Receptors with low affinity for neurosteroids and GABA contribute to tonic inhibition of granule cells in epileptic animals

Abstract

Neurosteroid sensitivity of GABA(A) receptor mediated inhibition of the hippocampal dentate granule cells (DGCs) is reduced in animal models of temporal lobe epilepsy. However, the properties and subunit composition of GABA(A) receptors mediating tonic inhibition in DGCs of epileptic animals have not been described. In the DGCs of epileptic animals, allopregnanolone and L-655708 sensitivity of holding current was diminished and δ subunit was retained in the endoplasmic reticulum and its surface expression was decreased the in the hippocampus. Ro15-4513 and lanthanum had distinct effects on holding current recorded from DGCs of control and epileptic animals suggesting that the pharmacological properties of GABA(A) receptors maintaining tonic inhibition in DGCs of epileptic animals were similar to those containing the α4βxγ2 subunits. Furthermore, surface expression of the α4 subunit increased and a larger fraction of the subunit co-immunoprecipitated with theγ2 subunit in hippocampi of epileptic animals. Together, these studies revealed that functional α4βxδ and α5βxγ2 receptors were reduced in the hippocampi of epileptic animals and that novel α4bxγ2 receptors contributed to the maintenance of tonic inhibition. The presence of α4βxγ2 receptors resulted in low GABA affinity and neurosteroid sensitivity of tonic currents in the DGCs of epileptic animals that could potentially increase seizure vulnerability. These receptors may represent a novel therapeutic target for anticonvulsant drugs without sedative actions.

Figure 1

Modulation of tonic inhibition by allopregnanolone is diminished in epileptic DGCs. A. (a) Representative current traces recorded from DGCs of control and epileptic animals before and after application of 30 nM allopregnanolone. (b) Decimated traces of (a) for better temporal resolution. In this and all subsequent traces, arrowheads represent the beginning of drug application. The straight dotted lines in these and subsequent traces indicate the mean holding current for the respective recordings. (c) Quantitative data of mean shift in holding current (I_Δ) in recordings from control and epileptic DGCs following application of 30 nM allopregnanolone. B. (a) Representative current traces recorded from DGCs of control and epileptic animals before and after application of 60 nM allopregnanolone. (b) Decimated traces of (i) for better temporal resolution. (c) Quantification of the mean I_Δ in recordings from control and epileptic DGCs following application of 60 nM allopregnanolone. All recordings were performed in the presence of 10 µM NO-711 and 3 µM GABA. *p<0.05, **p<0.01 compared to control, unpaired t test.

Figure 2

Surface expression of the δ subunit was reduced in chronic epileptic animals and more of it was retained in the ER. A. Surface expression of δ subunit was studied in control (lane C) and epileptic (lane E) animals by a biotinylation assay (lanes marked “Surface”). Expression of δ subunit was also studied using total cell lysates for normalization (lanes marked “Total”). The same blots were used for expression of β-actin used as a marker for cytoplasmic proteins. Absence of a β-actin signal in surface samples indicates specificity of the biotinylation assay. B. The total microsomal fraction was isolated from control (lane C) and epileptic (lane E) animals and blotted for expression of δ subunit (lanes marked “Microsomal Fraction”). Expression in total cell lysates was studied for normalization (lanes marked “Total”). A stronger signal in the microsomal fraction from epileptic animals indicates ER retention of δ subunit. Blots were also reprobed with the ER markers calnexin and Grp78/Bip to confirm equal loading of ER membranes.

Figure 3

The magnitude of tonic inhibition recorded in the presence or absence of 10 µM NO-711 and 3 µM GABA in control and epileptic DGCs was similar. (a) Representative current traces recorded from DGCs of control and epileptic animals before and after application of 100 µM bicuculline in slices bathed in ACSF with uptake blocker NO-711 and GABA. Bicuculline application is shown by same bar as in control. (b) Quantification of the mean I_Δ in recordings from control and epileptic DGCs following application of bicuculline. (c) Representative current traces recorded from DGCs of control and epileptic animals before and after application of 100 µM bicuculline in slices bathed in ACSF without uptake blocker NO-711 and GABA. Bicuculline application is shown by the same bar as in control. (d) Cumulative frequency plot of I_rms distribution before (solid line) and after (dotted line) application of bicuculline in control (upper panel) and epileptic (lower panel) DGC. Note the leftward shift of distribution plot in presence of bicuculline.

