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Journal of Neuroscience Research 93:454–465 (2015)

N-Methyl-D-Aspartate Receptor Channel Blockers Prevent PentylenetetrazoleInduced Convulsions and Morphological Changes in Rat Brain Neurons Aleksey V. Zaitsev,1* Kira Kh. Kim,1 Dmitry S. Vasilev,1 Nera Ya. Lukomskaya,1 Valeria V. Lavrentyeva,1 Natalia L. Tumanova,1 Igor A. Zhuravin,1 and Lev G. Magazanik1,2 1

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, Saint Petersburg, Russia 2 Saint Petersburg State University, Saint Petersburg, Russia

Alterations in inhibitory and excitatory neurotransmission play a central role in the etiology of epilepsy, with overstimulation of glutamate receptors influencing epileptic activity and corresponding neuronal damage. N-methyl-D-aspartate (NMDA) receptors, which belong to a class of ionotropic glutamate receptors, play a primary role in this process. This study compared the anticonvulsant properties of two NMDA receptor channel blockers, memantine and 1-phenylcyclohexylamine (IEM-1921), in a pentylenetetrazole (PTZ) model of seizures in rats and investigated their potencies in preventing PTZ-induced morphological changes in the brain. The anticonvulsant properties of IEM-1921 (5 mg/kg) were more pronounced than those of memantine at the same dose. IEM-1921 and memantine decreased the duration of convulsions by 82% and 37%, respectively. Both compounds were relatively effective at preventing the tonic component of seizures but not myoclonic seizures. Memantine significantly reduced the lethality caused by PTZ-induced seizures from 42% to 11%, and all animals pretreated with IEM-1921 survived. Morphological examination of the rat brain 24 hr after administration of PTZ revealed alterations in the morphology of 20–25% of neurons in the neocortex and the hippocampus, potentially induced by excessive glutamate. The expression of the excitatory amino acid transporter 1 protein was increased in the hippocampus of the PTZ-treated rats. However, dark neurons did not express caspase-3 and were immunopositive for the neuronal nuclear antigen protein, indicating that these neurons were alive. Both NMDA antagonists prevented neuronal abnormalities in the brain. These results suggest that NMDA receptor channel blockers might be considered possible neuroprotective agents for prolonged seizures or status epiC V 2014 Wiley lepticus leading to neuronal damage. Periodicals, Inc. C 2014 Wiley Periodicals, Inc. V

Key words: memantine; IEM-1921; epilepsy; glutamate receptor; dark neuron

Alterations in inhibitory and excitatory neurotransmission play a central role in the etiology and pathogenesis of epilepsy, with overstimulation of glutamate receptors potentially influencing the initiation, propagation, and maintenance of epileptic activity (Urbanska et al., 1998; Morimoto et al., 2004; Rogawski and Loscher, 2004). Excessive glutamate causes seizure-induced excitotoxic cell alterations, including rapid formation of dark neurons (Sloviter and Dempster, 1985) and even death of vulnerable neuronal populations (Meldrum, 1993). N-methyl-D-aspartate (NMDA) receptors, which belong to a class of ionotropic glutamate receptors, play a primary role in these alterations, as evidenced by the protective effects of NMDA receptor blockade reproduced in many different in vitro and in vivo models (for review see Kohl and Dannhardt, 2001; Ghasemi and Schachter, 2011). Unfortunately, the results of clinical trials with selective NMDA receptor antagonists for the chronic treatment of epilepsy have been mostly disappointing (Rogawski, 2011). Side effects of NMDA receptor antagonists, such as memory dysfunction, learning deficits, psychotomimetic effects, and motor disturbances, pose significant problems (Ghasemi and Schachter, 2011). When reproduced in animal models, these side effects are often accompanied by neurodegeneration in the cingulate and the

Contract grant sponsor: RFBR; Contract grant numbers: 12-04-01080a; 13-04-00453a; 13-04-00244a; Contract grant sponsor: Programs No. 5 and No. 7 of the Presidium of the RAS *Correspondence to: Aleksey V. Zaitsev, IEPhB, Toreza Prospect 44, Saint Petersburg 194223, Russia. E-mail: [email protected] Received 3 August 2014; Revised 18 September 2014; Accepted 19 September 2014 Published online 31 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23500

