Abstracts

RATIONALE: The concentration of intracellular chloride is critical for GABA[sub]A[/sub] receptor (GABA[sub]A[/sub]R) -mediated inhibition. Previous studies have demonstrated that chloride accumulation during activation of GABA[sub]A[/sub]R can result in a depolarizing response. We investigated the effects of synaptic and exogenous activation of GABA[sub]A[/sub]R on the concentration of intracellular chloride.
METHODS: Whole cell voltage clamp recordings were obtained from visually identified layer 2/3 pyramidal neurons and layer 1 interneurons in acutely prepared brain slices from rat neocortex. The intracellular solution contained 1 mM chloride. Exogenous GABA (1 mM) was locally applied with a pressure pipette placed at the soma. Enhanced synaptic responses were evoked in the presence of 4-aminopyridine (50 [mu]M 4-AP) and excitatory amino acid antagonists. Voltage steps (-100 mV to -60 mV, 5-10 mV increments) were used to measure the reversal potential the GABA response.
RESULTS: Electrical stimulation in the presence of 4-AP induced prolonged currents often with superimposed rapid transient inhibitory postsynaptic currents. The rapid events and the slower conductance change shared the same polarity indicating that they shared similar ionic dependencies. Furthermore, the reversal potentials of responses to exogenous GABA and synaptic release were not significantly different (exogenous GABA layer 2/3: -88.6 [plusminus] 2.2 mV, n=12; synaptic GABA layer 2/3: 83.8 [plusminus] 1.0 mV, n=9; synaptic GABA layer 1: -86.2 [plusminus] 1.5 mV, n=10), indicating that the synaptic response consists primarily of activation of GABA[sub]A[/sub]Rs. Biphasic currents were prominent in most recordings and consisted of an outward component followed by an inward component. The inward component persisted near the reversal potential indicating that it was independent of ion flux of the early component. In both responses to synaptic and exogenous GABA, large outward currents were present during steps 5-15 mV above the reversal potential. When the membrane potential was returned to the reversal potential during the GABA response, substantial inward currents were present consistent with a change in the driving force due to the accumulation of intracellular chloride. Similarly, large negative currents during steps 5-15 mV below the reversal resulted in substantial outward currents when the potential was stepped to the reversal potential consistent with depletion of intracellular chloride. This effect was time dependent and saturable.
CONCLUSIONS: Responses to synaptic and exogenous GABA are strongly dependent on intracellular chloride. Our results suggest that chloride flux during activation of GABA[sub]A[/sub]Rs can result in substantial changes in intracellular chloride. These dynamic changes in intracellular chloride may have profound effects on the efficacy of GABA[sub]A[/sub]R-mediated inhibition.
Support: NS22373 and AES Research Training Fellowship.