Lack of functional GABA receptors alters Kiss1 , Gnrh1 and Gad1 mRNA expression in the medial basal hypothalamus at postnatal day 4

Background/Aims: Adult mice lacking functional GABAB receptors (GABAB1KO) show altered Gnrh1 and Gad1 expressions in the preoptic area-anterior hypothalamus (POA-AH) and females display disruption of cyclicity and fertility. Here we addressed whether sexual differentiation of the brain and the proper wiring of the GnRH and kisspeptin systems were already disturbed in postnatal day 4 (PND4) GABAB1KO mice. Methods: PND4 wild-type (WT) and GABAB1KO mice of both sexes were sacrificed; tissues were collected to determine mRNA expression (qPCR), amino acids (HPLC), and hormones (RIA and/or IHC). Results: GnRH neuron number (IHC) did not differ among groups in olfactory bulbs or OVLT-POA. Gnrh1 mRNA (qPCR) in POA-AH was similar among groups. Gnrh1 mRNA in medial basal hypothalamus (MBH) was similar in WTs but was increased in GABAB1KO females compared to GABAB1KO males. Hypothalamic GnRH (RIA) was sexually different in WTs (males < females), but this sex difference was lost in GABAB1KOs; the same pattern was observed when analyzing only the MBH, but not in the POA-AH. Arcuate nucleus Kiss1 mRNA (micropunch-qPCR) was higher in WT females than in WT males and GABAB1KO females. Gad1 mRNA in MBH was increased in GABAB1KO females compared to GABAB1KO males. Serum LH and gonadal estradiol content were also increased in GABAB1KOs. Conclusion: We demonstrate that GABABRs participate in the sexual differentiation of the ARC/MBH, because sex differences in several reproductive genes, such as Gad1, Kiss1 and Gnrh1, are critically disturbed in GABAB1KO mice at PND4, probably altering the organization and development of neural circuits governing the reproductive axis. (c) 2013 S. Karger AG, Basel

Postnatal development of the endocrine pancreas in mice lacking functional GABAB receptors

Adult mice lacking functional GABAB receptors (GABAB1KO) have glucose metabolism alterations. Since GABAB receptors (GABABRs) are expressed in progenitor cells, we evaluated islet development in GABAB1KO mice. Postnatal day 4 (PND4) and adult, male and female, GABAB1KO, and wild-type littermates (WT) were weighed and euthanized, and serum insulin and glucagon was measured. Pancreatic glucagon and insulin content were assessed, and pancreas insulin, glucagon, PCNA, and GAD65/67 were determined by immunohistochemistry. RNA from PND4 pancreata and adult isolated islets was obtained, and Ins1, Ins2, Gcg, Sst, Ppy, Nes, Pdx1, and Gad1 transcription levels were determined by quantitative PCR. The main results were as follows: 1) insulin content was increased in PND4 GABAB1KO females and in both sexes in adult GABAB1KOs; 2) GABAB1KO females had more clusters (>500 mum(2)) and less islets than WT females; 3) cluster proliferation was decreased at PND4 and increased in adult GABAB1KO mice; 4) increased beta-area at the expense of the alpha-cell area was present in GABAB1KO islets; 5) Ins2, Sst, and Ppy transcription were decreased in PND4 GABAB1KO pancreata, adult GABAB1KO female islets showed increased Ins1, Ins2, and Sst expression, Pdx1 was increased in male and female GABAB1KO islets; and 6) GAD65/67 was increased in adult GABAB1KO pancreata. We demonstrate that several islet parameters are altered in GABAB1KO mice, further pinpointing the importance of GABABRs in islet physiology. Some changes persist from neonatal ages to adulthood (e.g., insulin content in GABAB1KO females), whereas other features are differentially regulated according to age (e.g., Ins2 was reduced in PND4, whereas it was upregulated in adult GABAB1KO females)

GABAB receptors and glucose homeostasis : evaluation in GABAB receptor knockout mice

GABA has been proposed to inhibit insulin secretion through GABA(B) receptors (GABA(B)Rs) in pancreatic beta-cells. We investigated whether GABA(B)Rs participated in the regulation of glucose homeostasis in vivo. The animals used in this study were adult male and female BALB/C mice, mice deficient in the GABA(B1) subunit of the GABA(B)R (GABA(B)(-/-)), and wild types (WT). Blood glucose was measured under fasting/fed conditions and in glucose tolerance tests (GTTs) with a Lifescan Glucose meter, and serum insulin was measured by ELISA. Pancreatic insulin content and islet insulin were released by RIA. Western blots for the GABA(B1) subunit in islet membranes and immunohistochemistry for insulin and GABA(B1) were performed in both genotypes. BALB/C mice preinjected with Baclofen (GABA(B)R agonist, 7.5 mg/kg ip) presented impaired GTTs and decreased insulin secretion compared with saline-preinjected controls. GABA(B)(-/-) mice showed fasting and fed glucose levels similar to WT. GABA(B)(-/-) mice showed improved GTTs at moderate glucose overloads (2 g/kg). Baclofen pretreatment did not modify GTTs in GABA(B)(-/-) mice, whereas it impaired normal glycemia reinstatement in WT. Baclofen inhibited glucose-stimulated insulin secretion in WT isolated islets but was without effect in GABA(B)(-/-) islets. In GABA(B)(-/-) males, pancreatic insulin content was increased, basal and glucose-stimulated insulin secretion were augmented, and impaired insulin tolerance test and increased homeostatic model assessment of insulin resistance index were determined. Immunohistochemistry for insulin demonstrated an increase of very large islets in GABA(B)(-/-) males. Results demonstrate that GABA(B)Rs are involved in the regulation of glucose homeostasis in vivo and that the constitutive absence of GABA(B)Rs induces alterations in pancreatic histology, physiology, and insulin resistance

Impaired GABAB receptor signaling dramatically up-regulates Kiss1 expression selectively in nonhypothalamic brain regions of adult but not prepubertal mice

Kisspeptin, encoded by Kiss1, stimulates reproduction and is synthesized in the hypothalamic anteroventral periventricular and arcuate nuclei. Kiss1 is also expressed at lower levels in the medial amygdala (MeA) and bed nucleus of the stria terminalis (BNST), but the regulation and function of Kiss1 there is poorly understood. gamma-Aminobutyric acid (GABA) also regulates reproduction, and female GABAB1 receptor knockout (KO) mice have compromised fertility. However, the interaction between GABAB receptors and Kiss1 neurons is unknown. Here, using double-label in situ hybridization, we first demonstrated that a majority of hypothalamic Kiss1 neurons coexpress GABAB1 subunit, a finding also confirmed for most MeA Kiss1 neurons. Yet, despite known reproductive impairments in GABAB1KO mice, Kiss1 expression in the anteroventral periventricular and arcuate nuclei, assessed by both in situ hybridization and real-time PCR, was identical between adult wild-type and GABAB1KO mice. Surprisingly, however, Kiss1 levels in the BNST and MeA, as well as the lateral septum (a region normally lacking Kiss1 expression), were dramatically increased in both GABAB1KO males and females. The increased Kiss1 levels in extrahypothalamic regions were not caused by elevated sex steroids (which can increase Kiss1 expression), because circulating estradiol and testosterone were equivalent between genotypes. Interestingly, increased Kiss1 expression was not detected in the MeA or BNST in prepubertal KO mice of either sex, indicating that the enhancements in extrahypothalamic Kiss1 levels initiate during/after puberty. These findings suggest that GABAB signaling may normally directly or indirectly inhibit Kiss1 expression, particularly in the BNST and MeA, and highlight the importance of studying kisspeptin populations outside the hypothalamus