Contribution of Subsaturating GABA Concentrations to IPSCs in Cultured Hippocampal Neurons

Abstract

The time course of EPSCs and IPSCs is at least partly determined by the concentration profile of neurotransmitter acting on postsynaptic receptors. Several recent reports have suggested that the peak synaptic cleft concentration of the inhibitory neurotransmitter GABA likely reaches at least 500 M, a level that saturates the GABA A receptor. In the course of investigating the experimental anticonvulsant 3,3-diethyl-2-pyrrolidinone (diethyl-lactam), we have observed an important contribution to IPSC decay by subsaturating concentrations of GABA. Diethyl-lactam augments currents elicited by the exogenous application of subsaturating concentrations of GABA in voltage-clamped, cultured hippocampal neurons and significantly prolongs the decay of autaptic IPSCs and miniature IPSCs in our cultures. In addition, diethyl-lactam potentiates currents in excised outside-out membrane patches elicited by the prolonged application of low concentrations of GABA. However, when patches are exposed to 1-2 msec pulses of 1 mM GABA, diethyl-lactam does not alter current decay. Tiagabine, which blocks GABA reuptake, does not prolong IPSCs, so it is unlikely that uptake inhibition accounts for the enhancement of IPSCs. EPSCs and miniature IPSC frequency are unaffected by diethyl-lactam, again consistent with a postsynaptic site of action. We propose that during an IPSC, a substantial number of postsynaptic receptors must be exposed to subsaturating concentrations of GABA. A simplified model of GABA A receptor kinetics can account for the effects of diethyllactam on exogenous GABA and IPSCs if diethyl-lactam has its main effect on the monoliganded states of the GABA A receptor. Key words: epilepsy; GABA; GABA A receptor; hippocampus; IPSC; outside-out patches; rapid application; synapseThe factors that regulate GABA A receptor-mediated inhibitory synaptic transmission are the focus of intense study because GABAergic inhibition plays a significant role in regulating electrical activity in the C NS. Furthermore, the GABA A receptor appears to be a target for anticonvulsants, sedative hypnotics, anesthetics, and anxiolytics. Benzodiazepines, barbiturates, neurosteroids, and the ␥-butyrolactones are compounds that are known to enhance GABAergic inhibition (Macdonald et al., 1989b;T w yman and Macdonald, 1992;Rogers et al., 1994;Mathews et al., 1996).The duration of the synaptic GABA transient is determined by diffusion and uptake, depending on experimental preparation and conditions Lambert, 1992, 1994;Isaacson et al., 1993;Draguhn and Heinemann, 1996). The time course of the IPSC is dictated by these factors and by the kinetics of the GABA A receptor. Several groups have studied the duration and magnitude of the synaptic GABA transient (Maconochie et al., 1994;Jones and Westbrook, 1995;Tia et al., 1996) and shown that IPSCs can be partly mimicked by exposing excised outside-out patches to high (Ն500 M) concentrations of GABA for a few milliseconds. These experiments support the idea that IPSCs result from a very brief exposure of postsyn...

Cyclothiazide potently inhibits γ-aminobutyric acid type A receptors in addition to enhancing glutamate responses

“…We further tested whether CTZ interferes with GABA binding by analyzing the effect of CTZ on the GABA dose-response curve. The EC 50GABA value was 18.8 M, similar to previous reports (20,21). In the presence of CTZ (100 M), the GABA dose-response curve was not apparently shifted (EC 50GABAϩCTZ ϭ 22.2 M), suggesting that CTZ may have only a slight effect, if any, on GABA binding (Fig.…”

Effects of the Anticonvulsant Retigabine on Cultured Cortical Neurons: Changes in Electroresponsive Properties and Synaptic Transmission

“…The experiments on IPSCs, spontaneous mIPSCs, and baseline noise support the hypothesis that retigabine, like diethyl-lactam, is most effective under conditions in which the synapse is not saturated by GABA (Hill et al, 1998;Perrais and Ropert, 1999;Hajos et al, 2000;Leao et al, 2000). Thus, the most robust effect of retigabine on IPSCs occurs during the decay phase of the IPSC, when concentrations of GABA in the synapse are diminishing.…”

Slow Desensitization Regulates the Availability of Synaptic GABAAReceptors

“…Diethyl-lactam, another experimental anticonvulsant, prolongs the IPSC decay, although it only potentiates responses to low concentrations of GABA (Ͻ30 M), leading to the conclusion that subsaturating GABA concentrations contribute to the generation of IPSCs (Hill et al, 1998). Our results indicate that such a subsaturating component of the GABA transient will promote channel desensitization.…”

“…Simulations of glutamate clearance also predict that micromolar levels can persist for relatively long periods at locations distant from the release site (Clements, 1996;Otis et al, 1996a;Barbour and Häusser, 1997;Rusakov and Kullmann, 1999). The consequences of sustained, subsaturating transmitter exposure (Hill et al, 1998) are not necessarily limited to receptor activation. For example, prolonged low concentrations of GABA result in pronounced desensitization of GABA A receptors (Celentano and Wong, 1994;Orser et al, 1994;Berger et al, 1998), possibly because of the presence of a singly bound desensitized state with slow entry and exit rates (Jones and Westbrook, 1995).…”