GnRH neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying GnRH neuronal differentiation, migration and axonal targeting in normal/transgenic animals, and nasal explants. Using these same models, our work also addresses the mechanisms regulating (intrinsic and trans-synaptic) GnRH gene expression, peptide synthesis and secretion in GnRH neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH neuronal movement. As GnRH neurons migrate they also mature and the two processes may in fact be linked. To investigate the maturation of GnRH neurons we use calcium imaging, electrophysiology and biochemical measures to examine GnRH neuronal activity and peptide secretion. In addition, we collaborate with labs performing human genetic screening of Kallman patients. Once a mutation is identified, we analyze the expression pattern in mice and perform biological assays to determine the outcome of the mutated gene on GnRH development. Over the past year, three studies were finished: 1) Peripheral feedback of gonadal estrogen to the brain is critical for reproduction. Bisphenol-A (BPA), an environmental pollutant with estrogenic actions, can disrupt this feedback and lead to infertility in both humans and animals. Since GnRH neurons express several receptors that bind estrogen, they are potential targets for endocrine disruptors. However, to date direct effects of BPA on GnRH neurons had not been shown. This study investigated the effects of BPA on GnRH neuronal activity. Since oscillations in intracellular calcium have been shown to correlate with electrical activity in GnRH neurons, calcium imaging was used to assay the effects of BPA. Exposure to BPA significantly decreased GnRH calcium activity. Blockage of GABAergic and glutamatergic input did not abrogate the inhibitory BPA effect, suggesting direct regulation of GnRH neurons by BPA. In addition to estrogen receptors (ERs), single-cell RT-PCR analysis confirmed GnRH neurons express GPR30 (GPER) and estrogen related receptor (ERR), all potential targets for BPA. Perturbation studies of the signaling pathway revealed that the BPA-mediated inhibition of GnRH neuronal activity occurred independent of ERs, GPER or ERR, via a non-canonical pathway. These results provide the first evidence of a direct effect of BPA on GnRH neurons. 2) Developmental pathologies affecting functional GnRH expression, nasal placode development or GnRH neuronal migration, can translate into various forms of hypogonadism, with lack or delay of reproductive function. In humans, pathology associated with defects in olfactory system development and impaired migration of GnRH neurons is classified as Kallmann syndrome (KS), characterized by anosmia and lack of pubertal onset. An earlier genetic screen of Kallmann Syndrome patients revealed a novel mutation in CCDC141. Little is known about CCDC141, which encodes a coiled-coil domain containing protein. This follow-up study showed that Ccdc141 is expressed in GnRH neurons and olfactory fibers and that knockdown of Ccdc141 reduces GnRH neuronal migration. Our findings in human patients and mouse models predict that CCDC141 takes part in embryonic migration of GnRH neurons enabling them to form a hypothalamic neuronal network to initiate pulsatile GnRH secretion and reproductive function. 3) GnRH neurons control reproduction, but lack estrogen receptor subtypes known to be critical for feedback from the ovaries to the CNS. However, GnRH neurons are regulated by kisspeptin neurons which do express this estrogen receptor subtype. Recently, galanin (a neuromodulator) was identified in a subpopulation of kisspeptin neurons. Although it is well known thats kisspeptin activates GnRH neurons, little was known about the effects of galanin on GnRH neurons. To investigate a potential role for galanin on GnRH neuronal activity, GnRH neurons were screened for three galanin receptor transcripts. GalR1 was found. A series of experiments was then performed to determine the action of galanin on kisspeptin activated GnRH neurons. Applied after kisspeptin activation, galanin rapidly suppressed GnRH neuronal activity. Applied with kisspeptin, galanin prevented kisspeptin activation until its removal. In fact, galanin inhibited spontaneously active GnRH neurons when applied without kisspeptin. We found that this galanin inhibition relied on Gi/o signaling, but was independent of cAMP levels and sensitive to blockers of G protein-coupled inwardly rectifying potassium channels. Using a newly developed bioassay for GnRH detection, we showed galanin decreased kisspeptin-evoked GnRH secretion. Together, this study showed that galanin is a potent regulator of GnRH neurons, possibly acting as a physiological break to kisspeptin excitation.
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