Figure 4

Tonic inhibition in epileptic DGCs is not maintained by α5 subunit GABA_A receptors. (a) Representative current traces recorded from control and epileptic DGCs before and after application of the selective α5 subunit antagonist L-655708 (1 µM). (b) Decimated traces of (a) for better temporal resolution. (c) Quantification of the mean I_Δ in recordings from control and epileptic DGCs following application of L-655708. All recordings were performed in the presence of 10 µM NO-711 and 3 µM GABA. *p<0.05 compared to control, unpaired t test.

Figure 5

Tonic inhibition in epileptic DGCs is mediated by GABA_A receptors assembled with α4γ2 subunits. A. (a) Representative current traces recorded from control and epileptic DGCs before and after application of 100 µM lanthanum (LaCl₃). (b) Decimated traces of (a) for better temporal resolution. (c) Quantitative analysis of the mean shift in holding current (I_Δ) in recordings from control and epileptic DGCs following application of lanthanum. B. (a) Representative current traces recorded from control and epileptic DGCs before and after application of Ro-154513 (300 nM), a partial inverse agonist against non-α4 subunit-containing GABA_A receptors. (b) Decimated traces of (i) for better temporal resolution. (c) Quantification of the mean I_Δ in recordings from control and epileptic DGCs following application of Ro-154513. All recordings were performed in the presence of 10 µM NO-711 and 3 µM GABA. *p<0.05 compared to control, unpaired t test.

Figure 6

Low concentration of GABA (1 µM) enhanced tonic inhibition in control but not epileptic DGCs. A. Representative current traces recorded from control and epileptic DGCs (TLE) before and after application of 1 µM GABA in the absence of an uptake blocker or prior introduction of exogenous GABA. B. Decimated traces of (A) for better temporal resolution. C. Slope conductance of the RMS noise measured at various holding potentials before and after application of 1 µM GABA in control and epileptic DGCs. The solid line represents the regression line through observations made during baseline in control DGCs, and the dashed line represents observations made after application of GABA.

Figure 7

Open channel blocker penicillin inhibits persistently open GABA_A receptors in control DGCs but not in epileptic DGCs. In all traces I_rms is shown by arrows between solid and dashed lines. A. Representative current trace recorded from control DGC before (baseline upper trace) and after (lower trace) application of 300 µM penicillin. B. Representative current traces recorded from an epileptic DGC, before (baseline top) and after (bottom) application of penicillin. C. Cumulative frequency plot of I_rms during 60 30 sec epochs during before (solid line) and after (dotted line) application of penicillin from a control DGC. Note the left shift of the distribution. D. Cumulative frequency plot of I_rms distribution before (solid line) and after (dotted line) application of penicillin in epileptic DGC. Note the minimal shift compared to that of control DGC.

Figure 8

Surface expression of the α4 subunit increased in epileptic animals and its association with the γ2 subunit was higher. A. Surface expression of the α4 subunit was studied from surface proteins isolated from naïve (lane C) and epileptic (lane E) animals using a biotinylation assay (panel “Surface”). Total expression of the α4 subunit was also studied for normalization (panel “Total”). B. Association between the α4 and γ2 subunits was studied in control and epileptic animals. Using an anti-γ2 antibody, the γ2 subunit and associated proteins were separated from hippocampi of control (lane C) and epileptic (lane E) animals. Expression of the γ2, α4, and α1 subunits was studied by Western blotting. Expression of these subunits was also studied from total proteins for normalization (panel total). To confirm specific pull-down of the γ2 and associated proteins using anti-γ2 IgG, normal rabbit IgG was also used to pull-down proteins (lane IgG).