GABA transaminase inhibition induces spontaneous and enhances depolarization-evoked GABA efflux via reversal of the GABA transporter

Abstract

The GABA transporter can reverse with depolarization, causing nonvesicular GABA release. However, this is thought to occur only under pathological conditions. Patch-clamp recordings were made from rat hippocampal neurons in primary cell cultures. Inhibition of GABA transaminase with the anticonvulsant gamma-vinyl GABA (vigabatrin; 0.05-100 microm) resulted in a large leak current that was blocked by bicuculline (50 microm). This leak current occurred in the absence of extracellular calcium and was blocked by the GABA transporter antagonist SKF-89976a (5 microm). These results indicate that vigabatrin induces spontaneous GABA efflux from neighboring cells via reversal of GABA transporters, subsequently leading to the stimulation of GABA(A) receptors on the recorded neuron. The leak current increased slowly over 4 d of treatment with 100 microm vigabatrin, at which time it reached an equivalent conductance of 9.0 +/- 4.9 nS. Blockade of glutamic acid decarboxylase with semicarbazide (2 mm) decreased the leak current that was induced by vigabatrin by 47%. In untreated cells, carrier-mediated GABA efflux did not occur spontaneously but was induced by an increase in [K(+)](o) from 3 to as little as 6 mm. Vigabatrin enhanced this depolarization-evoked nonvesicular GABA release and also enhanced the heteroexchange release of GABA induced by nipecotate. Thus, the GABA transporter normally operates near its equilibrium and can be easily induced to reverse by an increase in cytosolic [GABA] or mild depolarization. We propose that this transporter-mediated nonvesicular GABA release plays an important role in neuronal inhibition under both physiological and pathophysiological conditions and is the target of some anticonvulsants.

Fig. 1.

Schematic of recording setup. Recordings were made from neurons in primary cell cultures. When GABA was released from neighboring neurons and glia, a GABA_A receptor-mediated current was measured (Gaspary et al., 1998). Conditions were used in which vesicular GABA release was blocked. Therefore, these recordings were a rapid and highly sensitive bioassay of GABA release via reversal of the GABA transporter at physiologically relevant sites.

Fig. 2.

Vigabatrin caused spontaneous, tonic GABA_A receptor activation attributable to calcium-independent GABA release. A, Examples of recordings from two neurons on culture dishes that had been treated with 100 μm vigabatrin for 4 d. Recordings are in zero-calcium Ringer's solution. In both cases a large amount of negative current was required to hold the membrane potential at −60 mV in voltage clamp. At the bar, bicuculline (50 μm) decreased the holding current in both neurons. In some neurons, such as the top example, there was a sag in the response to bicuculline and a rebound increase in holding current after bicuculline application. This may have been attributable to a reduction in desensitization of the GABA_A receptor by bicuculline. B, The amount of leak current that was blocked by bicuculline (50 μm) was approximately the same in the presence and in the absence of extracellular calcium.Dark trace, Zero-calcium Ringer's solution; gray trace, normal Ringer's solution.

Fig. 3.

Tonic GABA release induced by vigabatrin caused the inhibition of neighboring neurons and was a result of the GABA transporter operating in reverse. A, Perforated patch recording from a neuron on a culture dish that had been treated with 100 μm vigabatrin for 165 hr. The recording was made at a holding potential of −60 mV in zero-calcium Ringer's solution that contained TTX, CNQX, AP-5, and CGP-55845. Application of bicuculline (100 μm) led to a decrease in an outward current (Bicuc), indicating that the tonic leak current induced by GABA_A receptor stimulation caused inhibition of this neuron. Application of SKF-89976a (5 μm) also caused a decrease in outward current (SKF), consistent with the blockade of GABA release by the transporter. After block of the outward leak current with bicuculline (100 μm) in the bath solution, the response to SKF-89976a (5 μm) was decreased (SKF + Bath Bicuc). The block of the response to SKF-89976a by bicuculline was reversible (SKF 2).B, Example of a second neuron with the same protocol. This neuron was treated with vigabatrin for 64 hr. Similar results were obtained from a total of 14 neurons.

Fig. 4.

