Lack of functional GABA_B receptors alters GnRH physiology and sexual dimorphic expression of GnRH and GAD-67 in the brain

Abstract

GABA, the main inhibitory neurotransmitter, acts through GABAA/C and GABAB receptors (GABABRs); it is critical for gonadotropin regulation. We studied whether the lack of functional GABABRs in GABAB1 knockout (GABAB1KO) mice affected the gonadotropin axis physiology. Adult male and female GABAB1KO and wild-type (WT) mice were killed to collect blood and tissue samples. Gonadotropin-releasing hormone (GnRH) content in whole hypothalami (HT), olfactory bulbs (OB), and frontoparietal cortexes (CT) were determined (RIA). GnRH expression by quantitative real-time PCR (qRT-PCR) was evaluated in preoptic area-anterior hypothalamus (POA-AH), medial basal-posterior hypothalamus (MBH-PH), OB, and CT. Pulsatile GnRH secretion from hypothalamic explants was measured by RIA. GABA, glutamate, and taurine contents in HT and CT were determined by HPLC. Glutamic acid decarboxylase-67 (GAD-67) mRNA was measured by qRT-PCR in POA-AH, MBH-PH, and CT. Gonadotropin content, serum levels, and secretion from adenohypophyseal cell cultures (ACC) were measured by RIA. GnRH mRNA expression was increased in POA-AH of WT males compared with females; this pattern of expression was inversed in GABAB1KO mice. MBH-PH, OB, and CT did not follow this pattern. In GABAB1KO females, GnRH pulse frequency was increased and GABA and glutamate contents were augmented. POA-AH GAD-67 mRNA showed the same expression pattern as GnRH mRNA in this area. Gonadotropin pituitary contents and serum levels showed no differences between genotypes. Increased basal LH secretion and decreased GnRH-stimulated gonadotropin response were observed in GABAB1KO female ACCs. These results support the hypothesis that the absence of functional GABABRs alters GnRH physiology and critically affects sexual dimorphic expression of GnRH and GAD-67 in POA-AH.

Postnatal development of the endocrine pancreas in mice lacking functional GABABreceptors

“…This same inversion of sexually dimorphic expression of Gad1 was also observed in anterior hypothalami of adult GABA B1 KO mice (16), suggesting that GABA B receptors are regulating the expression of this enzyme in a similar way in both tissues. Although Gad1 expression was increased in GABA B1 KO females and decreased in GABA B1 KO males, the GAD65/67-positive area was increased in both male and female GABA B1 KO islets.…”

“…Results on GAD expression indicate that at least in adult GABA B1 KO mice both gene transcription and protein expression are altered, indicating that this enzyme may be under GABA B receptor regulation, as we have also demonstrated in the hypothalamus (16).…”

“…It has been shown that Ins2 is more prevalent than Ins1 in mice at both the transcript and protein levels (2,22,44); therefore, a decrease in Ins2 transcription with an increase in insulin content in PND4 GABA B1 KO females is surprising, although regulation at posttranscriptional levels may occur. We also evaluated GAD expression in PND4 pancreas, since the absence of functional GABA B receptors may condition the expression of other components of the GABAergic system, and we had shown previously that Gad1 expression was altered in the hypothalamus of these transgenic mice (16). Gad1 mRNA (qPCR), coding for GAD67, the main isoform found in mice islets (40,53), and GAD65/67 protein (IHC) expression could not be determined consistently in pancreata from neonate mice in either genotype, as opposed to adults.…”

Variation in Progesterone Receptors and GnRH Expression in the Hypothalamus of the Pregnant South American Plains Vizcacha, Lagostomus maximus (Mammalia, Rodentia)1

“…Results were expressed in terms of rat LH standards, using the following standards: for LH iodination, r-LH-I10, reference preparation rat LH-RP-3 (AFP7187B) and anti-rat LH-S11 (AFPC697071P) [20]. Assay sensitivity was 0.31 ng/ml.…”

“…All procedures were carried out at 48C. GnRH concentration was determined as previously described [20]. Briefly, samples were analyzed in duplicate using anti-GnRH antiserum (rabbit polyclonal HU60, which recognizes mGnRH and cGnRH-II, final dilution 1:50 000) kindly provided by Dr. Urbanski (Division of Neuroscience, Oregon National Primate Research Center).…”