Heterogeneity of hippocampal GABA(A) receptors: regulation by corticosterone

Abstract

Chronic stressors produce changes in hippocampal neurochemistry, neuronal morphology, and hippocampal-dependent learning and memory processes. In rats, stress-induced changes in CA3 apical dendritic structure are mediated by corticosterone (CORT) acting, in part, on excitatory amino acid neurotransmission. CORT also alters GABA-mediated inhibitory neurotransmission, so the GABA(A) receptor system may also contribute to dendritic remodeling and other stress-related changes in hippocampal function. A previous study indicated that chronic CORT treatment produces complex changes in GABA(A) receptor subunit mRNA levels, so we hypothesized that CORT alters the pharmacological properties of hippocampal GABA(A) receptors. To test this, adult male rats were treated with CORT or vehicle pellets for 10 d, after which we quantified [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) and [(3)H]flunitrazepam binding to GABA(A) receptors using in vitro receptor autoradiography. Pharmacological properties of receptors were assessed by examining the allosteric regulation of binding at both sites by GABA and 5alpha-pregnane-3alpha,21-diol-20-one (THDOC), an endogenous anxiolytic steroid. We found striking regional differences in the modulation of [(35)S]TBPS binding, particularly between strata radiatum and strata oriens, suggesting a functional heterogeneity among hippocampal GABA(A) receptors even within the apical versus basal dendrites of pyramidal neurons. Furthermore, we found that CORT treatment decreased the negative modulation of hippocampal [(35)S]TBPS binding by both GABA and THDOC and increased the enhancement of [(3)H]flunitrazepam binding by GABA and THDOC in the dentate gyrus. Together, these data suggest that prolonged exposure to stress levels of corticosteroids may alter hippocampal inhibitory tone by regulating the pharmacological properties of GABA(A) receptors in discrete dendritic subfields.

Fig. 1.

Effect of CORT on the modulation of [³⁵S]TBPS binding by 100 nm THDOC in dorsal hippocampus. Male rats of mixed gonadal status received CORT pellets or vehicle pellets subcutaneously for 10 d. Data are expressed as the specific binding of [³⁵S]TBPS in the presence of THDOC as percentage of binding in the absence of THDOC, normalized for each animal. Shown are means and error bars representing SEM; n = 27 for CORT- and vehicle-treated animals. There was an overall significant main effect of CORT treatment on the mean percentage modulation by THDOC in hippocampus (F_(1,52) = 7.28; p= 0.0094), indicated by an asterisk. There were also significant regional differences between mean levels of modulation by THDOC, averaged across treatments, between CA3 stratum radiatum and CA3 stratum oriens (t = −34.65; p≤ 0.0001), and between CA3 stratum radiatum and CA1 stratum radiatum (t = −9.95; p ≤ 0.0001). See Table 3 for summary of statistical analysis.

Fig. 2.

Representative autoradiograms showing the modulation of 2 nm [³⁵S]TBPS binding by GABA in the dorsal hippocampus. In A, incubation buffer included no exogenous GABA; in B, 2.0 μm GABA; in C, 5 μm GABA. InA, CA1–CA3 in Ammon's horn and dentate gyrus are indicated with arrows pointing to the pyramidal or granule cell layers. In B, note the effect of GABA on [³⁵S]TBPS binding: an increase in (CA1) stratum oriens (white arrow) and a decrease in stratum radiatum (black arrow), relative to control binding inA. [³⁵S]TBPS binding was inhibited in all regions by 5 μm GABA.

Fig. 3.

Effect of CORT on the modulation of [³⁵S]TBPS binding by GABA in the dorsal hippocampus. Experiment 2 used brain sections from gonadally intact CORT- or vehicle-treated animals incubated with 2.3 [³⁵S]TBPS, with or without 1.5 μmGABA. Data are expressed as the specific binding of [³⁵S]TBPS, percentage of binding in the presence of GABA relative to basal binding in the absence of GABA. Shown are means and error bars representing SEM; n = 9. There was an overall significant main effect of CORT versus vehicle treatment in hippocampus (F_(1,16) = 4.80; p = 0.044), indicated by anasterisk. Set C contrasts indicated significant differences in the mean levels of modulation of [³⁵S]TBPS binding by GABA, averaged across treatments, between CA3 radiatum and CA3 oriens (t= −13.56; p ≤ 0.0001), and between CA3 radiatum and CA1 radiatum (t = 5.15; p≤ 0.0001).

Fig. 4.

Modulation of [³⁵S]TBPS binding by GABA in the dorsal hippocampus and cortex. In Experiment 3, brain sections were incubated with 2 nm[³⁵S]TBPS with or without 1.5 μmGABA. Data are expressed as [³⁵S]TBPS specific binding in the presence of 1.5 μm GABA as the percentage of binding in the absence of GABA. Shown are means and error bars representing SEM averaged across CORT- and vehicle-treated groups;n = 18. There were significant regional differences between the modulation of [³⁵S]TBPS by GABA in Ammon's horn and dentate gyrus (F_(1,16) = 127.6; p≤ 0.0001) and between the means of the individual dendritic subfields (F_(6,16) = 278.0; p≤ 0.0001) in Ammon's horn or dentate gyrus, or both. Set C contrasts indicated significant differences, indicated by anasterisk, between the mean modulation of [³⁵S]TBPS binding by GABA in CA3 stratum radiatum versus CA3 stratum oriens (t = −17.82;p ≤ 0.0001) and between CA3 stratum radiatum and CA1 stratum radiatum (t = 8.40;p ≤ 0.0001). Also note the striking differences in mean percentage modulation by GABA between layers I–III, IV, V, and VI of the parietal cortex.

Fig. 5.

Effect of CORT on the modulation of [³H]flunitrazepam binding by THDOC in the dorsal hippocampus. Brain sections from CORT- or vehicle-treated, gonadally intact animals were incubated with 0.5 nm[³H]flunitrazepam in buffer alone or buffer containing 100 nm THDOC. Data presented are [³H]flunitrazepam-specific binding in the presence of THDOC expressed as the percentage of basal binding in the absence of THDOC. Shown are means and error bars representing SEM;n = 9. The CORT- versus vehicle-treatment factor was significant (F_(1,16) = 4.79;p = 0.0437) but so was the interaction between treatment and hippocampal region (Ammon's horn vs dentate gyrus;F_(1,16) = 5.89; p = 0.0274). Set B contrasts indicated that the percentage modulation of [³H]flunitrazepam binding by THDOC differed between CORT- and vehicle-treated animals in the dentate gyrus (t = −2.65; p = 0.0173), indicated by an asterisk.

Fig. 6.

Effect of CORT on the modulation of [³H]flunitrazepam binding by GABA in the dorsal hippocampus. Brain sections from CORT- or vehicle-treated, gonadally intact animals were incubated with 0.5 nm[³H]flunitrazepam in buffer alone or buffer containing 3 μm GABA. Data presented are [³H]flunitrazepam-specific binding in the presence of GABA, expressed as the percentage of binding in the absence of GABA. Shown are means and error bars representing SEM; n= 9. There was a significant CORT- versus vehicle-treatment effect (F_(1,16) = 9.81; p= 0.0064), and a significant interaction between treatment and hippocampal region (F_(1,16) = 6.23;p = 0.0239). Set B contrasts indicated that mean percentage modulation of [³H]flunitrazepam binding by GABA differed between CORT- and vehicle-treated animals in the dentate gyrus (t = −3.98; p = 0.0011), indicated by an asterisk.