Abstracts

Rationale: Tonic activation of GABA_A receptors provides a potent inhibitory mechanism for hippocampal neurons. Chronic cellular and molecular changes seen in epilepsy are known to affect tonic inhibition. Less is known about the mechanisms regulating tonic inhibition over short time periods. Methods: Tonic GABA currents were measured from cultured rat hippocampal neurons using whole cell patch clamp techniques to evaluate how endogenous tonic currents are affected by (1) transient increases in extracellular [GABA], (2) depolarization during GABA application, or (3) depolarization of presynaptic terminals with 12 mM K+. Experiments were performed with symmetrical Cl- concentrations under conditions to isolate GABA_A receptor mediated currents. Rapid solution exchanges were produced with a microperfusion system.Results: Tonic currents due to endogenous GABA were measured before and after application of exogenous GABA (1 M). Tonic currents were reduced by 25 9% when measured 5 s after GABA application (mean SEM, p<0.05, n=11 neurons). Reductions in driving force due to GABA-induced shifts in E_GABA (-0.8 0.7 mV to 9.2 1.8 mV, n=8) accounted for this reduction in tonic current. Transient depolarization to +40 mV during the application of exogenous GABA converted the reduction in tonic current into a potentiation, tonic currents measured 6 s after repolarization were increased by 68 13% (n=11, p<0.01). When cells were depolarized during GABA application E_GABA shifted from 1.2 0.9 mV to +3.0 0.3 mV (n=8), this shift in E_GABA was too small to account for the observed potentiation (theoretical E_GABA shift required to produce this potentiation would be +41 8 mV, p<0.01). The voltage-dependent potentiation of tonic GABA currents represented postdepolarization potentiation (PDP), an intrinsic GABA_A receptor property. Reducing vesicular GABA release with zero extracellular Ca2+/EGTA did not affect tonic current density in cultured hippocampal neurons. To prevent vesicular release of GABA, cultures were treated with the vesicular H+-ATPase inhibitor concanamycin A (conA). Tonic current in conA-treated neurons was no different than that in control neurons (-58 12 vs. -39 4 pA, p=0.24, n=5-7). In conA-treated cells, transient application of 12 mM K+ to depolarize presynatptic terminals increased tonic current to 140 50 pA (p<0.05, n=5). The K+-induced increase in tonic current was reversibly inhibited by SKF89976a, indicating that this was due to GABA release mediated by reversal of GABA transporter type 1 (GAT1).Conclusions: Tonic GABA currents are rapidly regulated by GABA-induced changes in intracellular Cl- concentration, PDP of extrasynaptic GABA_A receptors, and nonvesicular GABA release. These mechanisms may influence tonic inhibition during seizures when neurons are robustly depolarized and extracellular GABA and K+ concentrations are elevated.