The spontaneous, tonic nonvesicular GABA release induced by vigabatrin required 3–4 d of treatment to develop fully and occurred at nanomolar concentrations. A, The nonvesicular GABA release induced by vigabatrin increased very slowly. Plotted is the component of leak current that was blocked by bicuculline (50 μm) in neurons that had been treated with 100 μm vigabatrin for variable times, up to 5 d. The response to bicuculline in treated neurons was significantly greater than in control neurons (CTL) for all time points (p < 0.001). B, GABA leak current was induced by low concentrations of vigabatrin. Plotted is the leak current blocked by bicuculline (50 μm) versus the concentration of vigabatrin to which cells in culture were exposed for 3–4 d. Spontaneous GABA release was induced by as little as 50 nm vigabatrin and increased progressively with increasing concentrations up to 100 μm. All points are significantly greater for vigabatrin-treated cultures as compared with control (p < 0.001).

Fig. 5.

Blockade of glutamic acid decarboxylase decreased the tonic GABA release induced by vigabatrin. Plotted is the magnitude of the response to bicuculline (50 μm) in neurons from culture dishes that were treated with 5 μm vigabatrin for 3–4 d as compared with the response of sister dishes that were treated with 5 μm vigabatrin for 3–4 d and also treated with semicarbazide (2 mm) starting 24 hr after treatment with vigabatrin. The response of neurons that were treated with semicarbazide was 53% of control (p < 0.005).

Fig. 6.

Vigabatrin enhanced heteroexchange GABA release.A, Comparison of the response to 10 mm NPA of a neuron on a dish treated overnight with vigabatrin (100 μm) with that of a control neuron from a sister dish.B, Composite average of the responses to 10 mm NPA for neurons treated with 100 μmvigabatrin overnight (n = 32) as compared with the responses for control neurons (n = 31). Baseline was leak-subtracted in A and B.C, NPA response versus [vigabatrin] at <1 d. The AUC (i.e., total charge transfer) of the response to 10 mm NPA was plotted as the percentage of control. Neurons were treated with different concentrations of vigabatrin for 12–24 hr. D, NPA response versus [vigabatrin] at 3–4 d. Plotted is the AUC (as percentage of control) of the mean responses to 1 mm NPA for neurons that were treated with different concentrations of vigabatrin for 3–4 d. Neurons in D are the same as those in Figure 4B (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 7.

The sensitivity of the GABA_A receptor was decreased by treatment with a high concentration of vigabatrin.A, Shown is the peak response to direct application of 2 μm GABA in neurons from cultures that had been treated with 100 μm vigabatrin for 3–4 d (**p < 0.001). These are the same neurons as shown in Figures 4B and 6D.B, Treatment with 100 μm vigabatrin overnight led to a trend toward a smaller peak response to direct application of 2 μm GABA that was not statistically significant (p = 0.06). Shown is the mean response from 31 vigabatrin-treated neurons and 28 control neurons. Baseline was leak-subtracted in B. This is the same set of neurons as in Figure 6B.

Fig. 8.

The GABA transporter reversed easily in untreated cells in response to potassium-induced depolarization.A, Responses of a single neuron to graded increases in [K⁺]_o. This neuron had not been exposed to vigabatrin. The recording was made in zero-calcium Ringer's solution. As shown previously with this protocol (Gaspary et al., 1998), the currents induced by increased [K⁺]_o result from GABA_Areceptor activation because of carrier-mediated GABA release.B, The amount of carrier-mediated GABA release was proportional to the level of [K⁺]_o, with a response induced by a rise in [K⁺]_o to as little as 6 mm. Plotted is the total charge transfer measured as the AUC of the response for data that were obtained from 24 neurons; all points significantly differ from each other at p < 0.05 or less.

Fig. 9.

Vigabatrin enhanced the carrier-mediated GABA release induced by elevated [K⁺]_o.A, Example of the response to 12 mm[K⁺]_o (in zero-calcium solution) of a neuron on a dish treated overnight with vigabatrin (100 μm) and a control neuron from a sister dish.B, Composite average of the responses to 12 mm [K⁺]_o for neurons treated with vigabatrin (100 μm; n = 29) and control neurons (n = 28). Baseline was leak-subtracted in A and B.C, Vigabatrin (100 μm overnight) induced an increase in the response to 12 mm[K⁺]_o to 193% of control. The response to GABA in vigabatrin-treated neurons was 59% of control (**p < 0.001).

Fig. 10.

Model for dual role of the GABA transporter. Under normal conditions (A) the GABA transporter works in the forward direction to clear the extracellular space of GABA. During high frequency firing and seizures (B) [K⁺]_o and [Na⁺]_i both rise, and the cells depolarize. This would induce a reversal of the GABA transporter, maintaining GABAergic inhibition at a time that vesicular release decreases. The source of GABA release (neurons or glia) is not known at present.