Effects of NMDA Receptor Blockers on Seizures

retrosplenial cortex (Kohl and Dannhardt, 2001; Ghasemi and Schachter, 2011). Nevertheless, NMDA receptor antagonists might be useful in the treatment of some types of seizures. For example, the well-known NMDA receptor channel blocker memantine, which is approved for chronic clinical use in the treatment of Alzheimer’s disease (Parsons et al., 1999; Lipton, 2006), exerted anticonvulsant effects in maximal electroshock seizures (MES; Chojnacka-Wojcik et al., 1983; Urbanska et al., 1992; Parsons et al., 1995) and seizures induced by various chemoconvulsants (Meldrum et al., 1986; Bisaga et al., 1993; Parsons et al., 1995; GeterDouglass and Witkin, 1999; Lukomskaya et al., 2004; Mares and Mikulecka, 2009). However, high-dose (20 mg/ kg) memantine induced spontaneous motor seizures in amygdala-kindled rats (Loscher and Honack, 1990). There is evidence that ketamine, another NMDA receptor channel blocker, is useful in the treatment of refractory status epilepticus (Dorandeu et al., 2013). A structurally similar compound, 1-phenylcyclohexylamine (IEM-1921), is a more potent anticonvulsant and causes less motor impairment than ketamine at anticonvulsant doses (Rogawski et al., 1989; Blake et al., 1992; Parsons et al., 1995; Lukomskaya et al., 2007; Kim et al., 2012). However, its anticonvulsant and neuroprotective properties have not been fully elucidated. Thus, further investigations are required to clarify the potential use of memantine and other NMDA receptor channel blockers as antiepileptic drugs. The present study compares the anticonvulsant properties of memantine and IEM-1921 in a pentylenetetrazole (PTZ) model of seizures in rats to investigate their potencies in preventing PTZ-induced morphological changes in the brain. MATERIALS AND METHODS Experiments were carried out with 5–6-week-old male Wistar rats (n 5 114) weighing 114 6 3 g that had free access to food and water. All experiments were performed between 11 AM and 2 PM. Experiments were conducted in compliance with the Rules of Animal Care and Use Committee of the Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, and comply fully with The Council of the European Communities Directive 86/609/ EEC. Naive animals were used for all the experiments. Drugs Memantine (Sigma-Aldrich, St. Louis, MO) and IEM1921 (synthesized by Dr. V.E. Gmiro, Institute of Experimental Medicine of the Russian Academy of Medical Sciences) at a dose of 5 mg/kg were freshly dissolved in distilled water. PTZ (Sigma-Aldrich) at a dose of 70 mg/kg was freshly dissolved in saline. The drugs were administered at an injection volume of 2 ml/kg. The molecular weights of both drugs are almost the same, 212 and 216, respectively. Anticonvulsant Effects of Memantine and IEM-1921 Seizures were induced by intraperitoneal injection of PTZ (70 mg/kg). Memantine or IEM-1921 (5 mg/kg) was Journal of Neuroscience Research

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administered intramuscularly 30 or 60 min before the PTZ injections. After injection of PTZ, the rats were placed singly in Plexiglas cages and videotaped for 30 min. The seizure phenotype and the different stages of seizures were assessed with scores based on a modified Racine’s scale (Racine, 1972; Luttjohann et al., 2009) as follows: 1, single to repeated myoclonic jerks; 2, partial clonic seizure in a sitting position; 3, generalized convulsions, including clonic and/or tonic–clonic seizures while lying on the belly; and 4, generalized convulsions, including pure tonic seizures with hind limb extension and/or tonic– clonic seizures while lying on the side that might start with wild jumping and running. The stages, latencies, duration, and incidence of convulsions were recorded. The maximal score recorded for the animals and the duration of the convulsions during 30 min after PTZ injection were averaged for the group and used as an indicator of the intensity of convulsive reactions. Morphology For the morphological study, four groups of animals were established: 1) naive control, no PTZ injection (n 5 7); 2) control group treated with PTZ injection (n 5 15); 3) experimental group pretreated with 5 mg/kg of memantine 1 hr before PTZ injection (n 5 12); and 4) experimental group pretreated with 5 mg/kg of IEM-1921 1 hr before PTZ injection (n 5 12). Before decapitation, the rats were deeply anesthetized with diethyl ether. The animals in groups 2–4 were decapitated 24 hr after the PTZ injection. Brain tissue blocks were fixed in 10% formalin in phosphate-buffered saline (PBS; 4 C, pH 7.4) for 1 week, frozen, and sectioned in the coronal plane with a Leica CM 1510S cryostat (Leica Microsystems, Wetzlar, Germany). The morphology of the cortical (bregma 10.20 mm) and the hippocampal (bregma 23.30 mm) sections of the brain was analyzed. Some brain sections (20 mm) were Nissl stained and analyzed with light microscopy with an ImagerA microscope (Zeiss, Oberkochen, Germany). The total number of cells in the neural tissue and the number of dark neurons were analyzed in 10 sections of the parietal cortex and in areas CA1, CA2, and CA4 of the hippocampus in each animal. For random sampling, the first of the analyzed sections in the tissue block was chosen randomly, and the number of dark neurons, signifying changes in cell morphology, in sequences of the sections (60 mm between previous and next section) was counted. The cell number was counted in a 500 3 500 mm counting window in cortical tissue and in a 1,000-mm-wide fragment of the pyramidal layer (measured in the middle of the layer) in the CA1, CA2, and CA4 areas of the hippocampus. At least 5,000 cells were analyzed in each cortical and hippocampal structure. Dark neurons were identified as strongly stained, usually shrunken cells, with an optical density of more than 300% that of the cell bodies of other cells in the tissue. The optical density of the cell bodies was measured in the Video TesT—Morphology (Video TesT, Saint Petersburg, Russia) software program. Immunochemistry Some brain tissue sections (20 mm) were processed for visualization of the Fox-3 protein and caspase-3 in neurons by using immunofluorescent labeling. The first of the sections in

