Regulation of Neurosteroid Biosynthesis by Neurotransmitters and Neuropeptides

Summary

It is now established that the brain has the capability of synthesizing biologically active steroids, termed neurosteroids, that participate in the regulation of various neurophysiological and behavioral processes. However, the neuronal mechanisms regulating the activity of neurosteroid-producing cells have not yet been elucidated. We recently found that, in the frog brain, three enzymes involved in steroid biosynthesis are actively expressed in hypothalamic neurons: 3β-hydroxysteroid dehydrogenase/△⁵-△⁴ isomerase (3β-HSD), cytochrome P450 17α-hydroxylase/C17,20-lyase (P450_C17) and hydroxysteroid sulfotransferase (HST). Concurrently, we showed that frog hypothalamic explants can convert tritiated pregnenolone ([³H]△⁵P) into various bioactive steroids, including 17- hydroxypregnenolone (17OH-△⁵P), progesterone (P), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA), △⁵P sulfate (△⁵PS) and DHEA sulfate (DHEAS). The hypothalamic nuclei, where the 3β-HSD-, P450_C17- and HST-expressing neurons are located, receive afferent fibers containing a variety of neurotransmitters and neuropeptides. Here, we show that GABA, endozepines and neuropeptide Y (NPY) regulate neurosteroid biosynthesis.

Double immunohistochemical labeling of hypothalamic slices with antisera against 3β-HSD and various subunits of the GABA_A receptor revealed that most 3β-HSD -positive neurons also express the α3 and β2/β3 subunits of the GABA_A receptor. Incubation of hypothalamic explants with graded concentrations of GABA induced a dose-dependent inhibition of the conversion of [³H]△⁵P into radioactive metabolites. The effect of GABA on neurosteroid biosynthesis was mimicked by the GABA_A receptor agonist muscimol and was blocked by the selective GABA_A receptor antagonists bicuculline and SR95531. The GABA_A receptor complex encompasses a central-type benzodiazepine receptor (CBR). Thus, we investigated the effect of the endozepine octadecaneuropeptide (ODN), an endogenous ligand of CBR, on neurosteroid biosynthesis. Using an antiserum against human ODN, we observed that ODN-positive glial cells send thick processes in the close vicinity of 3β-HSD-containing neurons. Incubation of hypothalamic explants with synthetic ODN induced a dose- dependent stimulation of the conversion of [³H]△⁵P into various neurosteroids. The β-carbolines β-CCM and DMCM, two inverse agonists of CBR, mimicked the stimulatory effect of ODN on neurosteroid biosynthesis, whereas the CBR antagonist flumazenil significantly reduced the stimulatory responses induced by ODN, β-CCM and DMCM. These data indicate that GABA, acting through GABAA receptors, inhibits 3β-HSD activity and that ODN, acting as an inverse agonist on the GABA_A/CBR complex, stimulates neurosteroid biosynthesis.

Labeling of brain sections revealed the existence of NPY-immunoreactive varicosities in close proximity to HST-containing perikarya. In situ hybridization studies showed that Y₁ and Y₅ receptor mRNAs are expressed in the anterior preoptic area and the dorsal magnocellular nucleus. Pulse-chase experiments with ³⁵S-labeled 3′-phosphoadenosine 5′-phosphosulfate as a sulfate donor, and [³H]△⁵P or [³H]DHEA as a steroid precursor, demonstrated that NPY inhibits the conversion of △⁵P into △⁵PS and DHEA into DHEAS by hypothalamic explants. The inhibitory effect of NPY on the formation of sulfated neurosteroids was mimicked by PYY, a non-selective NPY receptor agonist, and by [Leu³¹,Pro³⁴]NPY, an agonist for non-Y₂ receptors, and was completely suppressed by the Y₁ receptor antagonist BIBP3226. Conversely, the Y₂ receptor agonist NPY(13–36) and the Y₅ receptor agonist [D-Trp³²]NPY did not affect the biosynthesis of △⁵PS and DHEAS. These data indicate that NPY, acting through Y₁ receptors, exerts an inhibitory influence on the biosynthesis of sulfated neurosteroids. The present study provides evidence that, in the brain, neurostransmitters and neuropeptides regulate the activity of neurosteroid- producing neurons. Since neurosteroids have been implicated in the control of a number of behavioral and metabolic activities, these data strongly suggest that some of the neurophysiological effects of neurotransmitters and neuropeptides can be mediated through modulation of neurosteroid biosynthesis.