ISSN 0012-4966, Doklady Biological Sciences, 2017, Vol. 477, pp. 207–209. © Pleiades Publishing, Ltd., 2017. Original Russian Text © T.Yu. Postnikova, A.M. Trofimova, A.V. Zaitsev, L.G. Magazanik, 2017, published in Doklady Akademii Nauk, 2017, Vol. 477, No. 5, pp. 618–621.
PHYSIOLOGY
Status Epilepticus Induced by Pentylenetetrazole Increases Short-term Synaptic Facilitation in the Hippocampus of Juvenile Rats T. Yu. Postnikovaa, b, A. M. Trofimovaa ,b, A. V. Zaitseva,*, and Academician L. G. Magazanika, c,** Received July 3, 2017
Abstract—We studied the effect of status epilepticus (SE) on short-term synaptic plasticity. The amplitudes of field potentials in response to extracellular stimulation of the Schaffer collaterals were recorded in hippocampal slices. Subtle modifications were revealed on day 1 after SE, whereas on days 3 and 7 we did not find any differences from the control. These data show that, one day after SE, the probability of a transmitter release in hippocampal synapses decreases that serves as a compensatory mechanism, which prevents seizure activity. DOI: 10.1134/S0012496617060102
Temporal epilepsy is the most common type of human focal epilepsy and often develops due to the initial damage to the brain [1]. Febrile seizures, head traumas, birth traumas, or status epilepticus induced by various factors may serve as this initial damage [2]. Approximately 30% of temporal epilepsy cases are resistant to pharmacological treatment; therefore, prevention of epilepsy development after the initial damage is a prospective strategy for treatment of this disease [1]. In spite of thorough studies of the mechanisms of the damaging effect of seizures, processes triggering epileptogenesis, and compensatory capabilities of the brain, they remain unclear. In the present study, we examined the mechanisms of epileptogenesis and its compensation. During childhood the brain has great compensatory capabilities; therefore, it is more resistant to the damaging effect of seizures compared to the adulthood conditions [3]. In the present study, we used a model of pentileneterazole (PTZ) seizures in immature Wistar rats [4, 5]. In this model, PTZ induces single status epilepticus, after which the rats recover relatively well; no spontaneous seizures appear in the next period of life. a Sechenov
Institute of Evolutionary Physiology, Russian Academy of Sciences, St. Petersburg, Russia b Peter the Great St. Petersburg Polytechnical University, St. Petersburg, Russia c St. Petersburg State University, St. Petersburg, Russia *e-mail:
[email protected] **e-mail:
[email protected]
We hypothesized that one of the efficient mechanisms preventing epileptogenesis in the brain is a decrease in the probability of release of the excitatory transmitter glutamate in synapses of the hippocampus, the most vulnerable brain structure in temporal epilepsy. In order to revise this hypothesis, we studied the features of short-term synaptic plasticity in CA3– CA1 synapses of the hippocampus because this form of synaptic plasticity significantly depends on the probability of transmitter release [6]. Twenty- to twenty-two-day-old male Wistar rats weighting 35–40 g were used for the study. The rats were housed under the standard conditions at room temperature and free access to water and food. All experiments were performed according to the rules of experiments with laboratory animals approved by the Sechenov Institute of Evolutionary Physiology, Russian Academy of Sciences, and are in agreement with Russian and international regulations. Status epilepticus was induced in the experimental animals by intraperitoneal injection of PTZ (SigmaAldrich, United States) at a dose of 70 mg/kg. Only the animals which exhibited generalized clonico-tonic seizures for at least 45 min, i.e., status epilepticus, were selected for the experiment. The control animals were injected with isotonic saline solution. One, three, or seven days after seizure attack, the rats were decapitated. The slices were prepared by the method described in detail previously [7]. The brain was rapidly removed and the cerebellum, frontal pole, and a part of the dorsal brain surface were dissected. Horizontal 400-μm brain slices were cut using a Microm HM 650V vibratome (Microm, Germany) in an icecold (0°C) artificial cerebrospinal fluid (ACSF) con-
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0.3 mV 50 ms 0.3 mV 50 ms Fig. 1. Examples of the evoked field potentials in the CA1 field of the hippocampus in control and experimental rats one day after PTZ-induced status epilepticus at the interstimuli intervals of (a) 30 and (b) 100 ms. The facilitation value was calculated as a ratio of the amplitude of the first response to the amplitude of the second response. The values were 1.22 and 2.01 for the control and PTZ groups, respectively for the interval of 30 ms and 1.26 and 1.88 for the control and PTZ groups, respectively, for the interval of 100 ms.