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the analyzed sequence (100 mm between previous and next section) was selected randomly and used for immunolabeling. The Fox-3 and caspase-3 distributions were analyzed in 10 sections of the parietal cortex and in areas CA1 and CA4 of the hippocampus in the naive control and PTZ-treated rats. The sections were incubated overnight at 37 C in PBS containing 2% bovine serum albumin, 0.3% Triton X-100 (Merck, Darmstadt, Germany), and a mixture of two antibodies, mouse monoclonal anti-Fox3/neuronal nuclear antigen (NeuN) antibody (dilution 1:1,000; ab104224; Abcam, Bristol, United Kingdom) and rabbit polyclonal anticaspase-3 (1:400; ab4051; Abcam). After having been thoroughly rinsed, the sections were incubated for 1 hr at 37 C in a mixture of two secondary fluorescently tagged antibodies, fluorescein isothiocyanate (FITC)-conjugated goat polyclonal antibody against mouse IgG (1:200; ab97022; Abcam) and phycoerythrin (PE)-conjugated secondary antibody against rabbit IgG (1:200; ab7007; Abcam) diluted in the blocking serum. The same brain sections were counterstained with Hoechst 33342 (Invitrogen, Carlsbad, CA) to show cortical lamination and the total cell number. Microscopy was performed with a Leica DMR microscope connected to a confocal Leica TCS SL (Leica Microsystems) scanner with an He/Ar laser, which was used for excitation of FITC and PE (488 nm) and Hoechst 33342 (350 nm). The emission of FITC was observed in the 496–537 nm wavelength, PE in the 652– 690 nm wavelength, and Hoechst 33342 in the 430–461 nm wavelength. The percentage of Fox3-positive cells in the 1,000-mm-wide fragment of the CA1 pyramidal layer was compared with the naive control rats (n 5 7) and PTZ-treated rats (n 5 15). The total number of cells in each area was estimated based on the number of Hoechst 33342-positive cells. Western Blotting The brain tissue blocks were homogenized on ice in 0.5 M Tris-HCl buffer (pH 7.4) with 1% (of volume) of Triton X-100 and centrifuged for 5 min at 2,500g at 4 C. Excitatory amino acid transporter 1 (EAAT1) in the supernatant was analyzed by electrophoresis in 8% polyacrylamide gels in the presence of sodium dodecyl sulfate, followed by immunoblotting on polyvinylidene difluoride membranes. The membranes were incubated nightly at 4 C in anti-EAAT1 rabbit polyclonal antibody (1:10,000; ab416; Abcam) in 50 mM Tris-HCl, pH 7.4, with 150 mM NaCl, 0.05% Tween 20, and 5% dry nonfat milk. The actin protein in the same samples was analyzed with rabbit antiactin antibodies (1:5,000; A5060; Sigma). Immunoreactivity was detected with horseradish peroxidase-coupled goat anti-rabbit secondary IgG (1:5,000, 1 hr; ab6721; Abcam) and visualized with an Optiblot ECL Ultra Detect kit (1.2pg-2ng; ab133409; Abcam). The relative intensity of immunoreactive bands on the membranes was quantified by computer-assisted densitometry in Video TesT—Morphology. Rat hepatic tissue was used as a negative control. Colorimetric Analysis of Caspase-3 Activity A caspase-3 assay kit (ab39401; Abcam) was used according to the manufacturer’s recommended protocol. The brain tissue blocks were homogenized in 50 ml chilled cell lysis buffer, incubated on ice for 10 min, and centrifuged for 1 min at

10,000g at 4 C. The Bradford assay (Bradford, 1976) was used to analyze the protein concentration. In each assay, the samples were diluted in a ratio of 50 mg protein to 50 ml cell lysis buffer. Then, 50 ml of 23 reaction buffer (containing 10 mM dithiothreitol) and 5 ml of 4 mM DEVD-p-NA substrate (200 mM final concentration) were added to each sample. The optical density of each sample was measured at 405 nm in an MR96A microplate reader (Mindray, Shenzhen, China) just after the DEVD-p-NA substrate was added. The samples were incubated at 37 C for 1 hr. The optical density of each sample was measured after the incubation with the substrate. The increase in caspase-3 activity was determined by the changes in optical density. Statistical Analysis All statistical tests were performed in Statistica 8.0 software (Statsoft, Tulsa, OK). The statistical significance of the anticonvulsive effects of the NMDA receptor channel blockers was tested by ANOVA, followed by Fisher’s least significant difference post hoc tests (multiple-comparisons tests). Fisher’s exact test was used to compare the proportion of animals with different seizure scores and the rate of lethality. Because the seizure scores and the number of dark neurons did not fit a normal distribution, between-group differences in the data were compared with the Mann-Whitney test. An a level of 0.05 was used as the criterion for significance in all the tests. The data are given as mean 6 SEM.

