Distinct Functional and Pharmacological Properties of Tonic and Quantal Inhibitory Postsynaptic Currents Mediated by g-Aminobutyric AcidA Receptors in Hippocampal Neurons

g-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter, activates a persistent low amplitude tonic current in several brain regions in addition to conventional synaptic currents. Here we demonstrate that GABAA receptors mediating the tonic current in hippocampal neurons exhibit functional and pharmacological properties different from those of quantal synaptic currents. Patch-clamp techniques were used to characterize miniature inhibitory postsynaptic currents (mIPSCs) and the tonic GABAergic current recorded in CA1 pyramidal neurons in rat hippocampal slices and in dissociated neurons grown in culture. The competitive GABAA receptor antagonists, bicuculline and picrotoxin, blocked both the mIPSCs and the tonic current. In contrast, mIPSCs but not the tonic current were inhibited by gabazine (SR-95531). Coapplication experiments and computer simulations revealed that gabazine bound to the receptors responsible for the tonic current but did not prevent channel activation. However, gabazine competitively inhibited bicuculline blockade. The unitary conductance of the GABAA receptors underlying the tonic current (;6 pS) was less than the main conductance of channels activated during quantal synaptic transmission (;15–30 pS). Furthermore, compounds that potentiate GABAA receptor function including the benzodiazepine, midazolam, and anesthetic, propofol, prolonged the duration of mIPSCs and increased tonic current amplitude in cultured neurons to different extents. Clinicallyrelevant concentrations of midazolam and propofol caused a greater increase in tonic current compared with mIPSCs, as measured by total charge transfer. In summary, the receptors underlying the tonic current are functionally and pharmacologically distinct from quantally activated synaptic receptors and these receptors represent a novel target for neurodepressive drugs. g-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, modifies electrical activity in the brain by regulating membrane hyperpolarization and the “shunting” of excitatory input. GABA released from presynaptic terminal binds to GABAA receptors clustered at the postsynaptic membrane and activates inhibitory postsynaptic currents (IPSCs). In addition to conventional quantal synaptic transmission, a persistent form of GABAergic inhibition has been described in several brain regions. A small but significant tonic GABAergic current has been observed in the cerebellum (Brickley et al., 1996; Wall and Usowicz 1997), cortex (Salin and Prince, 1996), thalamus (Liu et al., 1995), and hippocampus (Otis et al., 1991). This tonic current has been best characterized in the cerebellum, where glomerular structures that surround synapses onto granule cells serve as a repository for transmitter released from neighboring synapses. Transmitter in the glomerulus may activate high-affinity GABAA receptors with minimal desensitization properties that are located in perisomatic and extrasynaptic regions of granule cells (Rossi and Hamann, 1998). The mechanisms that regulate the tonic GABAergic inhibition in other brain regions are not well understood. The tonic conductance in the hippocampus may result from the summation of overlapping miniature IPSCs (Soltesz et al., 1995; Salin and Prince, 1996), or the spill-over of vesicular transmitter released from neighboring synapses (Brickley et al., 1996; Rossi and Hamann, 1998). Recently, it was postulated that the tonic current results from the release of GABA from a surface matrix reservoir that becomes exposed during exocytosis (Vautrin et al., 2000). Also, reverse operation of GABA cotransporters (Gaspary et al., 1998) or release of GABA from astrocytes (Liu et al., 2000) might elevate GABA to concentrations sufficient to activate receptors. The in vivo ambient concentration of GABA in the extracellular space, measured using microdialysis (0.8–2.9 mM), is sufficient to ABBREVIATIONS: GABA, g-aminobutyric acid; IPSC, inhibitory postsynaptic currents; mIPSC, miniature inhibitory postsynaptic current; aCSF, artificial cerebrospinal fluid; TTX, tetrodotoxin. 