Fig. 2. GABAergic estrogen-receptive neurons (GERNs) localization, mRNA expression by ISH and somatic receptor expression by IHC.

a Serial coronal sections showing the ERα immunolabelling at the Bregma rostro-caudal coordinates (numbers in mm under the photomicrographs). Boxed area in A₆ at higher magnification showing the exclusive nuclear labeling pattern. The bold numbered levels (A₁ and A₈), were chosen to show that no positive labeling was found in either rostral or caudal directions. A₉: a horizontal view of the distribution of estrogen-receptive cells was symbolized by the red oval. A₁₀: sagittal view of rat brain atlas, modified from Paxinos & Watson⁸⁶, at lat. 0.90 mm, where lateral habenula (LHb) is symbolized with a gray shade and A9 plane was symbolized with a horizontal line. b In situ hybridization using multiple RNAscope methods. B₁: multiplex fluorescence method, Esr1, gene that encodes ERα (red punctuated labeling) co-expressed with Slc32a1, gene that encodes VGAT (green punctuated labeling); arrows indicate the double-labeled cells; B₂: duplex method, Esr1 (red punctuated labeling) co-localization with Slc17a6, gene that encodes VGLUT2 (green punctuated labeling); arrows indicate the double-labeled cells; B₃: with duplex method, Slc32a1 encoding VGAT (red punctuated labeling) shows complete co-localization with Slc17a6 encoding VGLUT2 (green punctuated labeling); arrows indicate the double-labeled cells. Inset of B₃, Slc32a1 expression in a sexually active (SA) rat LHb, Br. −3.72 mm (brown labeling, single chromogenic-Brown method-RNAscope). *Note the similarity with ERα expression in A₆. c Indirect immunohistochemistry showing the GABAergic nature of the ERα+ neurons (red) in a SA rat brain. The GABA antibody (green, Sigma, A0310) produced characteristic surface labeling (C₁, C₂: the strip-like image was produced by Vibratome slicing leaving the brain section with an uneven surface). d ERα+ neurons co-express receptor/receptor subtypes for vasopressin, orexin, dopamine, and serotonin. D₁: In situ hybridization using RNAscope-multiplex method targeting Esr1 (red dots), Slc32a1 (white dots) and Hcrtr2, gene encodes the orexin receptor 2 (green dots). D₂, D₃: Indirect immunofluorescence reactions, showing that the ERα-IR cells co-expressed vasopressin receptor V1a (inset showing the V1a antibody labeling pattern in temporal hippocampus CA2 cells body layer. See also SI Fig. 6 for more information about this antibody), dopamine receptor D5R (also called D1Rb) and serotonin receptor 5-HTr2a, respectively. Scale bars: A₅ and b: 500 µm and rest: 10 µm