RESULTS Anticonvulsant Effect of Memantine and IEM-1921 on PTZ-Induced Seizures in Wistar Rats We compared the anticonvulsant potency of two NMDA receptor channel blockers, memantine (5 mg/kg) and IEM-1921 (5 mg/kg), on PTZ-induced seizures in Wistar rats. This dose was chosen because the MES-test ED50 of both compounds was reported to be about 5– 7 mg/kg (Rogawski et al., 1989; Parsons et al., 1995, 1999). In the control group (n 5 36), the PTZ injection (70 mg/kg, i.p.) induced partial and/or generalized seizures, with scores ranging from 2 to 4 in 94% of the tested animals (Fig. 1A), and it caused death in 42% of the animals (Fig. 1B). Pretreatment with memantine did not change the proportion of animals exhibiting seizures (32 of 35 rats, 91%), and it did not affect the maximal seizure score distribution (Kolmogorov-Smirnov test, P > 0.10). Pretreatment with IEM-1921 reduced the proportion of rats with such seizures to 75% (27 of 36 rats; Fisher’s exact test, P < 0.05). Pretreatment also significantly changed the maximal seizure score distribution (Fig. 1A) and decreased the average seizure score (Fig. 1C). Pretreatment with the NMDA receptor blockers also reduced lethality (Fig. 1B). There were no animal deaths with IEM-1921 at the concentration used. Neither blocker had a significant effect on the latency of the seizures. Memantine had no effect on the latency of any seizure stage, although the latency of generalized convulsions showed a tendency to increase (seizure stages 3, 4) in the rats pretreated with IEM-1921 Journal of Neuroscience Research


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Fig. 1. Anticonvulsant effects of memantine (5 mg/kg) and IEM-1921 (5 mg/kg) on PTZ-induced seizures in rats. A: Distribution of rats by maximal seizure score in three groups: PTZ, rats with PTZ injection (n 5 36); 1Memantine, rats pretreated with memantine before PTZ injection (n 5 35); and 1IEM-1921, rats pretreated with IEM-1921 before PTZ injection (n 5 36). B: Pretreatment with NMDA receptor channel blockers decreased lethality after PTZ-induced seizures in rats.

C: Graph shows the average maximal score of the animals in each group. D,E: Graphs show latency to jerks (D) and to generalized convulsions (E) in the three groups of rats. F: Pretreatment with NMDA receptor channel blockers decreased the duration of seizures. All measurements were completed within 30 min of the PTZ injection. *P 0.05, **P 0.01, ***P 0.001.

(P 5 0.08; Fig. 1D,E). Both blockers suppressed the duration of tonic convulsions, thereby reducing the total time of the convulsions overall, but only the effect of IEM1921 was statistically significant (Fig. 1F). To compare the distinct seizure intensity stages during PTZ-induced seizures in the control and experimental animals, we established a seizure time-course pattern for each animal. For this purpose, we divided the 30-min observation period into 30 consecutive 1-min intervals

and determined the maximal seizure score during each interval. Two exemplary seizure patterns of the PTZtreated rats (black line) and the rats pretreated with memantine before the PTZ injection (gray line) are presented in Figure 2A, which illustrates the starting points of the different seizure stages along the time axis, with time in minutes expressed on the X-axis and seizure scores on the Y-axis. We then determined the proportion of animals with different seizure stages in each group (Fig. 2C)

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Fig. 2. Temporal dynamics of PTZ-induced seizures in the control and experimental groups of rats. A: Two exemplary seizure timecourse patterns after PTZ administration. The black line denotes a rat in the control group, and the gray line denotes a rat pretreated with memantine. B: Temporal dynamics of average seizure score in the three groups. *P < 0.05 statistical significance of the differences

between the control group and the group pretreated with memantine, Mann-Whitney test; #P < 0.05 statistically significant differences between control group and the group pretreated with IEM-1921, Mann-Whitney test. C: Graphs show the percentage of rats at different seizure stages during the first 30 min following the PTZ injections.

within each 1-min interval and calculated changes in the average seizure score during the observation period in the different groups (Fig. 2B). The graphs clearly show the diverse seizure types in the different animal groups. Most

animals in the control group exhibited convulsions during the first 10–15 min after the PTZ injection, and the percentage of rats with convulsions then decreased, with only a few animals still having convulsions after 25 min. Journal of Neuroscience Research


Fig. 3. Morphology of different brain regions after PTZ-induced seizures. Temporal cortex layers II and III (A–C), the stratum pyramidale of CA1 (D–F), CA2 (G–I), CA4 (J–L), and DG (M–O) in naive control rats (A,D,G,J,M), rats treated with PTZ (70 mg/kg; B,E,H,K,N), and rats pretreated with 5 mg/kg of IEM-1921 1 hr before PTZ injection (C,F,I,L,O). In the naive control rats, pyramidal neurons and interneurons appear round or oval, with blue


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cytoplasm and a light blue nucleus with clear visible borders (A,D). After PTZ-induced seizures, damaged (compacted) pyramidal neurons (arrows) appear dark purple with shrunken cytoplasm and nucleus (B,E). Morphological changes were minor in the rats pretreated with 5 mg/kg of IEM-1921 1 hr before the PTZ injection. Scale bars 5 20 mm.

