УДК 612.816;612.014.423

intracellular blockade of GABAa receptors in the rat hippocampal neurons

© 2014 I. Khalilov1, 2, 3, X. Leinekugel4, M. Mukhtarov1, R. Khazipov1, 2, 3*

laboratory of neurobiology, IFMB, Kazan Federal University, Kremlevskaya ul., 18, Kazan, 420008, Russia 2INMED-Inserm U901, 163 avenue de Luminy, 13273 Marseille Cedex 09, France 3Université de la Méditerranée, UMR S901 Aix-Marseille 2, 13009 Marseille, France; *e-mail: roustem.khazipov@inserm.fr 4IMN, CNRS UMR 5293, Université Bordeaux 1, Bat B2, Avenue des Facultés, 33405 Talence, France

Received April 29, 2013

The intracellular blockade of GABAA-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABAa receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABAA receptor channels substitute for Cl- and in which Mg2+ and ATP are omitted to induce rundown of the GABAA receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg2+- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABAA-receptor-mediated synaptic currents and responses induced by the GABAA receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg2+, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities.

Keywords: GABA receptor, hippocampus, glutamate, AMPA receptor, NMDA receptor.

DOI: 10.7868/S023347551401006X

Cortical neuronal networks generate complex patterns of activity that are primarily driven by glutamatergic and GABAergic synaptic connections [1]. Analysis of the contribution of these synaptic connections to network-driven activities can be carried out by various approaches. One of the most efficient approaches for isolation of a specific conductance is based on pharmacological blockade of other types of conductances using specific antagonists. For example, GABAergic transmission can be studied after pharmacological blockade of glutamate receptors [2, 3], while glutamatergic conductances can be isolated after blockade of GABA receptors [4]. Although this approach is useful for isolation of a specific conduc-

tance, it is not suitable for investigation of network-driven activity, because pharmacological blockers affect the entire network. Indeed, blockade of glutamate receptors blocks network-driven activities, whereas blockade of GABAa receptors results in epileptiform activities [5]. Therefore, alternative approaches are required allowing separation of glutamatergic and GABAergic conductances in a given cell without affecting the network. An approach most commonly used is based on different voltage dependence of glutamatergic cationic conductance and GABAergic chloride conductance in the conditions of whole-cell recordings using low-chloride pipette solution [6—10]. With this approach, GABAergic postsynaptic currents

can be recorded by holding the membrane voltage at the reversal potential for glutamatergic currents, whereas glutamatergic postsynaptic currents can be recorded at the reversal potential for GABAergic postsynaptic currents. However, this approach has several disadvantages. Firstly, holding the cell membrane voltage exactly at the reversal potential for a given conductance may be difficult because of spatial clamp problems causing a membrane potential difference along the dendritic tree. Secondly, intracellular chloride concentration may not be the same in different cell compartments because of nonuniform distribution of chloride transporters and nonuniform dialysis of the intracellular milieu along the distance from the recording site [11]. In addition, even in the absence of transmembrane currents at the reversal potential for GABAergic conductance, opening of a large number of GABAa channels may shunt glutamatergic postsynaptic currents [12]. Another disadvantage is that glutamatergic currents can be separately recorded at one and only membrane potential (typically, AMPA receptor-mediated postsynaptic currents are recorded at around —70 mV); thus, determining the current—voltage relationships of glutamatergic conductance and participation of NMDA receptors that need to be recorded at voltages depolarizing the membrane to alleviate magnesium block is not feasible.

These problems can be overcome by blocking GABAA receptors from the inside of the cell. At present, specific antagonists of GABAA receptors acting at the intracellular side of the membrane are not available. However, blockade of GABAA currents can be achieved using two approaches. The first approach is based on the different permeability of GABAA channels to various anions. Fluoride is one of the anions to which GABAA channels are least permeable [13]. Therefore, replacement of intracellular chloride by fluoride during whole-cell recordings minimizes the outflow of anions (that corresponds to inward transmembrane currents) through GABA channels. At the same time, currents mediated by the influx of chloride from the extracellular solution into the cell are little affected. The second approach to block GABAA-re-ceptor-mediated responses intracellularly may be based on the phenomenon of rundown of GABAA receptor channels that develops in the absence of intracellular Mg2+ and ATP [14]. Several reports have employed a combination of fluoride-based, Mg2+- and ATP-free intracellular solution to suppress the GABAA receptor function in the recorded cell and to isolate the glutamatergic component in the rat visual cortex [15] and hippocampus [16—20]. However, the efficiency and kinetics of the intracellular blockade of GABAA-receptor-mediated synaptic currents with F--based and Mg2+, ATP-free solution and the effects of such dialysis on glutamate (AMPA and NMDA) re-

ceptor-mediated postsynaptic currents have not been described.


Experiments were performed on hippocampal slices obtained from 1-5-day-old male Wistar rats. Following decapitation, the brain was rapidly removed and placed in oxygenated ice-cooled artificial cerebrospinal fluid (ACSF); hippocampal transverse slices (400-600-|m thick) were cut with Mcllwain tissue chopper and kept in oxygenated (95% O2 and 5% CO2; pH 7.3) ACSF (in mM: 126 NaCl; 3.5 KCl; 2.0 CaCl2; 1.3 MgCl2; 25 NaHCO3; 1.2 NaH2PO4; 1L glucose) at room temperature, at least one hour before use. Individual slices were then transferred to the recording chamber where they were fully submerged and super-fused with oxygenated ACSF at 30—32°C, at a rate of 2—3 mL/min.

Recordings were performed using patch-clamp technique in whole-cell configuration [21] using an Axo-patch 200 (Axon Instrument, USA) patch-clamp amplifier. Microelectrodes had a resistance of 5—7 MOm. Pipette-filling solution was of the following composition (in mM): 140 CsF; 1 CaCl2; 10 HEPES; 10 EGTA; pH 7.3; osmolarity 270—280 mOsm. In all experiments QX314 (1mM) was added to the pipette solution to block sodium-dependent action potentials and GABAB receptor-mediated currents [22]. Slices were stimulated by a bipolar electrode placed in stratum radia-tum or hilus; stimulation parameters were: amplitude, 10-50 V; duration, 30 |s; frequency, 0.02-0.05 Hz. Synaptic currents and agonist-evoked responses were digitized (10 kHz) and acquired into the memory of a computer for further analysis. Data were then analyzed using pClamp (Axon Instruments, USA), Mini-analysis (Synaptosoft, USA). Group measures are expressed as means ± SEM. Statistical significance of differences between means was assessed with the Student's t-test. The level of significance was set atp < 0.05.

Biocytin and tetrodotoxinwere were purchased from Sigma; isoguvacine, bicuculline, CNQX (6-cyano-7-ni-troquinoxaline-2,3 dione), and APV (d-2-Amino-5-phosphopentanoate, from Tocris Neuramin (UK).


Effects on GABAA-receptor-mediated responses.

The effect of dialysis with F--based and Mg2+, ATP-free solution on GABAA-receptor-mediated responses was monitored by recording spontaneous and evoked GABAA-receptor-mediated postsynaptic currents (PSCs) and responses induced by GABAA receptor agonist isoguvacine. Spontaneous GABAA-PSCs recorded within 5 min after the formation of whole-cell configuration were inwardly directed at a holding poten-


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