GnRH-1 (also known as LHRH) 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-1 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-1 neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH-1 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-1 system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH-1 neuronal movement. As GnRH-1 neurons migrate they also mature and the two processes may in fact be linked. To investigate the maturation of GnRH-1 neurons we use calcium imaging, electrophysiology and biochemical measures to examine GnRH-1 neuronal activity and peptide secretion. Over the past year two studies were finished: 1) Progesterone (P4), a steroid hormone, modulates reproductive behavior and is associated with rapid changes in GnRH secretion. However, a direct action of P4 on GnRH neurons has not been previously described. Receptors in the Progestin/adipoQ Receptor family (PAQRs), as well as Progesterone receptor membrane component 1 (PgRMC1) and its partner SERPINE1 mRNA binding protein 1 (SERBP1), have been shown to mediate rapid progestin actions in various tissues, including the brain. This study shows that PgRMC1 and SERBP1, but not PAQRs, are expressed in prenatal GnRH neurons. Expression of PgRMC1 and SERBP1 was verified in adult mouse GnRH neurons. To investigate the effect of P4 on GnRH neuronal activity, calcium imaging was used on primary GnRH neurons maintained in explants. Application of P4 significantly decreased the activity of GnRH neurons, independent of GABAergic and glutamatergic input, suggesting a direct action of P4 on GnRH neurons. Inhibition was not blocked by RU486, an antagonist of the classic nuclear P4 receptor. Inhibition was also maintained after uncoupling of the inhibitory regulative G-protein (Gi/o), the signal transduction pathway used by PAQRs. However, AG-205, a PgRMC1 ligand and inhibitor, blocked the rapid P4-mediated inhibtion, and inhibition of protein kinase G, thought to be activated downstream of PgRMC1, also blocked the inhibitory activity of P4. These data show for the first time that P4 can act directly on GnRH neurons through PgRMC1 to inhibit neuronal activity. 2) Sensory neurons in vertebrates are derived from two embryonic transient cell sources: neural crest and ectodermal placodes. The placodes are thickenings of ectodermal tissue that are responsible for the formation of cranial ganglia as well as complex sensory organs that include the lens, inner ear, and olfactory epithelium . The neural crest cells have been indicated to arise at the edges of the neural plate/dorsal neural tube, from both the neural plate and the epidermis in response to reciprocal interactions. neural crest cells migrate throughout the organism, and give rise to a multitude of cell types that include melanocytes, cartilage and connective tissue of the head, components of the cranial nerves, the dorsal root ganglia, and Schwann cells. The embryonic definition of these two transient populations and their relative contribution to the formation of sensory organs has been investigated and debated for several decades. Historically, all placodes have been described as exclusively derived from non-neural ectodermal progenitors. Recent genetic fate-mapping studies suggested a neural crest contribution to the olfactory placodes as well as the otic (auditory) placodes in rodents. This review analyzes and discusses some recent developmental studies on the olfactory placode, placodal derivatives, and olfactory system. New investigations using Cre-lox-mice to specifically remove molecules of interest from GnRH-1 cells during development will be continued this year with specific studies on the early development of the GnRH-1 neurons and the location of their progenitor cells in relation to nasal placodal cells and neural crest. A study on the interaction of cytokines and GABA during GnRH neuronal migration is being completed. Here we find that Stromal derived growth factor (SDF-1) and Gamma-aminobutyric acid (GABA) are two extracellular cues that regulate the rate of neuronal migration during development and may, in fact, act synergistically. GABA and SDF-1 are shown to exert opposite effects on the speed of cell movement by activating depolarizing or hyperpolarizing signaling pathways, GABA via changes in chloride and SDF-1 via changes in potassium. GABA and SDF-1 were also found to act synergistically to promote linear rather than random movement. The simultaneous activation of these signaling pathways therefore results in tight control of cellular speed and improved directionality along the migratory pathway of GnRH neurons. Other studies in progress are examining the role of growth factor receptors (FGFR1, PDGF), and NELF (a migrational molecule), in GnRH-1 development as well as in situ characterization of the migration of GnRH-1 neurons (real time microscopy). In addition, we continue to study the physiology of GnRH neurons. Currently we are evaluating the mechanism for GABA remaining excitatory to GnRH by evaluating chloride transporters and GABA receptor subtypes and their distribution in GnRH neurons. Other studies include examining/identifying 1) molecules that modulate GnRH-1 neuronal activity that participate in reproductive functions such as adiponectin, 2) midline cues which influence olfactory axon outgrowth and 3) the dynamics of GnRH-1 neuronal migration.

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Constantin, Stephanie (2017) Progress and Challenges in the Search for the Mechanisms of Pulsatile Gonadotropin-Releasing Hormone Secretion. Front Endocrinol (Lausanne) 8:180
Turan, Ihsan; Hutchins, B Ian; Hacihamdioglu, Bulent et al. (2017) CCDC141 Mutations in Idiopathic Hypogonadotropic Hypogonadism. J Clin Endocrinol Metab 102:1816-1825
Constantin, Stephanie; Wray, Susan (2016) Galanin Activates G Protein Gated Inwardly Rectifying Potassium Channels and Suppresses Kisspeptin-10 Activation of GnRH Neurons. Endocrinology 157:3197-212
Klenke, Ulrike; Constantin, Stephanie; Wray, Susan (2016) BPA Directly Decreases GnRH Neuronal Activity via Noncanonical Pathway. Endocrinology 157:1980-90
Hutchins, B Ian; Kotan, L Damla; Taylor-Burds, Carol et al. (2016) CCDC141 Mutation Identified in Anosmic Hypogonadotropic Hypogonadism (Kallmann Syndrome) Alters GnRH Neuronal Migration. Endocrinology 157:1956-66
Forni, Paolo E; Wray, Susan (2015) GnRH, anosmia and hypogonadotropic hypogonadism--where are we? Front Neuroendocrinol 36:165-77
Taylor-Burds, Carol; Cheng, Paul; Wray, Susan (2015) Chloride Accumulators NKCC1 and AE2 in Mouse GnRH Neurons: Implications for GABAA Mediated Excitation. PLoS One 10:e0131076
Klenke, Ulrike; Taylor-Burds, Carol; Wray, Susan (2014) Metabolic influences on reproduction: adiponectin attenuates GnRH neuronal activity in female mice. Endocrinology 155:1851-63
Hutchins, B Ian; Wray, Susan (2014) Capture of microtubule plus-ends at the actin cortex promotes axophilic neuronal migration by enhancing microtubule tension in the leading process. Front Cell Neurosci 8:400
Kotan, L Damla; Hutchins, B Ian; Ozkan, Yusuf et al. (2014) Mutations in FEZF1 cause Kallmann syndrome. Am J Hum Genet 95:326-31

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