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Fig. 4. Percentage of dark neurons with decreased turgor in different brain regions (layers II and III, Cx II–III) and V and VI (Cx V–VI) of parietal cortex, ff1, ff2, and ff4 areas of the hippocampus in naive control rats (n 5 7), PTZ-treated rats (n 5 15), and rats pretreated with the NMDA blockers IEM1921 (PTZ 1 IEM-1921, n 5 12) or memantine (PTZ 1 memantine, n 5 12) 1 hr before PTZ injection. Data are presented as mean 6 SEM. *P < 0.05 difference from naive control, two-tailed Mann-Whitney U-test; #P < 0.05 difference from PTZ-treated rats.

Pretreatment with the NMDA receptor blockers reduced the percentage of animals with convulsions and strongly decreased the time interval during which the convulsions were observed. Moreover, the effect was much stronger in the rats pretreated with IEM-1921 (Fig. 2). Influence of Memantine and IEM-1921 on Morphological Changes in the Brain After PTZ Injections Seizures cause widespread neuronal abnormalities, including the formation of dark (compacted) neurons (Jortner, 2006; Baracskay et al., 2008), and morphological changes, which depend on the duration and intensity of the seizures. Memantine and IEM-1921 significantly attenuated seizures. Thus, we selected PTZ-treated animals that exhibited mostly mild seizures (seizure score 2.6 6 0.2, seizure duration 2.3 6 1.5 min, n 5 15) for morphological examination. In these properties the group of PTZ-treated rats did not differ from the memantine-pretreated rats (seizure score 2.4 6 0.3, seizure duration 2.0 6 1.6 min, n 5 12). The examination of Nissl-stained brain tissue from naive control, PTZ-treated, and experimental groups of rats pretreated with the NMDA receptor blockers revealed that PTZ caused significant changes in neuronal morphology (Figs. 3, 4). The brains of all the PTZ-treated rats contained dark pyramidal neurons, which were most abundant (about 20–25% of the total neuron population) in the superficial layers of the parietal cortex (Fig. 3B), in the CA1 (Fig. 3E) and the CA4 areas (Fig. 3K), and in the dentate gyrus (DG) of the hippocampus (Fig. 3N). No changes were observed in the CA2 area of the hippocampus (Fig. 3H) or in the deep cortical layers. Pretreatment with memantine or IEM-1921 1 hr before the PTZ injection decreased the number of dark pyramidal neurons.

There were no observable differences in the effects of memantine and IEM-1921 (Fig. 4). The effect of the NMDA receptor channel blockers was greatest in the DG, where no dark neurons were observed (Fig 3O). Analysis of the correlation between the latency and the duration of the PTZ-induced convulsions and the percentage of dark neurons revealed no significant correlations either in the hippocampus or in the cortex (Spearman rank-order correlations in all tests, P > 0.05). Representative scatterplots are shown in Figure 5A,B. The number of dark neurons did not differ between PTZ-treated rats with generalized convulsions (seizure score 4, n 5 4) and PTZ-treated rats that exhibited only short myoclonic seizures lasting less than 45 sec (seizure score 2, n 5 7; CA1 33.5% 6 6.0% vs. 23.6% 6 5.6%, two-tailed Mann-Whitney U-test, P 5 0.19; CA4 17.6% 6 3.5% vs. 18.6% 6 2.8%, P 5 0.85; cortex 18.6% 6 1.7% vs. 20.1% 6 0.9%, P 5 0.34). Even seizures with mild behavioral components prompted the appearance of dark cells in the cortex and the hippocampus. The NMDA receptor channel blockers decreased the number of dark neurons in rats that exhibited convulsions of different severity. For example, one memantine-pretreated rat had tonic–clonic seizures lasting almost 20 min. However, morphological examination of the brain did not reveal an increased number of dark neurons (CA1 3.1%, CA4 8.5%, parietal cortex 11.7%) compared with other animals in that group (Fig. 5B). These results suggest that inhibition of NMDA receptors during seizures effectively prevents the formation of dark neurons independently of the antagonists’ anticonvulsant effectiveness. The immunohistochemical analysis of the distributions of caspase-3 and Fox3 (NeuN) neuronal proteins in the neocortex and the hippocampus of the PTZ-treated rats (Fig. 6) revealed that almost all the neurons, including dark ones, were Fox3 positive, and only a few were caspase-3 positive. There were no differences between the PTZ-treated and the naive control rats either in the number of Fox3-positive neurons (Fig. 7A) or in the number of caspase-3-positive cells in the CA1 area of the hippocampus (4.6% 6 4.2% vs. 4.6% 6 4.0%, two-tailed Mann-Whitney U-test, P < 0.05). Analysis of caspase-3 activity in the hippocampus of the PTZ-treated rats showed no difference compared with that in the naive control (Fig. 7B). The total number of cells in the CA1 area of the hippocampus in the PTZ-injected rats was the same as that in the naive control animals (Fig. 7C). Thus, our results indicate that all neurons, including the dark ones, were alive and that there was no neuronal cell loss 24 hr after the PTZ-induced seizures. Levels of Glutamate Transporter EAAT1 Protein Expression Seizures increase the extracellular endogenous glutamate concentration (Millan et al., 1991; Minamoto et al., 1992; Pena and Tapia, 2000; Meurs et al., 2008; Szyndler et al., 2008; Kanamori and Ross, 2011), and excessive glutamate induces rapid upregulation of astrocyte glutamate transport as a compensatory Journal of Neuroscience Research