sisting of (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, 24 NaHCO3, 10 glucose, which was aerated with carbogen (95% CO2/5% O2). The slices were placed into a bath filled with aerated ACSF and warmed at 35°C for 1 h, after which they were maintained at room temperature. After incubation, the slices were placed into the experimental chamber and maintained for 15–20 min prior to the start of electrophysiological study. The ACSF solution in the chamber was maintained at room temperature and constantly oxygenated at a rate no less than 5 mL/min. One to five slices from each animal were used for the experiment. Field excitatory postsynaptic potentials (fEPSP) were recorded in the radial layer of the CA1 hippocampal field using a glass microelectrode filled with ACSF with a resistance of 0.2–1.0 MΩ. Stimulating bipolar twisted electrode made of a nichrome wire in commercial isolation with a diameter of 0.1 mm was placed in the Schaffer collateral path of the radial layer on the border between the CA2 and CA1 fields of the rat hippocampus [4]. Stimulation was performed by paired rectangular electrical pulses with a duration of
0.1 ms applied every 20 s. The intensity of the stimulus used in the experiment was selected in such a way that the amplitude of fEPSP was 40–50% of the amplitude, which first evoked population spike. During the experiments, the strength of stimulation remained constant; it was 50–150 μA, and the amplitude of fEPSP was within the range of 0.5–1 mV. Short-term synaptic plasticity, i.e., facilitation, was examined using different inter-stimuli intervals, specifically, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, and 500 ms. The electrophysiological data were analyzed using the Clampfit 10.2 software (Axon Instruments, United States). The amplitude of each fEPSP was measured. The value of short-term synaptic facilitation was calculated as the ratio of the amplitude of the second fEPSP to the amplitude of the first fEPSP. Statistical analysis was performed and figures were prepared using the Statistica 8.0 (StatSoft, United States) and OriginPro 8 (OriginLab Corporation, United States) software. The significance of differences was calculated using analysis of variances (ANOVA) followed by the post hoc Fisher LSD test. We compared the ratios of the amplitudes of fEPSP pairs at various inter-stimuli intervals within the range of 10–500 ms in the control and experimental animals one, three, or seven days after PTZ-induced status epilepticus. Status epilepticus resulted in a statistically significant increase in short-term facilitation in synapses of pyramidal cells of the hippocampal CA1 field compared to the control level (F3, 26 = 3.27, p < 0.05; Figs. 1 and 2). Maximum facilitation of the responses was observed 1 day after status epilepticus; the averaged value within the whole range of inter-stimuli intervals studied was 1.46 ± 0.05 (n = 6). This value was 16% higher as compared to the facilitation level in the control group, in which it was 1.26 ± 0.04 (n = 12, p < 0.01). The averaged values of facilitation three or seven days after status epilepticus were 1.35 ± 0.05 (n = 7) and 1.34 ± 0.06 (n = 5), respectively, and they did not significantly differ from that in the control group. Note that the facilitation values in all groups depended on the duration of the inter-stimuli intervals (F14, 224 = 66.4, p < 0.001) and maximum facilitation was at the intervals of 30–80 ms whereas facilitation practically disappeared at an interval of 200 ms (Fig. 2). After status epilepticus, the degree of facilitation at different inter-stimuli intervals changed differently (F42, 224 = 1.82; p < 0.01). On the first day a significant increase in facilitation was observed within the range inter-stimuli intervals of 10–150 ms (except 50 ms). On the seventh day a significant increase in facilitation was found only for the shortest inter-stimuli intervals of 10–20 ms. On the third day, no significant differences in the value of facilitation were found as compared to control for all inter-stimuli intervals. The increased facilitation indicated a lower probability of the release of presynaptic glutamate in synDOKLADY BIOLOGICAL SCIENCES
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STATUS EPILEPTICUS
pus may be a promising antiepileptogenic approach, which potentiates the compensatory mechanisms of the brain and prevents the development of temporal lobe epilepsy after the primary damage. The drugs may, e.g., include antagonists of the calcium channels of P/Q- or N-types, which promote calcium input into the presynaptic terminal [9]; agonists of group II metabotropic glutamate receptors [10], which inhibit functioning of calcium channels through several intermediaries; or substances modulating the functions of proteins involved in the molecular mechanisms of vesicle fusion with the presynaptic membrane [11].
