Hyperpolarizing GABAergic transmission depends on KCC2 function and membrane potential

Abstract

KCC2 comprises the major Cl(-) extruding mechanism in most adult neurons. Hyperpolarizing GABAergic transmission depends on KCC2 function. We recently demonstrated that glutamate reduces KCC2 function by a phosphorylation-dependent mechanism that leads to excitatory GABA responses. Here we investigated the methods by which to estimate changes in E(GABA), as well as the processes that lead to depolarizing GABA responses and their effects on neuronal excitability. We demonstrated that current-clamp recordings of membrane potential responses to GABA can determine upper and lower limits of E(GABA). We also further characterized depolarizing GABA responses, which both excited and inhibited neurons. Our analyses revealed that persistently active GABA(A) receptors contributed to loading Cl(-) during the glutamate exposure, indicating that tonic inhibition can facilitate the development of depolarizing GABA responses and increase excitability after pathophysiological insults. Finally, we demonstrated that hyperpolarizing GABA responses could temporarily switch to depolarizing responses when they coincided with an afterhyperpolarization.

Figure 1

The canonical role of GABA. (A) GABA (10 µM-black bar) shunted membrane conductances causing a hyperpolarization of the membrane potential and inhibited spontaneous action potentials. Recordings were obtained in current-clamp mode and in the absence of any antagonists. (B) Spontaneous IPSP hyperpolarized the membrane potential. The E_GABA value obtained by voltage-clamp protocols for this neuron is indicated by the dotted line (−85.4 mV). These data were obtained in the presence of DNQX and AP5.

Figure 2

Depolarizing GABA was more versatile than hyperpolarizing GABA. Recordings were obtained from a neuron that was exposed to glutamate (20 µM) for 2 min. (A) Two minutes after the end of the glutamate pulse spontaneous GABAergic excitatory postsynaptic potentials elicited multiple action potentials. A pulse of exogenous GABA (10 µM) elicited a single action potential before shunting further activity. (B) Four minutes after the end of the glutamate pulse only the phasic GABA responses triggered action potentials. (C) Eight minutes after the end of the glutamate pulse neither the phasic responses (arrow) nor the exogenous GABA triggered action potentials. All recordings were obtained in the presence of DNQX and AP5. Black bars indicate the duration of the GABA applications. Scale bar in (C) is for all traces.

Figure 3

Tonic currents contributed to Cl⁻ loading during the exposure to glutamate. (A) Spontaneous IPSP (arrows) were observed prior to and during the depolarizing glutamate pulse. Activity quickly ceased after several seconds due to depolarizing block. Glutamate was applied without any antagonists. (B) Bicuculline (solid line) caused a depolarizing potential during the glutamate pulse. Two consecutive traces are superimposed to illustrate the effect of bicuculline: the trace in black contains the glutamate and bicuculline/glutamate pulse, the trace in gray was glutamate alone. Recordings were obtained in the presence of TTX, DNQX and AP5, while glutamate and bicuculline were applied alone.

Figure 4

The polarity of the GABA responses depended on the membrane potential. Neurons were exposed to a 500 ms GABA pulse (10 µM, arrows) before and after a 5 s glutamate pulse (20 µM, black bar). (A) Several neurons exhibited hyperpolarizing GABA responses both before and during the glutamate-induced AHP. (B) Several neurons exhibited a weaker hyperpolarizing GABA response, and a depolarizing GABA response during the glutamate-induced AHP. Consecutive traces (black and gray, 1/60 s) are overlaid to demonstrate that transient shifts in E_GABA did not occur. Recordings were obtained in the presence of TTX, DNQX and AP5.