Istvan Mody

Bridging the cleft at GABA synapses in the brain

A fragile balance between excitation and inhibition maintains the normal functioning of the CNS. The dominant inhibitory neurotransmitter of the mammalian brain is GABA, which acts mainly through GABAA and GABAB receptors. Small changes in GABA-mediated inhibition can alter neuronal excitability profoundly and, therefore, a wide range of compounds that clearly modify GABAA-receptor function are used clinically as anesthetics or for the treatment of various nervous system disorders. Recent findings have started to unravel the operation of central GABA synapses where inhibitory events appear to result from the synchronous opening of only tens of GABAA receptors activated by a saturating concentration of GABA. Such properties of GABA synapses impose certain constraints on the physiological and pharmacological modulation of inhibition in the brain.

Characterization of synaptically elicited GABAB responses using patch-clamp recordings in rat hippocampal slices.

1. Tight‐seal, whole‐cell voltage clamp recording techniques were used to characterize monosynaptically evoked GABAB currents in adult rat brain slices maintained at 34‐35 degrees C. Responses were recorded from granule cells of the dentate gyrus following the blockade of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX)‐, D‐2‐amino‐5‐phosphonovaleric acid (D‐AP5)‐ and picrotoxin‐sensitive fast synaptic transmission, so that the remaining synaptic currents could be studied in isolation. 2. Under these conditions, stimulation in the molecular layer elicited a slow outward current which was blocked by the selective GABAB antagonist CGP 35348 in a concentration‐dependent manner (200‐800 microM). This current was absent in recordings made with pipettes containing 10‐15 mM of the lidocaine derivative QX‐314 or when caesium was substituted for K+. 3. Increasing the [K+]o e‐fold (from 2.5 to 6.8 mM) shifted the reversal potential of the GABAB current from ‐97.9 to ‐73.2 mV, as predicted by the Nernst equation. Peak conductance was constant, but in 6.8 mM [K+]o at voltages hyperpolarized to EK (equilibrium potential for potassium), a small outward rectification was evident. 4. The time course of the current could be described by fourth‐power exponential activation kinetics with double exponential inactivation. At 34‐35 degrees C, the average activation time constant (tau m) was 45.2 ms, while the two inactivation time constants (tau h1 and tau h2) were 110.2 and 516.2 ms, with corresponding weighting factors (wh1 and wh2) of 0.84 and 0.16, respectively. The Q10 (temperature coefficient) values for these time constants were between 1.82 and 2.31. Neither tau m, nor tau h1 and tau h2 were voltage dependent in the range from ‐45 to ‐95 mV. 5. Paired‐pulse depression of the GABAB current was studied by giving identical conditioning and test stimuli over a wide range (50‐5000 ms) of interstimulus intervals (ISIs). The maximal depression (48%) occurred at 200 ms ISI, and the depression lasted for over 5 s. The magnitude of paired‐pulse depression was not dependent on the postsynaptic membrane potential. 6. Application of the competitive antagonist CGP 35348 such that the peak current was diminished by approximately 50% had no effect on the activation or inactivation kinetics of the current. Similarly, during paired‐pulse depression the kinetics of test currents were identical to those of conditioning currents. These findings support the hypothesis that the mechanism responsible for paired‐pulse depression involves a reduction in neurotransmitter release without postsynaptic alterations in K+ channel activation/inactivation kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)

Perpetual inhibitory activity in mammalian brain slices generated by spontaneous GABA release.

Miniature spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by GABAA receptors were recorded using whole-cell patch clamp recordings in rat brain slices maintained in vitro at 34 +/- 1 degree C. We have found that firing of action potentials by principal neurons or by GABAergic interneurons is not necessary to the generation of sIPSCs since they persist in the presence of 1-5 microM tetrodotoxin (TTX). The average frequency of the discrete sIPSCs exhibits a large cell-to-cell variability and is between 5-15 Hz. The amplitudes of the sIPSCs depend on the difference between the membrane potential and the equilibrium potential for Cl- (ECl). Generally, 70-80 mV away from ECl, sIPSCs have a mean amplitude of 30-80 pA (i.e. peak conductance of 400-1000 pS) with an average decay time constant of 5.8 ms. Accordingly, unitary single sIPSCs arise from the simultaneous activation of no more than 20 GABAA receptor/channels. The perpetual barrage of spontaneous GABAergic activity is very likely to be a critical factor in the regulation of neuronal excitability and the mechanism of action of several neuroactive compounds.

