gamma-Aminobutyric acid, acting through gamma -aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus

Abstract

Most of the actions of neurosteroids on the central nervous system are mediated through allosteric modulation of the gamma-aminobutyric acid type A (GABA(A)) receptor, but a direct effect of GABA on the regulation of neurosteroid biosynthesis has never been investigated. In the present report, we have attempted to determine whether 3beta-hydroxysteroid dehydrogenase (3beta-HSD)-containing neurons, which secrete neurosteroids in the frog hypothalamus, also express the GABA(A) receptor, and we have investigated the effect of GABA on neurosteroid biosynthesis by frog hypothalamic explants. Double immunohistochemical labeling revealed that most 3beta-HSD-positive neurons also contain GABA(A) receptor alpha(3) and beta(2)/beta(3) subunit-like immunoreactivities. Pulse-chase experiments showed that GABA inhibited in a dose-dependent manner the conversion of tritiated pregnenolone into radioactive steroids, including 17-hydroxy-pregnenolone, progesterone, 17-hydroxy-progesterone, dehydroepiandrosterone, and dihydrotestosterone. The effect of GABA on neurosteroid biosynthesis was mimicked by the GABA(A) receptor agonist muscimol but was not affected by the GABA(B) receptor agonist baclofen. The selective GABA(A) receptor antagonists bicuculline and SR95531 reversed the inhibitory effect of GABA on neurosteroid formation. The present results indicate that steroid-producing neurons of the frog hypothalamus express the GABA(A) receptor alpha(3) and beta(2)/beta(3) subunits. Our data also demonstrate that GABA, acting on GABA(A) receptors at the hypothalamic level, inhibits the activity of several key steroidogenic enzymes, including 3beta-HSD and cytochrome P450(C17) (17alpha-hydroxylase).

Figure 1

Confocal laser scanning microscope photomicrographs comparing the distribution of 3β-HSD- and GABA_A receptor α₃ subunit-like immunoreactivity in the frog hypothalamus. (A and B) Adjacent frontal sections through the anterior preoptic area are labeled with the antiserum against 3β-HSD (A) or the polyclonal antibodies against the α₃ subunit (B). (C and D) Adjacent frontal sections through the posterior tuberculum labeled with the antiserum against 3β-HSD (C) or the polyclonal antibodies against the α₃ subunit (D). Open arrows, neurons expressing only the 3β-HSD-like immunoreactivity; arrowheads, neurons expressing only the GABA_A receptor α₃ subunit-like immunoreactivity; filled arrows, neurons expressing both 3β-HSD- and GABA_A receptor α₃ subunit-like immunoreactivities. (Bars = 10 μm.)

Figure 2

Dual-channel confocal laser scanning microscope photomicrographs comparing the distribution of 3β-HSD and GABA_A receptor β₂/β₃ subunit-like immunoreactivity in the frog hypothalamus. (A–C) Frontal section through the posterior tuberculum labeled with the antiserum against 3β-HSD revealed with DAR_S/TXR (A; arrows) or the monoclonal antibody against the β₂/β₃ subunits revealed with GAM_S/Alexa-488 (B; arrowheads). Combination of the two images acquired in A and B revealed the presence of β₂/β₃ subunit-like immunoreactivity in a subset of 3β-HSD-positive neurons (C; arrows). (Bars = 10 μm.) (D–F) Frontal section through the nucleus of the periventricular organ labeled with the antiserum against 3β-HSD revealed with DAR_S/TXR (D) or the monoclonal antibody against the β₂/β₃ subunits revealed with GAM_S/Alexa-488 (E). Combination of the two images acquired in D and E revealed the coexistence of 3β-HSD- and β₂/β₃ subunit-like immunoreactivity in the same neuron (F). (Bars = 2 μm.)

Figure 3

Labeling of consecutive coronal sections through the anterior preoptic area, showing the specificity of the immunocytochemical reactions. (A and B) Specificity control of the 3β-HSD immunostaining. Adjacent sections were incubated with the antibodies against 3β-HSD (A) or with the antibodies preabsorbed with purified human type I 3β-HSD (10⁻⁶ M) (B). (C and D) Specificity control of the α₃ subunit immunostaining. Adjacent sections were incubated with the antibodies against the α₃ subunit (C) or with the antibodies preabsorbed with the purified peptide hapten (10⁻⁶ M) (D). (Bars = 10 μm.)

Figure 4

Analysis of radioactive steroids extracted from the tissue (A and B) or medium (C and D) after a 2-h incubation of frog hypothalamic explants with [³H]pregnenolone in the absence (A and C) or presence (B and D) of 10⁻⁶ M GABA. The ordinate indicates the radioactivity measured in the HPLC eluent. The dashed lines represent the gradient of secondary solvent (% solution B). The arrows indicate the elution position of standard steroids: 17OH-Δ⁵P, 17-hydroxypregnenolone; 5α-DHT, dihydrotestosterone; DHEA, dehydroepiandrosterone; F, cortisol; S, 11-deoxycortisol; T, testosterone; 17OH-P, 17-hydroxyprogesterone; P, progesterone; Δ⁵P, pregnenolone.

Figure 5

Effect of graded concentrations of GABA on the conversion of [³H]pregnenolone into 17-hydroxypregnenolone and dihydrotestosterone (17OH-Δ⁵P/5α-DHT; A), dehydroepiandrosterone (DHEA; B), 17-hydroxyprogesterone (17OH-P; C) and progesterone (P; D) by hypothalamic slices. The values were obtained from experiments similar to those presented in Fig. 4. Results are expressed as percentages of the amount of each steroid formed in the absence of GABA. Values are the mean (±SEM) of four independent experiments.

Figure 6

Effects of GABA (10⁻⁵ M), the GABA_A agonist muscimol (10⁻⁵ M), and the GABA_B agonist baclofen (10⁻⁵ M) on the conversion of [³H]pregnenolone into 17-hydroxypregnenolone and dihydrotestosterone (17OH-Δ⁵P/5α-DHT; A), dehydroepiandrosterone (DHEA; B), 17-hydroxyprogesterone (17OH-P; C), and progesterone (P; D) by hypothalamic slices. The values were obtained from experiments similar to those presented in Fig. 4. Results are expressed as percentages of the amount of each steroid formed in the absence of drugs. Each value is the mean (±SEM) of four independent experiments. **, P < 0.01 by one-way ANOVA followed by a post hoc Dunnett's test; NS, not statistically different vs. control.

Figure 7

Effects of GABA (10⁻⁶ M) in the absence or presence of the GABA_A antagonists SR95531 (10⁻⁵ M) and bicuculline (10⁻⁵ M) on the conversion of [³H]pregnenolone into 17-hydroxypregnenolone and dihydrotestosterone (17OH-Δ⁵P/5α-DHT; A), dehydroepiandrosterone (DHEA; B), 17-hydroxyprogesterone (17OH-P; C), and progesterone (P; D) by hypothalamic slices. The values were obtained from experiments similar to those presented in Fig. 4. Results are expressed as percentages of the amount of each steroid formed in the absence of drugs. Each value is the mean (±SEM) of four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA followed by a post hoc Bonferroni's test; NS, not statistically different.