Anesthetic drugs are known to interact with GABA A receptors, both to potentiate the effects of low concentrations of GABA and to directly gate open the ion channel in the absence of GABA; however, the site(s) involved in direct gating by these drugs is not known. We have studied the ability of alphaxalone (an anesthetic steroid) and pentobarbital (an anesthetic barbiturate) to directly activate recombinant GABA A receptors containing the ␣1, ␤2, and ␥2L subunits. Steroid gating was not affected when either of two mutated ␤2 subunits [␤2(Y157S) and ␤2(Y205S)] are incorporated into the receptors, although these subunits greatly reduce the affinity of GABA binding. These observations indicate that steroid binding and subsequent channel gating do not require these particular residues, as already shown for barbiturates. Bicuculline or gabazine (two competitive antagonists of GABA binding) reduced the currents elicited by alphaxalone and pentobarbital from wild-type GABA A receptors; however, gabazine produced only a partial block of responses to pentobarbital or alphaxalone, and bicuculline only partially blocked responses to pentobarbital. These observations indicate that the blockers do not compete with alphaxalone or pentobarbital for a single class of sites on the GABA A receptor. Finally, at receptors containing ␣1␤2(Y157S)␥2L subunits, both bicuculline and gabazine showed weak agonist activity and actually potentiated responses to alphaxalone. These observations indicate that the blocking drugs can produce allosteric changes in GABA A receptors, at least those containing this mutated ␤2 subunit. We conclude that the sites for binding steroids and barbiturates do not overlap with the GABA-binding site. Furthermore, neither gabazine nor bicuculline competes for binding at the steroid or barbiturate sites. The data are consistent with a model in which both gabazine and bicuculline act as allosteric inhibitors of channel opening for the GABA A receptor after binding to the GABA-binding site.Key words: GABA A receptor; GABA; neurosteroids; bicuculline; inverse agonist; anesthetics; allosteric inhibitor GABA activates a ligand-gated ion channel (the GABA A receptor), which underlies most rapid inhibition in the brain. Various other compounds also bind to the GABA A receptor and can gate the channel or modulate channel function (Macdonald and Olsen, 1993). In particular, steroids and barbiturates are each able to directly gate the GABA A receptor channel (in the absence of GABA), and they can also enhance the activation produced by low concentrations of GABA. It is not known whether the same sites are involved in producing direct gating and in potentiating the effects of GABA. For the sites involved in potentiation, however, the steroid-binding site and the barbiturate-binding site are distinct from each other and are also distinct from the GABAbinding site (Macdonald and Olsen, 1993). Because the characterized sites for steroid and barbiturate binding differ from the GABA-binding site, it is puzzling that a competit...

1. We used whole cell recordings to compare passive membrane properties and synaptic properties of postnatal rat hippocampal neurons grown for 7-15 days in either conventional mass cultures or on physically restricted microisland cultures. Despite matching microisland and mass culture cell across several variables, there were significant differences between neurons in the two groups regarding passive membrane characteristics and synaptic properties. 2. Microisland neurons displayed significantly faster charging of the membrane capacitance than mass culture counterparts matched with microisland neurons for age, somal diameter, and transmitter phenotype. When we used a two-compartment equivalent circuit model to quantify this result, microisland neurons displayed approximately half the distal capacitance of mass culture neurons. These data suggest that microisland neurons elaborate less extensive neuritic arborizations than mass culture neurons. 3. Evoked synaptic responses were enhanced on microislands compared with mass cultures. Excitatory and inhibitory autaptic currents were more frequent and displayed larger amplitudes on single-neuron microislands than in matched mass culture neurons. 4. In recordings from pairs of neurons in the two environments, we observed a significantly higher probability of obtaining a monosynaptic response on two-neuron microislands than in matched mass culture pairs (85% vs. 42%). Evoked excitatory postsynaptic currents were also significantly larger in the microisland environment, with evoked excitatory synaptic currents from two-neuron microislands exhibiting a mean amplitude 20-fold larger than mass culture monosynaptic responses. 5. The differences in evoked synaptic responses were not reflected in differences in the amplitude or frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). Analysis of mEPSC rise times, decay times, and peak amplitudes within individual cells suggests that electrotonic filtering is not an important contributor to the variability of peak amplitudes and decay times of synaptic currents in cells of either culture environment. However, composite data across neurons in both cultures reveal a significant correlation between mEPSC rise and decay times. 6. Out results suggest that the microisland preparation may be a useful tool for exploring factors that influence synapse formation and development. Additionally, the preparation is a particularly convenient model for the study of single-neuron-mediated synaptic events.

Although glia are known to be sensitive to exogenously applied neurotransmitter substances, little is known about how glia respond to neuronal activity on the millisecond time scale of individual synaptic events. We have explored the electrophysiological effects of excitatory neuronal signaling on glial cells in rat hippocampal microcultures. Exogenous applications of glutamate analogs to islands of glia revealed a large ionotropic AMPA receptor-mediated current and a smaller current mediated by electrogenic glutamate uptake. Glia demonstrated no evidence of NMDA or metabotropic receptor-mediated currents or membrane conductance changes. Despite the dominant contribution of AMPA receptors to exogenous glutamate applications in glia, AMPA receptor currents contributed only approximately 20% to the response of glia to endogenous glutamate release from solitary excitatory neurons. Electrogenic glutamate uptake contributed strongly to glial responses to neuronal stimulation. In addition, some glia exhibited a residual current is response to neuronal stimulation that was not attributable to calcium-dependent transmitter release or to neuronal potassium efflux. These results help provide a context for understanding the role of glial transporters and receptors in nervous system signaling.

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Background and purpose: Some neurosteroids, notably 3a-hydroxysteroids, positively modulate GABAA receptors, but sulphated steroids negatively modulate these receptors. Recently, other lipophilic amphiphiles have been suggested to positively modulate GABA receptors. We examined whether there was similarity among the actions of these agents and the mechanisms of neurosteroids. Significant similarity would affect theories about the specificity of steroid actions. Experimental approach: Xenopus laevis oocytes were challenged with Triton X-100, octyl-b-glucoside, capsaicin, docosahexaenoic acid and sodium dodecyl sulphate (SDS), along with different GABA concentrations. Key results: These compounds have both positive and negative effects on GABA currents, which can be accentuated according to the degree of receptor activation. A low GABA concentration (1 mM) promoted potentiation and a high concentration (20 mM) promoted inhibition of current, except for SDS that inhibited function even at low GABA concentrations. Amphiphile inhibition was characterized by enhanced apparent desensitization and by weak voltage dependence, similar to pregnenolone sulphate antagonism. We then tested amphiphile effects on mutated receptor subunits that are insensitive to negative (a1V256S) and positive (a1Q241L or a1N407A/Y410F) steroid modulation. Negative regulation by amphiphiles was nearly abolished in a1V256S-mutated receptors, but potentiation was unaffected. In a1Q241L-or a1N407A/Y410F-mutated receptors, potentiation by amphiphiles remained intact. Conclusions and implications: Structurally diverse amphiphiles have antagonist actions at GABAA receptors very similar to those of sulphated neurosteroids, while the potentiating mechanisms of these amphiphiles are distinct from those of neurosteroidpositive modulators. Thus, such antagonism at GABAA receptors does not have a clear pharmacophore requirement.