Dantrolene-Na(Dantrium) blocks induction of long-term potentiation in hippocampal slices

Long-term potentiation (LTP) is characterized by a long lasting increase in the efficacy of neurotransmission which may consist of two phases. First an induction phase, with an absolute requirement for post-synaptic activation. Second, a maintenance phase, possibly involving pre-synaptic mechanisms. An essential function for calcium ions in the induction of LTP has been established and a particular emphasis has been placed on the role of N-methyl-D-aspartate (NMDA) receptor activation in gating a postsynaptic influx of calcium. We now report that pharmacological blockade of intraneuronal calcium release with 20 microM dantrolene-sodium (dantrium) completely blocks the induction of LTP in the CA1 region of the rat hippocampal slice. This drug inhibits calcium release from the sarcoplasmic reticulum and also diminishes the rise in intraneuronal calcium ion concentrations elicited by NMDA receptor activation in cultured CA1 pyramidal cells. Dantrolene does not block NMDA gated membrane currents or voltage activated Ca2+ currents in these cells. We suggest that release of intraneuronal calcium, rather than calcium influx may be the critical post-synaptic feature underlying LTP induction. We do not however exclude a pre-synaptic involvement in the specificity and/or maintenance of long-term potentiation.

Calbindin−D28K (CaBP) levels and calcium currents in acutely dissociated epileptic neurons

SummaryNerve cells that lack the cytoplasmic Ca2+ binding protein Calbindin-D28K (CaBP) appear to be selectively vulnerable to Ca2+-related injury consistent with a postulated intraneuronal Ca2+-buffering role of CaBP. We have confirmed the selective loss of CaBP from the dentate gyrus during kindling-induced epilepsy in acutely dissociated granule cells (GCs) from kindled rats. Immunohistochemically stained kindled neurons showed a significant loss of CaBP when compared to controls (p < 0.001; ANOVA). The Ca2+-buffering role of CaBP was assessed in acutely dissociated control and kindled GCs by examining a physiological process highly sensitive to intracellular Ca2+-buffering: the Ca2+ -dependent inactivation of high-voltage activated (HVA or L-type) Ca2+ currents in the absence (or presence) of exogenous Ca2+-chelators. Whole-cell patch clamp recordings in kindled GCs demonstrated a markedly enhanced Ca2+-dependent inactivation of Ca2+-currents. After brief conditioning Ca2+ currents, in the absence of an exogenous intraneuronal Ca2+-chelator, subsequent test Ca2+ currents were inactivated by 58.3% in kindled GCs, a significant increase from the 37.4% inactivation observed in control GCs (p< 0.005; ANOVA). The differential Ca2+ current decay and Ca2+-dependent inactivation were prevented in both control and kindled GCs upon loading the neurons with the exogenous Ca2+-chelator BAPTA. These experiments demonstrate a high correlation between the loss of CaBP and changes in Ca2+ current inactivation and are consistent with the hypothesis that CaBP contributes to the physiological Ca2+-buffering in mammalian neurons.

Halothane enhances tonic neuronal inhibition of elevating intracellular calcium

Whether the major action of anesthetics is to depress the central nervous system (CNS) by reducing excitation or enhancing inhibition remains unknown. Using whole cell patch-clamp recording in hippocampal slices, halothane and pentobarbital were found to prolong the decay time constant (TAU(D)) of GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). Intracellular administration of the Ca2+ chelator BAPTA or the Ca2+ release inhibitor dantrolene significantly (ANOVA, P less than 0.005) reduced halothanes effect; in contrast, the pentobarbital effect was unchanged. Halothane induced depression of population spike amplitude was blocked by the GABAA antagonist bicuculline. Together, these findings suggest that a major depressant effect of halothane involves enhancement of GABAA-mediated inhibition through release of intraneuronally stored Ca2+.

A role for N-methyl-D-aspartate receptors in norepinephrine-induced long-lasting potentiation in the dentate gyrus.