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Fig. 5. Proportion of dark neurons in the neocortex and the hippocampus, showing a lack of correlation with seizure latency and duration. A: Scatterplot shows no correlation between the proportion of dark neurons in superficial layers of parietal cortex and seizure latency (Spearman rank-order correlations (RS); PTZ group RS 5 0.30, P 5 0.28; memantine group RS 5 0.21, P 5 0.55). B: Scatterplot shows no correlation between the proportion of dark neurons in field CA1 of the rat hippocampus and seizure duration (PTZ group

RS 5 0.37, P 5 0.18; memantine group RS 5 –0.15, P 5 0.64). Note that the proportion of dark neurons in both structures was significantly smaller in the memantine-pretreated group than in the control PTZtreated group. Squares denote the PTZ-treated group, and circles indicate the memantine-pretreated group. Open plots denote a maximal seizure score of 1, light gray a seizure score of 2, gray a seizure score of 3, and black a seizure score of 4.

mechanism (Duan et al., 1999; Sheldon and Robinson, 2007; Doi et al., 2009). Comparison of the expression level of EAAT1 in hippocampal tissue of the PTZtreated rats and those pretreated with IEM-1921 with

that of naive control animals by Western blot analysis revealed increased expression in the PTZ-treated rats (148.2% 6 12.0% of naive control, P < 0.05; Fig. 8). In the rats treated with PTZ and the NMDA receptor

Fig. 6. Results of immunohistochemical analysis of hippocampal caspase-3 and Fox-3/NeuN expression in the rats 24 hr after PTZinduced seizures. A: Microphotography of a caspase-3-positive neuron in the stratum pyramidale of CA1 in a PTZ-treated rat. Such neurons were observed very infrequently. B: Antibody staining for Fox-3/ NeuN in the hippocampus. The lack of gaps between the neurons

indicates that all express NeuN and are functioning. C–E: Immunostaining for Fox-3 protein: green channel (C), blue nuclear counterstain with Hoechst 33342 (D), and a merged image (E). Note that all the neurons, including the compacted ones, are Fox-3 positive. Scale bars 5 20 lm in A; 100 lm in B; 30 lm in C,D.


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Fig. 7. Results of morphometry and colorimetric analysis of caspase-3 activity of PTZ-treated group and control group. A: Percentage of Fox-3/NeuN-positive cells in the parietal cortex and in the stratum pyramidale of the ff1 and ff4 hippocampus areas of the naive control (n 5 7) and PTZ-treated rats (n 5 15). The total number of cells in each area was estimated based on the number of Hoechst 33342-

positive cells. No differences were found between naive control and PTZ-treated rats. P > 0.05, two-tailed Mann-Whitney U-test. B: Graph shows no difference in caspase-3 activity in the hippocampus tissue of the PTZ-treated rats and the naive control. C: The density of cells in the CA1 area of the hippocampus was the same in the PTZ-injected rats and the naive control animals.

channel blocker IEM-1921, the expression was only 126.8% 6 12.1% of the control level (P > 0.05), although the difference was not statistically significant compared with the PTZ-treated rats.

noncompetitive NMDA receptor channel blockers, memantine and IEM-1921. These types of antagonists act in a “use-dependent” manner, meaning that they usually block the channel only when it is open (Traynelis et al., 2010). NMDA receptor channel blockers might be particularly useful in cases of increased glutamate concentration (Parsons et al., 1995). Although structurally different, IEM-1921 and memantine showed almost the same usedependent activity against NMDA receptor-mediated currents in in vitro models. Previous authors have proposed similar mechanisms of action for these channel blockers (Blake et al., 1992; Parsons et al., 1995; Nikolaev et al., 2012). Our data on the anticonvulsant effects of memantine (5 mg/kg) and IEM-1921 (5 mg/kg) are in agreement with previously reported data (Rogawski et al., 1989; Blake et al., 1992; Parsons et al., 1999). In the PTZ test, both compounds significantly decreased the duration of the tonic component of seizures, whereas they were ineffective in preventing myoclonic seizures. Earlier studies described the high efficacy of memantine against tonic seizures induced by electroshock (Chojnacka-Wojcik et al., 1983; Urbanska et al., 1992; Kleinrok et al., 1995; Parsons et al., 1995; Czuczwar et al., 1996) or various chemoconvulsants, such as PTZ, bicuculline, picrotoxin, 3-mercaptopropionic acid, and NMDA, in rats and mice (Meldrum et al., 1986; Bisaga et al., 1993; Parsons et al., 1995; Geter-Douglass and Witkin, 1999; Lukomskaya et al., 2004; Mares and Mikulecka, 2009). These earlier studies showed that the ED50 for memantine in different models varied from 5 to 12 mg/kg. IEM-1921 exerted anticonvulsant effects in MES with an ED50 of 7 mg/kg when administered intraperitoneally (Rogawski et al., 1989; Thurkauf et al., 1990) or 14 mg/kg when administered orally in mice (Blake et al., 1992). Memantine and IEM-1921 also effectively prevented audiogenic seizures in Krushinski-Molodkina rats genetically prone to these seizures (Vataev et al., 2010; Lukomskaya et al., 2014).

DISCUSSION Anticonvulsant Properties of Memantine and IEM-1921 The present study used the PTZ-seizure test to estimate and compare the anticonvulsant properties of two

Fig. 8. Comparison of the expression of the EAAT1 protein in the hippocampus tissue in the three groups of rats (naive control group, rats treated with PTZ, and rats pretreated with IEM-1921 before PTZ injection) by Western blot analysis. The ratio of the optical density of EAAT1 bands to actin bands was counted in each tissue sample. *P < 0.05 statistically significant difference, two-tailed Mann-Whitney U-test.



Previously reported data on the protective efficacy of IEM-1921 in PTZ tests are scarce. One group reported that IEM-1921 protected only four of 10 mice at a dose of 30 mg/kg in a PTZ seizure test (s.c. injection at the CD97 convulsant dose [85 mg/kg] of PTZ; Rogawski et al., 1989) and that it was highly effective at low concentrations against the development of PTZ kindling (Lukomskaya et al., 2007). Although IEM-1921 and memantine have the same ED50 according to the MES test (Rogawski et al., 1989; Parsons et al., 1995, 1999), in the PTZ test described here IEM-1921 anticonvulsant properties were more pronounced than those of memantine. IEM-1921 decreased the average duration of convulsions by 82%, whereas memantine did so by only 37%. Memantine (5 mg/kg) reduced the lethality caused by PTZ-induced seizures from 42% to 11% (P < 0.01), and all animals pretreated with IEM-1921 (5 mg/kg) survived. Several hypotheses could explain why these two blockers exhibited different anticonvulsant potency against PTZ-induced seizures. Because the molecules are structurally different, their pharmacokinetics, mechanisms of absorption and distribution, and ability to cross the blood–brain barrier might differ. For example, according to some observations, the speed with which memantine (5 mg/kg) exerts anticonvulsant activity seems to be much slower than that of IEM-1921 at the same dose in Krushinski-Molodkina rats genetically prone to audiogenic seizures (Lukomskaya et al., 2014). In the present study, animal death observed after severe tonic–clonic or pure tonic seizures in the PTZ test most likely was due to fatal respiratory failure or cardiac arrhythmia (Tomson et al., 2008; Surges and Sander, 2012). Memantine and IEM-1921 reduced or completely prevented the tonic–clonic component of seizures and, accordingly, reduced the rate of animal deaths. However, we cannot exclude the possibility that NMDA receptor channel blockers directly affect the centers controlling respiration and the circulatory system, inasmuch as NMDA receptors are involved in their regulation, and they might be specifically active under conditions of stress (Waters and Machaalani, 2005; Pelosi et al., 2012). Neuroprotective Effects of NMDA Receptor Channel Blockers In the present study, PTZ-induced seizures caused widespread neuronal abnormalities in the rat neocortex and hippocampus. One day after PTZ injection, we observed an increased number of dark pyramidal neurons in the brains of all rats. In striking contrast, there were relatively few dark cells in the brains of the control animals. The formation of dark neurons has been previously reported in ischemia (Jenkins et al., 1981; Kovesdi et al., 2007), hypoglycemia (Auer et al., 1985a,b), epilepsy (Sloviter, 1983; Soderfeldt et al., 1983), and exposure to excitatory amino acids (Sloviter and Dempster, 1985). There is some discrepancy in observations of the formation of dark neurons caused by PTZ-induced seizures. In one study, a single administration of a convulsive PTZ dose Journal of Neuroscience Research