0026-895X/01/5904-814–824$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 4 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 479/891373 Mol Pharmacol 59:814–824, 2001 Printed in U.S.A. 814 at A PE T Jornals on M ay 7, 2017 m oharm .aspeurnals.org D ow nladed from activate GABAA receptors (Lerma et al., 1986). Alternatively, the tonic current might result from spontaneous openings of constitutively active GABAA channels (Neelands et al., 1999; Birnir et al., 2000). Regardless of the source of GABA responsible for the tonic current, receptors that mediate this persistent GABAergic conductance are of considerable physiological and pharmacological interest. Small but persistent increases in chloride conductance alter input resistance and membrane time constants; these changes, in turn, modulate synaptic efficacy and synaptic integration. The tonic GABAergic current may also play an important role in the manifestation of disease processes. Certain types of seizures are associated with a decrease in ambient concentrations of GABA and seizure control improves with treatments that increase the concentration of GABA. Modulation of tonic receptors represents a promising strategy for the development of new anticonvulsant, anxiolytic, and anesthetic drugs. Notably, allosteric modulation of GABAA receptor function by many compounds strongly depends on the occupancy of the receptor by GABA, as well as the state of receptor activation. The greatest increase in GABAA receptor activity by benzodiazepines and anesthetics occurs when receptors are activated by low concentrations of GABA (Harris et al., 1995). Accordingly, it is predicted that receptors underlying the tonic current (activated by low concentrations of GABA) would respond to pharmacological agents differently from receptors activated during quantal synaptic transmission. Given the potential physiological and therapeutic importance of GABAA receptors that mediate the tonic GABAergic inhibition, we investigated the tonic current in hippocampal neurons. We demonstrate the differential pharmacological properties of tonic and synaptic currents mediated by GABAA receptors. Midazolam and propofol produced a greater increase in charge transfer associated with the tonic current compared with that associated with miniature IPSCs. At concentrations that produce equivalent prolongation of IPSCs, the anesthetic propofol had a greater effect on the tonic current than the sedative midazolam. We speculate that modulation of the tonic current may account for differences in the clinical actions of these two classes of compounds. Some of the results were published in abstract form (Bai et al., 1998). Materials and Methods Cell Culture and Electrophysiological Techniques. Primary cultures of hippocampal neurons were prepared from embryonic Swiss White mice using aseptic techniques (MacDonald et al., 1989). Cells were maintained in culture for 13 to 18 days before use. Conventional whole-cell patch clamp recordings were performed at room temperature (21 to 23°C), at a holding potential of 260 mV. The extracellular recording solution contained 140 mM NaCl, 1.3 mM CaCl2, 5.4 mM KCl, 2 mM MgCl2, 25 mM HEPES, and 33 glucose, with pH adjusted to 7.4 with 1 M NaOH. Tetrodotoxin (TTX, 300 nM) was added to the extracellular solution to block voltagesensitive Na channels, and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (10 mM) and 2-amino-5-phosphonovalerate (40 mM) were added to inhibit ionotropic glutamate receptors. Recording electrodes were filled with a solution containing 120 mM CsCl, 30 mM HEPES, 11 mM EGTA, 2 mM MgCl2, 1 mM CaCl2, and 4 mM MgATP; pH was adjusted to 7.3 with CsOH. Currents were recorded simultaneously on a chart recorder and videotape recorder through a digital converter and a PC computer using Strathclyde Electrophysiological Software (SCAN or SPAN; Strathclyde Electrophysiological Software, courtesy of Dr. J. Dempster, Strathclyde University, United Kingdom; http://www.strath.ac.uk/Departments/PhysPharm/ ses.htm). Control and drug-containing solutions were delivered to the cultured neurons through glass barrels that were positioned close to the soma of the neuron. Propofol was prepared from Diprivan 1% (Zeneca Pharma, Mississauga, Ontario, Canada) and the solutions for the control experiments contained equivalent concentrations of Intralipid (KabiVitrum Canada Inc., Toronto, Canada). Intralipid did not influence the mIPSCs or tonic current. Midazolam was prepared from a commercial preparation of Versed (Hoffman-LaRoche Ltd., Mississauga, Ontario, Canada). We observed no differences in the actions of midazolam prepared from Versed compared with the pure compound (generously provided by Hoffman-La Roche, Nutley, NJ) dissolved in dimethyl sulfoxide. Bicuculline methobromide was purchased from Sigma (Oakville, Ontario, Canada) and