SummaryMechanisms of action of norepinephrine (NE) on dentate gyrus granule cells were studied in rat hippocampal slices using extra- and intracellular recordings and measurements of stimulus and amino acid-induced changes in extracellular Ca2+ and K+ concentration. Bath application of NE (10–50 μM) induced long-lasting potentiation of perforant path evoked potentials, and markedly enhanced high-frequency stimulus-induced Ca2+ influx and K+ efflux, actions blocked by β-receptor antagonists and mimicked by β agonists. Enhanced Ca2+ influx was primarily postsynaptic, since presynaptic Δ [Ca2+]0 in the stratum moleculare synaptic field was not altered by NE. Interestingly, the potentiation of both ionic fluxes and evoked population potentials were antagonized by the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV). Furthermore, NE selectively enhanced the Δ[Ca2+]0, Δ[K+]0 and extracellular slow negative field potentials elicited by iontophoretically applied NMDA, but not those induced by the excitatory amino acid quisqualate. These results suggest that granule cell influx of Ca2+ through NMDA ionophores is enhanced by NE via β-receptor activation. In intracellular recordings, NE depolarized granule cells (4.8±1.1 mV), and increased input resistance (RN) by 34±6.5%. These actions were also blocked by either the β-antagonist propranolol or specific β1-blocker metoprolol. Moreover, the depolarization and RN increase persisted for long periods (93±12 min) after NE washout. In contrast, while NE, in the presence of APV, still depolarized granule cells and increased RN, APV made these actions quickly reversible upon NE washout (16±9 min). This suggested that NE induction of long-term, but not short-term, plasticity in the dentate gyrus requires NMDA receptor activation. NE may be enhancing granule cell firing by some combination of blockade on the late Ca2+-activated K+ conductance and depolarization of granule cells, both actions that can bring granule cells into a voltage range where NMDA receptors are more easily activated. Furthermore, NE also elicited activity-independent long-lasting depolarization and RN increases, which required functional NMDA receptors to persist.

The Molecular Basis of Kindling

Kindling is an experimental model of epilepsy that involves activity‐dependent changes in neuronal structure and function. During kindling, pathological changes may occur at several organizational levels of the nervous system, from alterations in gene‐expression in individual neurons to the loss of specific neuronal populations and rearrangement of synaptic connectivity resulting from sustained stimulation of major excitatory pathways. This review summarizes recent developments in alterations at single neuronal and molecular levels that may be responsible for kindling epileptogenesis.

A method for isolating and patch-clamping single mammalian taste receptor cells.

Individual taste receptor cells were isolated from the tongue of the mouse by enzymatic treatment followed by mechanical dissociation. The cells were morphologically identical with taste cells from amphibians. Whole-cell voltage-clamp recordings indicated that the murine taste cells possess a variety of voltage-dependent inward and outward currents. Delayed rectifier currents were blocked by denatonium benzoate, one of the most bitter compounds known. This preparation should permit a detailed electrophysiologcal investigation of taste transduction in mammals at the level of taste receptor cells.

Kindling increases N−methyl−D−aspartate potency at single N−methyl−D−aspartate channels in dentate gyrus granule cells

Dose-response studies of N-methyl-D-aspartate channel openings were carried out using cell-attached patches in dentate gyrus granule cells acutely isolated from control and kindled rats. The tips of the patch electrodes were first filled with regular extracellular solution, followed by backfilling through the shank with the agonist containing solution. As the two solutions joined, the agonist (N-methyl-D-aspartate, 25 microM) steadily diffused to the cell membrane, and the concentration gradually built up resulting in the progressive increase in the opening probability of N-methyl-D-aspartate channels. The reliability of this cell-attached diffusional drug delivery method was tested by determining the concentration dependence of competitive antagonism of N-methyl-D-aspartate induced channel activity by D(-)-2-amino-5-phosphonopentanoic acid. The Ki for D(-)-2-amino-5-phosphonopentanoic acid in the presence of 25 microM N-methyl-D-aspartate was found to be 6.8 microM. Twenty-four hours following the last seizure, N-methyl-D-aspartate channels on kindled neurons were consistently activated by lower N-methyl-D-aspartate concentrations than channels on control granule cells, indicating a higher potency of agonist at epileptic N-methyl-D-aspartate channels. The higher potency of the agonist is most likely a reflection of the long-term alterations in the modulation of N-methyl-D-aspartate receptor function in epileptic neurons.