БИОЛОГИЧЕСКИЕ МЕМБРАНЫ, 2009, том 26, № 2, с. 94-103
УДК 577.352
SYNAPTIC MECHANISMS OF USE-DEPENDENT POTENTIATION AND USE-DEPENDENT INHIBITION OF EVOKED BURST DISCHARGES
IN RAT HIPPOCAMPAL SLICES
© 2009 r. N. V. Kalashnikova, V. G. Motin, B. I. Khodorov
Institute of General Pathology and Pathophysiology RAMS, Baltiiskaya ul, 8, Moscow, 125315 Russia;
Tel/Fax: (495)1511756; e-mail: boriskhodorov@mtu-net.ru Received 10.11.2008
The mechanisms of activity-dependent modulation of burst discharges in rat hippocampal slices have been studied. The extracellular registration of field responses (population spike, PS, and field excitatory postsynaptic potential, fEPSP) induced by repetitive electrical stimulation (1-4 Hz) of Schaffer collaterals (with 30 pulses trains separated by 5-min resting intervals) was performed in cellular and dendritic layers of CA1 area. It has been established that repetitive orthodromic stimulation exerts biphasic modulation of burst discharges in Mg2+-free medium: use-dependent potentiation (UDP) and use-dependent inhibition (UDI). The former was manifested as an increase in the number of PS in the burst discharge associated with a corresponding lengthening of the fEPSP. During the UDI development the number of NMDA-dependent PS in the burst diminished despite the continuing increase in the fEPSP duration. In some cases UDI was followed by spreading depression. Both UDP and UDI were reversible. The development of UDP and UDI could be effectively suppressed either by the NMDA antagonists or by the GABAergic inhibition enhancer, diazepam. The picrotoxin (PTX)-induced burst discharges did not undergo either UDP or UDI development. However, removal of Mg2+ from PTX-containing solution during continuing repetitive stimulation led to the appearance of NMDA-dependent UDI. Analysis of the data obtained indicates that: 1) UDP results from a progressive decrease in GABA-medi-ated inhibition in the course of low-frequency (1-4 Hz) repetitive stimulation (the so-called "fatigue of synaptic inhibition"); 2) UDI is caused by excessive Ca2+ influx into the neurons due to overactivation of NMDA receptors.
Key words: burst discharge, use-dependent potentiation, use-dependent inhibition, repetitive stimulation, epileptiform activity, hippocampal slices, CA1 area.
Removal of Mg2+ from the external solution super-fusing hippocampal slices is known to create the conditions for an appearance of the seizure-like neuronal activity in response to electrical stimulation of afferent fibres [1-4]. It is also shown that this stimulation not only triggers the epileptiform burst discharges but can also strongly affect their characteristics [5-9]. However, the exact mechanisms of epileptiform bursts generation and their activity-dependent modulation are not fully understood yet.
The experimental approach used in the present work to study this problem was motivated by the following finding. We revealed [10] that in Mg2+-free medium the low-frequency stimulation of Shaffer collaterals by trains of stimuli (30 stimuli; 1-4 Hz) induced biphasic changes in the burst discharge including their "use-dependent potentiation" (UDP) followed by a transient "use-dependent inhibition" (UDI) and spreading depression (SD). The objective of this study was to clarify
Abbreviations: PS - population spike, fEPSP - field excitatory postsynaptic potential, UDP - use-dependent potentiation, UDI -use-dependent inhibition, SD - spreading depression, PTX - picrotoxin, DZ - diazepam.
the origin of these events and, in particular, a contribution of AMPA, NMDA, and GABA receptors to their development.
EXPERIMENTAL
Preparation of brain slices. Experiments were performed on transverse hippocampal slices (350-^m thick) from male Wistar rats (150-180g). After decapitation under ether anesthesia, the hippocampal slices were cut using a vibratom tissue chopper and then transferred to a half-submerged type recording chamber. Slice preparation was described in detail in a previous publication [11]. Slices were maintained at 35 ± ± 0.5°C in the artificial cerebrospinal fluid ( ACSF; perfusion rate 2 ml/min) oxygenated with 95% O^5% CO2. The composition of ACSF was as follows (mM): NaCl 124, KCl 5, NaH2PO4 1.25, MgSO4 2, CaCl2 2, NaHCO3 26, D-glucose 10. ACSF was equilibrated with 95% O2/5% CO2 to a final pH of 7.4. In Mg2+-free ACSF MgSO4 was omitted.
Electrophysiological experiments. The slices were allowed to equilibrate for at least 2 h before the record-
Fig. 1. a, Schematic diagram of the rat hippocampal slice preparation showing the position of stimulating and recording electrodes in CA1. b, Simultaneous extracellular recordings of population spikes, PS (top traces), from stratum pyramidale (Pyr) and field excitatory postsynaptic potentials, fEPSP (bottom traces), from stratum radiatum (Rad). Stimulation of Schaffer collateral-commis-sural pathway (Stim) applied every 5 min with low-frequency long trains of stimuli (30 pulses; 1 Hz). Superposition of field potentials traces (PS, fEPSP) elicited by 1st, 15th, and 30th stimuli in the train in the presence of external Mg (control) and in Mg -free medium (0 Mg).
ings. Standard extracellular field recording technique was employed using single-barrel electrodes filled with 150 mM NaCl. The population spike (PS) of CA1 pyramidal neurons in the stratum pyramidale and a field excitatory postsynaptic potential (fEPSP) from stratum radiatum in the apical dendritic tree layer were recorded simultaneously in response to electrical stimulation of afferent fibres. Stimulus-evoked changes in extracellular Ca2+ concentration ([Ca2+]o) were recorded in the stratum pyramidale of CA1 by using double-barreled reference Ca2+-sensetive microelectrodes prepared from theta glass and filled with Ca2+ cocktail as was previously described [12, 13]. The repetitive electrical stimulation (0.5-4 Hz) of Schaffer collaterals (by trains of 30 pulses separated by 5-min resting intervals) was performed using bipolar platinum wire electrodes placed into the CA1 area for orthodromic stimulation [14, 15]. All stimulation intensities were set at halfmaximal levels. The responses recorded with an amplifier were digitized at 20 kHz and analyzed with original software developed for the spike activity processing.
Drug administration. Epileptiform activity was induced by removal of Mg2+ from the perfusion solution and application of the gamma-aminoaminobutyric acid (GABA)A antagonist, picrotoxin (PTX, 20 |M). All drugs were dissolved in the ACSF to the desired final concentrations and applied by switching the perfusion from control solution to the drug-containing medium. All solutions were equilibrated with 95% O2/5% CO2 to final pH of 7.4. DL-2-amino-5-phosphonovaleric acid (APV, 50 |M) and 6-cyano-7-nitroquino-xaline-2,3-dione (CNQX, 10 or 30 |M) were used as selective antagonists of NMDA- and AMPA/kainite glutamate receptors, respectively. In some experiments, MK-801
(50 |M) was used to block the NMDA channels and, to elucidate the role of GABAergic system, diazepam (DZ, 10 | M) was added.
Data analysis. The results were obtained in experiments carried out on 84 hippocampal slices derived from 76 rats. Results in the text and in the figures are expressed as mean ±SEM. Statistical evaluations were performed using ANOVA or Student's t-test.
RESULTS
Evoked Activity in Mg2+-Containing Medium
Under control conditions (in the presence of external Mg2+) each stimulus in the train of 30 pulses (1 Hz) led to the appearance of a single response: the primary population spike (primary PS) in stratum pyramidale (the cell body layer), and correspondingly the field excitatory postsynaptic potential (fEPSP) in stratum radiatum (the dentritic layer) (Fig.1). In agreement with the existing data [16, 17] these responses proved to be resistant to both competitive and non-competitive NMDA antagonists (50 |M APV and 50 |M MK-801, respectively) and sensitive to non-NMDA-receptor antagonist CNQX (30 |M) (not shown). In contrast, CNQX (30 |M) abolished both these responses, indicating the involvement of the AMPA-receptors.
Effect of Mg2+ Removal from the Medium
Use-dependent potentiation (UDP). Replacement of the normal perfusate by Mg2+-free medium induced an increase in the magnitude of the primary PS and the appearance of multiple additional PS (so-called
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PS fEPSP
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Fig. 2. Use-dependent enhancement of epileptiform discharges in Mg2+-free medium is reversible. The upper trace (a) shows the changes in the number of PS in the burst discharge (NPS) during two sequential trains of pulses (30 stimuli, 1Hz) separated by a 5-min resting interval in nominally Mg2+-free medium. b, Selected examples of extracellular responses (PS and fEPSP) evoked by repetitive orthodromic stimulation. Both PS (upper trace) and fEPSP (lower trace) induced by the 1st, 15th, and 30th stimuli in the train are shown. The numbers in a and b correspond to the number in the pulse in the train. Stimulus artifacts were truncated for clarity. All stimulation intensities were set at half-maximal levels.
"polyspiking") [10, 14] associated with prolonged fEPSP.
Both fEPSP duration and the number of additional PS (post-PS) in the burst progressively increased during repetitive stimulation, as is shown in Fig. 2. This was UDP retained during the whole period of the superfusion with Mg2+-free solution (40-60 min) and disappeared only after a return to the Mg2+-containing medium. Thus, the PS amplitude and EPSP duration estimated 40 min after Mg2+ removal from external solution were 196.7 ± 12.1% (n = 6, p < 0.05) and 162.9 ± 9.61% (n = 6, p < 0.05) as compared to the respective control values. In all experiments (n = 79) UDP was reversible. It diminished or even disappeared within a 5-min resting interval despite the absence of Mg2+ in the medium, and was resumed during the
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