Immunofluorescence Staining of Neuronal Markers

Immunofluorescence staining was performed using the method described in our previous publication^{4 (link),24 (link)}. The primary antibodies were: rabbit anti-GHRH (1:100, catalog No: ab187512, Abcam, Cambridge, MA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:50, catalog number BM0055; Boster Bioengineering, Wuhan, China), mouse anti-Gephyrin (1:50, Abcam, Cambridge, MA, USA), guinea pig anti-microtubule-associated protein 2 (MAP2) (1:200, Sysy, Goettingen, Germany), mouse anti-glutamate decarboxylase 67 (GAD67) (1:100, Abcam, Cambridge, MA, USA), and guinea pig anti-vesicular GABA transporter (VGAT) (1:200, Sysy, Goettingen, Germany). The secondary antibodies were: an Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (1:50, Zhongshan Golden Bridge, Inc., Beijing, China), Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (1:200, Zhongshan Golden BridgeInc., Beijing, China), and Alexa Fluor 633-conjugated goat anti-guinea pig IgG antibody (1:50, Abcam, Cambridge, MA, USA). Finally, the samples were treated with 4′,6-diamidino-2-phenylindole dihydrochloride (Sigma, St. Louis, MO, USA) for 5 min to identify the nuclei. Immunofluorescently labeled sections were examined with a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) and an Olympus IX 70 inverted microscope (Olympus America, Melville, NY, USA).