Facilitation 1.8 Control 1 day 3 days 7 days
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Fig. 2. Values of short-term synaptic facilitation at various inter-stimuli intervals in control (n = 12) and experimental rats one, three, and seven days (n = 6, n = 7, and n = 5, respectively) after PTZ-induced status epilepticus. M ± m. (*) Differences between the control and experimental groups one day after PTZ-induced status epilepticus are significant at p < 0.05.
apses of the CA1 Schaffer collaterals [6] one day after status epilepticus. The release of a transmitter is a result of a set of processes which start from the arrival of action potential depolarizing the presynaptic terminal. This allows the calcium input via the voltagegated channels that triggers the mechanisms of vesicle fusion to the presynaptic membrane. The transmitter release depends on three main factors: the number of the vesicles that are ready to immediate release, calcium concentration in the presynaptic terminal, and efficient operating of all molecular mechanisms responsible for calcium sensing and vesicle fusion [8]. Thus, the probability of the transmitter release depends on each of these factors. Our data support the hypothesis that one of the compensatory mechanisms that prevent epileptogenesis in the model of PTZ-induced of seizure conditions can be a reduced probability of the neurotransmitter release. These results allow us to suggest that application of pharmacological tools directed to attenuation of a probability of glutamate release in the hippocam-
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ACKNOWLEDGMENTS This study was supported by the Russian Science Foundation, project no. 16-15-10202. REFERENCES 1. Curia, G., Lucchi, C., Vinet, J., et al., Curr. Med. Chem., 2014, vol. 21, pp. 663–688. 2. Bruton, C.J., The Neuropathology of Temporal Lobe Epilepsy, Oxford: Oxford Univ. Press, 1988. 3. Ben-Ari, Y., Dialog. Clin. Neurosci., 2008, vol. 10, pp. 17–27. 4. Postnikova, T.Y., Zubareva, O.E., Kovalenko, A.A., Kim, K.K., Magazanik, L.G., and Zaytsev, A.V., Biochemistry (Moscow), 2017, vol. 82, pp. 282–290. 5. Zaitsev, A.V., Kim, K.K., Vasilev, D.S., Lukomskaya, N.Y., Lavrentyeva, V.V., Tumanova, N.L., Zhuravin, I.A., and Magazanik, L.G., J. Neurosci. Res., 2015, vol. 93. 6. Zucker, R.S. and Regehr, W.G., Annu. Rev. Physiol., 2002, vol. 64, pp. 355–405. 7. Kryukov, K.A., Kim, K.K., Magazanik, L.G., and Zaitsev, A.V., Neuroreport, 2016, vol. 27, pp. 1191– 1195. 8. Branco, T. and Staras, K., Nat. Rev. Neurosci., 2009, vol. 10, pp. 373–383. 9. Rogawski, M.A. and Loscher, W., Nat. Rev. Neurosci., 2004, vol. 5, pp. 553–564. 10. Alexander, G.M. and Godwin, D.W., Epilepsy Res., 2006, vol. 71, pp. 1–22. 11. Lynch, B.A., Lambeng, N., Nocka, K., et al., Proc. Natl. Acad. Sci. U.S.A., 2004, vol. 101, pp. 9861–9866.
Translated by M. Stepanichev
