alpha5 Subunit-containing GABA(A) receptors mediate a slowly decaying inhibitory synaptic current in CA1 pyramidal neurons following Schaffer collateral activation

Abstract

GABA(A) receptors that contain the alpha5 subunit (alpha5GABA(A)Rs) are highly expressed in the hippocampus, and have been implicated in learning and memory processes. They generate a tonic form of inhibition that regulates neuronal excitability. Recently it was shown that alpha5GABA(A)Rs also contribute to slow phasic inhibition of CA1 pyramidal neurons following local stimulation in the stratum lacunosum moleculare. However, it is unknown whether alpha5GABA(A)Rs can also be recruited indirectly by stimulation of Schaffer collaterals. Here, we studied GABAergic currents evoked by stimulation in the stratum radiatum of CA1 in the presence and absence of CNQX to block AMPA receptor-mediated excitation. We tested their sensitivity to gabazine and two drugs acting at the benzodiazepine site of alpha1/alpha2/alpha3 or alpha5GABA(A)Rs (400 nM zolpidem and 20 nM L-655,708, respectively). IPSCs evoked by stimulation in the stratum radiatum in the presence of CNQX were potentiated by zolpidem, blocked by 1 muM gabazine and were relatively insensitive to L-655,708 consistent with the lack of alpha5GABA(A)Rs. In contrast, IPSCs evoked by stimulation of Schaffer collaterals had a significant gabazine-insensitive component. This component was attenuated by L-655,708 and enhanced by burst stimulation. Furthermore, the L-655,708-sensitive current was absent in recordings from mice lacking alpha5GABA(A)Rs (gabra5(-/-) mice). These results show that alpha5GABA(A)R-mediated phasic inhibition is activated by the Schaffer collateral pathway and provide evidence for activity pattern-dependent participation of alpha5GABA(A)Rs in inhibition.

Fig. 1

Locally-evoked and Schaffer collateral-stimulated GABA_AR-mediated currents are differentially sensitive to gabazine. (Ai) Current traces showing voltage dependence of GABA_AR currents evoked by synaptic stimulation in two different recording conditions. Top traces: inhibitory currents recorded at 0 mV; GABA_A,local in the presence of 10 μM CNQX and GABA_A,SC. Bottom traces: Synaptic currents recorded at different holding potentials between −80 and +40 mV in steps of 20 mV. (Aii) Expanded traces for Schaffer collateral-stimulated currents (holding potentials of −70, −50, −30, −10, +10 and +40 mV) and current voltage relationship for estimated AMPAR (inverted triangles) and GABA_AR (squares) mediated components (n = 5). Currents were measured at times indicated by vertical lines in example traces. (B, C) GABA_AR currents in the presence of increasing concentrations of gabazine. (Bi) Concentration–response plot for GABA_A,local and GABA_A,SC. Response is the charge transfer normalised to pre-gabazine control values. GABA_A,SC(gz) indicates the 1 μM gabazine-resistant component of GABA_A,SC. Inset: Example traces of Schaffer collateral-stimulated currents in 1 μM gabazine with holding potentials as in Ai. (Bii) Superimposed current traces, with and without 1 μM gabazine. Normalised traces for GABA_A,local, GABA_A,SC, and GABA_A,SC(gz). (Ci) Concentration–response plot for the early and late components of GABA_A,SC elicited with burst stimulation (3 stimuli at 100 Hz). GABA_A,SC(5–35 ms) and GABA_A,SC(50–750 ms) were the measured charge transfers in the indicated time windows after stimulation. (Cii) Example traces of GABA_A,SC(burst) recorded at 0 mV with and without gabazine. Normalised traces on the right. Scale bars in Bi-ii and Cii: 250 ms, 400 pA.

Fig. 2

The α5 inverse-agonist L-655,708 inhibits GABA_A,SC(gz). (A) Superimposed example traces of GABA_AR currents before (grey) and after (black) application of L-655,708. (B) Normalised charge transfer (i) and peak current (ii) measured during baseline and after bath application of L-655,708 at time = 0. ***P < 0.001.

Fig. 3

L-655,708 (20 nM) is specific for the GABA_A receptor α5 subunit. (A) Superimposed example traces of GABA_A,SC (i) and GABA_A,SC(gz) (ii) before and after application of 20 nM L-655,708 in wild type and gabra5^−/− mice. (B) Normalised values for GABA_AR charge transfer measured during baseline and following application of L-655,708 at time 0 for GABA_A,SC (i) and GABA_A,SC(gz) (ii). (C) Concentration-response plot of GABA_A,SC for 20, 50 and 1000 nM L-655,708 in wild type and gabra5^−/− mice. ***P < 0.001.

Fig. 4

GABA_A receptor-mediated currents in gabra5^−/− mice and WT mice are differentially sensitive to 1 μM gabazine. (A) Superimposed example traces of GABA_A,SC currents before (grey) and after (black) application of 1 μM gabazine. Recordings from WT (i), and gabra5^−/− mice (ii). (B) Normalised values for charge transfer (i) and peak current (ii) measured during baseline and following application of 1 μM gabazine at time 0. *P < 0.05; **P < 0.01.

Fig. 5

The enhancing effect of zolpidem is larger on GABA_A,local and GABA_A,SC than on GABA_A,SC(gz). (A) Superimposed example traces of GABA_AR currents before and after application of zolpidem (400 nM). From left to right: GABA_A,local, GABA_A,SC, and GABA_A,SC(gz) with corresponding symbols in B. (B) Normalised values of charge transfer (i) and peak current (ii) measured during baseline and following application of zolpidem at time 0. Significant difference of charge transfers but not peak currents between baseline and the last 4 min of recording for the three recording conditions. *P < 0.05; ***P < 0.001.