INTRODUCTION: Neurosteroids are synthesized de novo in the brain from cholesterol. This process resembles steroidogenesis in the gonads, adrenal glands, and placenta and involves P450scc, 3β-HSD, 17β-HSD, and aromatase. Dysregulated neurosteroid levels are linked to several neuropsychiatric disorders. Major brain regions involved in neurosteroid synthesis include the neocortex, striatum, hypothalamus, thalamus, hippocampus, amygdala, brainstem, and cerebellum. Benzodiazepines, by crossing the blood–brain barrier, can directly affect these brain structures.

AIM(S): To evaluate the effect of clonazepam on neurosteroidogenesis in the rat brain.

METHOD(S): Two-month-old male SPD rats were housed at 22°C under a 12/12-h light/dark cycle with free access to food and water. Animals received NaCl (control) or clonazepam (0.5 mg/kg, i.p.) for 21 days. After decapitation, eight brain regions were isolated. RNA was extracted with TRIZOL, quality assessed spectrophotometrically, and reverse-transcribed to cDNA. Expression of P450scc, 3β-HSD, 17β-HSD, aromatase, and B2M was quantified by qRT-PCR using SYBR Green.

RESULTS: Clonazepam increased 3β-HSD gene expression in the amygdala and p450scc mRNA in the hippocampus. Increased 3β-HSD is associated with enhanced allopregnanolone synthesis, whereas elevated hippocampal p450scc suggests increased pregnenolone levels. Clonazepam decreased P450scc protein levels in the neocortex, striatum, amygdala, thalamus, hypothalamus, cerebellum, and brainstem, and reduced 3β-HSD in the neocortex, striatum, hippocampus, thalamus, hypothalamus, cerebellum, and brainstem. It also inhibited 17β-HSD and aromatase gene expression in all regions.

CONCLUSIONS: Clonazepam modulates neurosteroidogenesis in a region-specific manner, providing insight into its pharmacological mechanisms and supporting further investigation of benzodiazepine effects on brain steroid pathways.

FINANCIAL SUPPORT: This research was funded by the Medical University of Silesia grant for the Department of Histology No; BNW-1-005/N/4/I.