| Gonadotropin-releasing hormone(GnRH)neurons,scattered in the forebrain,play a pivotal role in reproductive function.GnRH is released in distinct pulses that are regulated by neurotransmitters or neuromodulators.γ-amino butyric acid(GABA)is typically an inhibitory neurotransmitter in the central nervous system.However GABA which has long been implicated as one of the major players in the regulation of GnRH neurons can excite most mature GnRH neurons.GABA is produced in the CNS via decarboxylation of glutamate,in a reaction catalyzed by glutamic acid decarboxylase(GAD).GABA regulate GnRH neurons through GABAa and GABAb receptors expressing on GnRH neurons.With immunohistochemistry and GAD67-GFP knockin mice,this study shows for the first time that a subset of GnRH neurons in the forebrain of adult mouse is GABA-ergic.Firstly,in our work,using the in situ hybridization and immunohistochemistry confirm the specificity of GFP expression in GAD67-GFP knock-in mice.The results showed that GAD67 in the central nervous system widely distributed,such as the cerebral cortex,thalamic reticular nucleus,globus pallidus and other areas.All the green fluorescent protein(GFP)immunoreactive neurons also expressed GAD67 mRNA.This indicates that GAD67-GFP knock-in mice expressing of GFP is specific.So,GFP can be used as a marker of GAD67.With immunohistochemistry,we find a subpopulation of GnRH neurons in the forebrain of adult mouse express GAD67.There is a gender difference in the percentage of GnRH neurons expressing GAD67-GFP(male:34%±5%,female: 62%±7%).In order to study the differences between the sexes,males and females mice are castrated and sex hormone replacement therapy is carried out.We find that the percentage of GnRH neurons expressing GAD67-GFP decreased after castration of female mice(40%±4%)7 days after surgery and increased to the normal female level after estradiol(17β-estradiol,E2)treatment(64%±5%).However,the percentage of GnRH neurons expressing GAD67-GFP did not change significantly in intact(34%±5%),castrated(36%±5%),and castration+testosterone propionate(TP)-treated(35%±4%)male mice.These data implied that the expression of GAD67 in GnRH nurons is infiuenced by estrongen.Therefore,females estrous cycle were studied.First,a vaginal smear of female mice was carried out to identify estrous and diestrous mice.Based on the results of immunohistochemistry,during the female estrous cycle,the percentage of GnRH neurons expressing GAD67-GFP was higher during the estrous stage(P30:71%±4%,P45:75%±4%)than that during the diestrous stage(P30:34%±5%,P45:34%±5%).Once again,we proved that estrogen regulate the expresion of GAD67 in the GnRH neurons.A subset of GnRH neurons in the forebrain of adult mouse express GAD67.But in embryonic 12 days(E12),in the vomeronasal organ and migration path of GnRH neurons,all of the GnRH neurons express GAD67.As time goes on,in the migration path and central nervous system,the number of GnRH neurons of expressing GAD67 decrease gradually.During sexual maturation of postnatal development,GnRH neurons did not express GAD67-GFP until P15,and the gender differences were first detected at P30,which corresponds to the maturation stage.In conclusion,our data suggest that 1)a subset of GnRH neurons in mouse forebrain is GABA-ergic,2)expression of GAD67-GFP in GnRH neurons is at least in part regulated by estrogen,and 3)the present data implicated that GnRH neurons could secrete GABA to regulate themselves. |