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(60 mg/kg) caused the appearance of dark neurons in the rat hippocampus after 24 hr (Ahmed et al., 2005), whereas, in another study, dark neurons in the hippocampus were not observed after a single administration of either a subconvulsive (37.5 mg/kg) or a convulsive (80 mg/kg) PTZ dose. However, dark neurons appeared after repeated administrations of PTZ (Aniol et al., 2011). The discrepancy in the results might be due to differences in experimental conditions, such as the intensity and duration of epileptic seizures. Currently, dark neurons are not considered dying neurons but rather are considered neurons at risk of undergoing subsequent cell death, inasmuch as recovery and volumetric expansion might occur en masse in dark neurons (Auer et al., 1985a; Csordas et al., 2003). In agreement with previous findings, this study confirmed that the dark neurons in the present study were alive, with most condensed neurons expressing the Fox3 neuronal protein (NeuN positive) and being caspase-3 negative. Both antigens have been widely used in neuropathological studies to highlight the physiological status of neurons. Healthy neurons show intense NeuN expression, and decreased NeuN positivity can be indicative of degeneration of differentiated neurons (Lavezzi et al., 2013). Caspase-3 has a major role in apoptosis, playing an essential part in the initiation and regulation of downstream proteolytic events leading to cell death (D’Amelio et al., 2012). The current data showed no changes in caspase-3 activity in hippocampal tissue 1 day after PTZ-induced seizures, suggesting the absence of widespread apoptotic cell death. The exact mechanism of dark cell formation is still unknown. However, it has been suggested that depolarization, augmented glutamate release, and glutamate receptor activation are likely mechanisms of dark neuron production (Kherani and Auer, 2008). In animals treated with PTZ, all these conditions can be met. Indeed, PTZ is a g-aminobutyric acid type A (GABAA) receptor antagonist (IC50 5 0.6 mM) and has been shown to reduce GABAA-mediated inward chloride currents by interacting with the GABAA receptor complex in the adult brain (Ramanjaneyulu and Ticku, 1984; Huang et al., 2001), thus facilitating depolarization of the neurons. Studies have also demonstrated that the extracellular endogenous glutamate concentration increases in the cortex and the hippocampus in response to PTZ injection (Feng et al., 2005; Szyndler et al., 2008). Our observation that the expression level of the EAAT1 protein increased in rats treated with PTZ is in line with these findings. We found that dark pyramidal neurons were abundant in layers II and III of the parietal cortex, in the stratum pyramidale of the CA1 and CA4 areas of the hippocampus, and in the DG of the hippocampus. There were very few such neurons in the deep cortical layers and in the CA2 area of the hippocampus. Thus, we suggest that PTZ-induced seizures cause hyperactivation of a restricted population of neurons. It is worth noting that the strongest neuronal loss in different models of epilepsy has been observed in similar areas (Holmes, 2002). For example, during PTZ-induced kindling, the number of damaged

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neurons in the DG and hilus of the rat hippocampus progressively increased (Aniol et al., 2011). After 2–10 weeks of postkindling, the total number of neurons decreased in the following order of regional vulnerability CA1>DG>CA4>CA21CA3 (Franke and Kittner, 2001). Repeated PTZ insults also specifically damaged somatosensory cortical layers III and IV (Park et al., 2006). The current study shows that the NMDA receptor channel blockers memantine and IEM-1921 effectively prevent the appearance of dark neurons in the rat brain. The inhibition of the NMDA receptors led to a decrease of intracellular Ca21 influx. In the PTZ-treated rats, the elevation in intracellular Ca21 resulting from hyperactivation of the NMDA receptors might alter the neurons via Ca21-dependent neuronal injury. These results are in line with previous observations that NMDA antagonists MK801 (Kherani and Auer, 2008) and ketamine (LopezGalindo et al., 2008) prevent the formation of dark neurons. Inasmuch as NMDA receptor antagonists reduced the quantity of dark neurons to control values in the hippocampus and neocortex in PTZ-induced seizures, these results suggest that NMDA receptor antagonists could be considered possible neuroprotective agents for prolonged seizures or status epilepticus leading to neuronal damage. ACKNOWLEDGMENTS The authors thank Eugeny P. Zhabko for his excellent technical assistance. REFERENCES Ahmed MM, Arif M, Chikuma T, Kato T. 2005. Pentylenetetrazolinduced seizures affect the levels of prolyl oligopeptidase, thimet oligopeptidase, and glial proteins in rat brain regions, and attenuation by MK-801 pretreatment. Neurochem Int 47:248–259. Aniol VA, Stepanichev MY, Lazareva NA, Gulyaeva NV. 2011. An early decrease in cell proliferation after pentylenetetrazole–induced seizures. Epilepsy Behav 22:433–441. Auer RN, Kalimo H, Olsson Y, Siesjo BK. 1985a. The temporal evolution of hypoglycemic brain damage. I. Light- and electron-microscopic findings in the rat cerebral cortex. Acta Neuropathol 67:13–24. Auer RN, Kalimo H, Olsson Y, Siesjo BK. 1985b. The temporal evolution of hypoglycemic brain damage. II. Light- and electron-microscopic findings in the hippocampal gyrus and subiculum of the rat. Acta Neuropathol 67:25–36. Baracskay P, Szepesi Z, Orban G, Juhasz G, Czurko A. 2008. Generalization of seizures parallels the formation of “dark” neurons in the hippocampus and pontine reticular formation after focal–cortical application of 4-aminopyridine (4-AP) in the rat. Brain Res 1228:217–228. Bisaga A, Krzascik P, Jankowska E, Palejko W, Kostowski W, Danysz W. 1993. Effect of glutamate receptor antagonists on N-methyl-Daspartate- and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced convulsant effects in mice and rats. Eur J Pharmacol 242:213–220. Blake PA, Yamaguchi S, Thurkauf A, Rogawski MA. 1992. Anticonvulsant 1-phenylcycloalkylamines: two analogues with low motor toxicity when orally administered. Epilepsia 33:188–194. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.

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