase in Ri in CA3 pyramidal cells and dentate granule cells (Tamir et al., 2017). In a kainate model, both the 1 d and 7 d post-SE entorhinal pyramidal neurons had significantly increased Ri compared to their respective controls (Shah et al., 2004). Another study detected no changes in Ri in hippocampal CA1 pyramidal cells 3–4 h after kainate-induced SE in mice (Minge and Bahring, 2011). In experiments conducted 7 d after electrical induction of SE, increased Ri in entorhinal stellate neurons was reported (Hargus et al., 2013). Focal laser lesions in the visual cortex of the rat significantly increased Ri in neurons located ipsilateral to the lesion (Imbrosci and Mittmann, 2013). Therefore, Ri might be one of the main electrophysiological characteristics involved in mechanisms of homeostatic or nonhomeostatic plasticity (Beck and Yaari, 2008). The Ri of a neuron reflects the number of open channels in the resting state. Possible reasons for an increase in Ri are a reduction in tonic synaptic currents, leak currents, specific changes in the properties of voltage-gated channels, or morphological changes (e.g., decrease in the membrane area). In the present study, we did not especially evaluate the mechanisms of Ri

changes. According to our previous observations, PTZinduced seizures temporally increased the expression level of transporters of glutamate (EAAT1) and GABA (GAT1) in the hippocampus (1 d post-SE) (Vasilev et al., 2018). Up-regulation of transporters may decrease the synaptic availability of glutamate and GABA, as well as extrasynaptic signaling, thereby decreasing tonic currents. However, application of antagonists of GABA and glutamate ionotropic receptors resulted in a relatively small increase in Ri in neurons from control animals (Povysheva et al., 2008; Smirnova et al., 2018), thereby suggesting that changes in tonic synaptic currents may not be the primary cause of the increase in Ri. Profound reduction in Ih current was found to be the primary cause of Ri increase in pyramidal neurons after kainate-induced seizures (Shah et al., 2004). We suggest that this possibility is most likely but additional experiments are required for the conclusions. The increase in Ri resulted in higher excitability of neurons because of a smaller synaptic input required to induce the firing of a neuron. The rising parts of f/I curves were shifted to the left for post-SE pyramidal neurons. Normalization of the current by the Ri value for each cell eliminated the differences in excitability between control and post-SE neurons, suggesting that the increase in Ri affected neuronal excitability. PTZ-induced SE altered basal excitatory synaptic transmission and transiently decreased seizure susceptibility In other epilepsy models, epileptic activity appeared to result in changes in the efficacy of excitatory or inhibitory synaptic connections between neurons (Beck and Yaari, 2008). In the present study, basal excitatory synaptic transmission, as assessed by the average slope of I/O curves, was significantly reduced in post-SE rats during the first week. These results are the exact opposite to those observed in a lithium-pilocarpine model (Amakhin et al., 2017). In the lithium-pilocarpine model, similar measurements of the I/O curve slope for fEPSPs recorded in

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CA1 pointed to an increase in basal excitatory synaptic transmission on the first day following SE (Amakhin et al., 2017). Repeated 4-aminopyridine induced seizures diminished the efficacy of glutamatergic transmission in the neocortex (Vilagi et al., 2009) but increased it in the hippocampus (Borbely et al., 2009). The mechanisms of alterations in synaptic transmission are diverse and include regulation of changes in the probability of presynaptic release, numbers of synaptic receptors, and postsynaptic transmitter sensitivity. To determine whether PTZ-induced SE contributed to a decrease in presynaptic neurotransmitter release, we investigated presynaptic facilitation in hippocampal slices. The observed PPR increase in post-SE slices may indicate a transient decrease in the probability of release of the excitatory mediator glutamate in hippocampal synapses (Dobrunz and Stevens, 1997; Buonomano, 1999; Zaitsev and Anwyl, 2012). We hypothesize that a decrease in the probability of release of the excitatory transmitter glutamate in hippocampal synapses is one efficient mechanism for preventing seizures and epileptogenesis. Indeed, 1 d after PTZ-induced seizures, in addition to a significant increase in the PPR, the threshold of the rats in the PTZ group to electroshock-induced hindlimb extension was twice higher than that in the control animals. Seven days after PTZ-treatment when the PPR did not differ from that of the control rats, the current threshold for hindlimb extension for post-SE and control animals was similar. Based on the results of the present study, we suggest that observed decrease in the release probability of glutamate and the prolonged decline in excitatory synaptic transmission are the main compensatory mechanisms in the brain that prevent the development of temporal lobe epilepsy after PTZ-induced SE. Transmitter release depends on three main factors: the number of vesicles that are ready for immediate release, the calcium concentration in the presynaptic terminal, and the efficient operation of all molecular mechanisms responsible for calcium sensing and vesicle fusion (Branco and Staras, 2009). Thus, pharmacological tools affecting any of these factors may possess an antiepileptogenic effect. Antagonists of high-voltageactivated calcium channels of R-, P/Q- or N-types, which promote calcium input into the presynaptic terminal, represent potential antiepileptogenic drugs, as blockade of these channels inhibits neurotransmitter release (Turner, 1998; Rogawski and Loscher, 2004; Rajakulendran and Hanna, 2016). Agonists of group II metabotropic glutamate receptors with an antiepileptic action have already been reported (Klodzinska et al., 2000; Alexander and Godwin, 2006). These inhibit calcium channel functioning through several intermediaries or substances, which modulate the functions of proteins involved in the molecular mechanisms of vesicle fusion with the presynaptic membrane (Lynch et al., 2004).

CONFLICTS OF INTEREST No relevant conflicts of interest.

ACKNOWLEDGMENTS This work was supported by the Russian Science Foundation (project 16-15-10202).

AUTHOR CONTRIBUTIONS TP, DA, and AZ designed the study. TP, DA, AT, and IS performed experiments and analyzed data. TP, DA, AT, IS, and AZ interpreted data for the work. TP, DA, IS, and AZ wrote the manuscript. TP, DA, AT, IS, and AZ approved the final version.

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(Received 28 July 2018, Accepted 17 December 2018) (Available online 26 December 2018)