gabazine (also known as SR-95531) was obtained from Research Biochemical International (RBI, Natick, MA). Whole-cell recordings were also made from the CA1 region of hippocampal slices obtained from 2to 3-week old Wistar rats. Coronal slices were prepared with a vibratome (VT1000E; Leica, Wetzlar, Germany) and incubated at room temperature for a minimum of 1 h in oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid containing 124 mM NaCl, 3 mM KCl, 4 mM CaCl2, 4 mM MgCl2, 26 mM NaHCO3, 1.25 mM NaH2PO4, and 10 mM glucose. Slices were then transferred to a tissue chamber as needed and maintained at 31°C 6 0.5°C at the interface between humidified and oxygenated (95% O2/5% CO2) aCSF perfused through the chamber at a rate of 0.5 to 1 ml/min. Tight-seal (.5 GV) whole-cell recordings were obtained from CA1 pyramidal cells using a “blind” approach. The internal pipette solution consisted of 140 mM CsCl, 10 mM HEPES, 2 mM MgCl2 (pH 7.2–7.3 using CsOH; osmolarity, 270–280 mOsM). Spontaneous miniature IPSCs (see below) were isolated by the addition of 0.5 mM TTX, 10 mM 6-cyano-2,3-dihydroxy-7-nitroquinoxaline and 40 mM 2-amino-5-phosphonovalerate to the aCSF. Drugs tested were dissolved in aCSF and superfused over slices. Spontaneous mIPSCs were recorded using an Axopatch-1D (Axon Instruments, Foster City, CA), filtered at 2 kHz and stored on videotape for subsequent off-line analysis using a digital data recorder (VR-10B; InstruTECH Corp., Port Washington, NY). Data Analysis. Current recordings that demonstrated a stable baseline and distinct mIPSCs were used for the analysis. All experiments were digitized (2 kHz) with a pulse-code modulator and stored on VHS videotapes. For analysis, the recordings were played back and re-digitized using an event detection program (SCAN). For detection of IPSCs, the trigger level was set at approximately three times higher than the level of the baseline noise (; 3.4 pA). All events greater than the threshold level were recorded for frequency analysis including those infrequent compound events (,2%) with multiple peaks. When multiple peaks were clearly evident during the visual inspection of the records, the additional peaks were counted as mIPSCs. However, compound events were excluded from the analysis of rise time or decay of synaptic currents. In addition, we manually scrolled through files of detected events to reject spurious events that were caused by excessive noise. Spontaneous postsynaptic currents recorded in the presence of tetrodotoxin (TTX) are referred to as miniature IPSCs (mIPSCs). Miniature IPSCs with a rapid onset (10 to 90% rise time , 5 ms) and decay phase that were not contaminated by other mIPSCs were selected for further kinetic analysis. At least 100 individual mIPSC events were recorded under each experimental condition. Peak amplitude, charge transfer (Q, the integrated area under mIPSCs), and the time constant of current decay (toff) were analyzed. The decay phase was well described by a single exponential equation in the form I(t) 5 Aoexp (2t/toff) 1 C, where I(t) is the current amplitude at any given time t, C is the residual current, and Ao is the current amplitude at time 0. Change in the charge transfer (DQmIPSC) assoTonic GABA-ergic Inhibition 815 at A PE T Jornals on M ay 7, 2017 m oharm .aspeurnals.org D ow nladed from ciated with mIPSC was analyzed according to Brickley et al. (1996) using the equation DQmIPSC 5 fdrug 3 Qdrug 2 fcon 3 Qcon, where fdrug and fcon are the frequencies (Hz) of mIPSCs and Qdrug and Qcon are the average charge transfer (pC) per mIPSC during drug and control conditions, respectively. Under our experimental conditions, we assumed that the change in charge transfer reflected a proportional change in membrane conductance. The amplitude of the tonic current was calculated as the difference between the holding current measured before and after the application of bicuculline (10 mM) (Brickley et al., 1996; Wall and Usowicz, 1997). The increase in the tonic current that was observed after the application of midazolam or propofol was measured from the chart record (Astro-Med, West Warwick, RI). The charge transfer associated with the tonic current was calculated according the equation: DQTC 5 ITC 3 Dt, where DQTC is the charge transfer produced by the tonic current, ITC is the current amplitude at steady-state, and Dt is time. Variance analysis was used to estimate the single channel current (i) from the mean current (Imean) and current variance (s ). Variance (s) was calculated according to the formula: