A modulatory role of ASICs on GABAergic synapses in rat hippocampal cell cultures

Abstract

Rapid acidification occurring during synaptic vesicle release can activate acid-sensing ion channels (ASICs) both on pre- and postsynaptic neurons. In the latter case, a fraction of postsynaptic current would be mediated by cation-selective acid-sensing ion channels. Additionally, in both cases, activation of acid-sensing ion channels could modulate synaptic strength by affecting transmitter release and/or sensitivity of postsynaptic receptors. To address potential involvement of acid-sensing ion channels in mediation/modulation of synaptic transmission at hippocampal GABAergic synapses, we studied effects of three structurally different blockers of acid-sensing ion channels on evoked postsynaptic currents using the patch-clamp technique. We found that GABAergic postsynaptic currents, recorded below their reversal potential as inward currents, are suppressed by all the employed blockers of acid-sensing ion channels. These currents were suppressed by ~ 20 % in the presence of a novel blocker 5b (1 μM) and by ~30 % in the presence of either amiloride (25 μM) or diminazene (20 μM). In the same cells the suppression of postsynaptic currents, recorded above their reversal potential as outward currents was statistically insignificant. These results imply that the effects of blockers in our experiments are at least partially postsynaptic. On the other hand, in the case of mediation of a fraction of postsynaptic current by acid-sensing ion channels, an increase of outward currents would be expected under our experimental conditions. Our analysis of a bicuculline-resistant fraction of postsynaptic currents also suggests that effects of the blockers are predominantly modulatory. In this work we present evidence for the first time that acid-sensing ion channels play a functional role at hippocampal GABAergic synapses. The suppressing effect of the blockers of acid-sensing ion channels on GABAergic transmission is due, at least partially, to a postsynaptic but (predominantly) modulatory mechanism. We hypothesize that the modulatory effect is due to functional crosstalk between ASICs and GABA_A-receptors recently reported in isolated neurons, however, verification of this hypothesis is necessary.

Keywords: Amiloride; Diminazene; GABA; Protons; Synaptic transmission.

Fig. 1

Experimental protocols used in most of the experiments. During each sweep a presynaptic neuron was stimulated twice, firstly when the membrane potential in the postsynaptic cell was clamped 10-15 mV below PSC reversal potential for a given synaptic connection (typically –45 mV), and secondly when the membrane potential was shifted by 20 mV (typically to –25 mV). Sweeps were collected every 4 seconds. Stimulations of a presynaptic neuron are marked by arrows. Voltage in the postsynaptic neuron is schematically shown in upper panels, and currents in the lower. a Experimental protocol used in most of the experiments with 5b. b Experimental protocol used in the experiments with amiloride and diminazene. This protocol is similar to that shown in A, but during each sweep a pre-synaptic neuron was stimulated three times – two currents were recorded as inward and one as outward. The upper panel illustrates voltage protocol, and the lower panel currents recorded in a postsynaptic neuron

Fig. 2

5b (novel blocker of ASICs) suppresses inward GABAergic currents in ‘HEPES 2’ solution. In the same series of experiments during each sweep evoked PSCs were recorded below their reversal potential as inward currents (a), and above the reversal potential, as outward ones (b) - see Methods for details. Superimposed traces of original current traces (averages of 10 sequential PSCs) before (solid lines) and after (dotted lines) 5b (1 μM) application are shown on the right panel, summary graphs (n = 8) are shown on the left panel. PSC-amplitudes were normalized to control values (average of 20 PSCs preceding drug application). In control, absolute amplitudes of inward and outward currents (mean ± S.D) in this series of experiments were: -194.35 ± 93,1 pA; 214.8 ± 175.4 pA

Fig. 3

ASICs do not mediate substantial fraction of PSCs at hippocampal GABAergic synapses. Top panel: only a small fraction of evoked GABAergic PSCs is bicuculline-resistant. a. An example of original current traces (averages of 10 sequential PSCs) before and after bicuculline (20 μM) application. This is an example of a relatively large residual current. For the experiments described below, we selected cells with relatively large residual currents. b. Summary graph (n = 20). In control (before bicuculline), absolute amplitude of currents (mean ± S.D) was: -336.5 ± 144.7 pA. Bottom panel: 5b does not strongly affect bicuculline-resistant (residual) currents at GABAergic connections. c. Superimposed traces of original current traces (averages of 10 sequential PSCs) before and after 5b (1 μM) application. d. Summary graph (n = 5). In control (before 5b application), absolute amplitude of currents (mean ± S.D) was: -45.8 ± 21.6 pA

Fig. 4

5b (novel blocker of ASICs) has little effect on inward GABAergic currents in ‘HEPES 10’ solution. In the same series of experiments during each sweep evoked PSCs were recorded below their reversal potential as inward currents (a), and above the reversal potential, as outward ones (b). Superimposed traces of original current traces (averages of 10 sequential PSCs) before (solid lines) and after (dotted lines) 5b (1 μM) application are shown on the right panel, summary graphs (n = 5) are shown on the left panel. PSC-amplitudes were normalized to control values (average of 20 PSCs preceding drug application). In control, absolute amplitudes of inward and outward currents (mean ± S.D) in this series of experiments were: -196.3 ± 92.6 pA; 193.1 ± 119.6 pA

Fig. 5

A non-selective blocker of ASICs amiloride suppresses inward GABAergic currents. In the same series of experiments during each sweep evoked PSCs (2 PSCSs with 100 ms interval) were recorded below their reversal potential as inward currents (a), and above the reversal potential, as outward ones (b), see Methods for details. Superimposed traces of original current traces (averages of 10 sequential PSCs) before (solid lines) and after (dotted lines) amiloride (25 μM) application are shown on the right panel, summary graphs (n = 5) are shown on the left panel. PSC-amplitudes and paired-pulse ratios for inward PSCs (PSC2/PSC1) were normalized to control values (average for 20 PSCs preceding drug application). Normalized paired-pulse ratio is plotted in (c). The experiments were done in ‘HEPES 2’ solution. In control, absolute amplitudes of inward and outward currents (mean ± S.D) in this series of experiments were: -174.4 ± 75.0 pA; 110.6 ± 60.8 pA

Fig. 6

ASIC blocker diminazen suppresses inward GABAergic currents. In the same series of experiments during each sweep evoked PSCs (2 PSCSs with 100 ms interval) were recorded below their reversal potential as inward currents (a), and above the reversal potential, as outward ones (b), see Methods for details. Superimposed traces of original current traces (averages of 10 sequential PSCs) before (solid lines) and after (dotted lines) diminazene (20 μM) application are shown on the right panel, summary graphs (n = 6) are shown on the left panel. PSC-amplitudes and paired-pulse ratios for inward PSCs (PSC2/PSC1) were normalized to control values (average for 20 PSCs preceding drug application). Normalized paired-pulse ratio is plotted in (c). The experiments were done in ‘HEPES 2’ solution. In control, absolute amplitudes of inward and outward currents (mean ± S.D) in this series of experiments were: -188.5 ± 97.0 pA; 178.4 ± 58.4 pA