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Hydrogen sulfide (H2S) is a novel neurotransmitter that has been shown to influence cardiovascular function as well as other autonomic and endocrine functions by targeting a wide range of ion channels. Using whole-cell electrophysiology, I have investigated the potential role of H2S in the regulation of neuronal excitability in the paraventricular nucleus of the hypothalamus (PVN), which is a central relay centre for autonomic and endocrine function. In current-clamp recordings, sodium hydrosulfide hydrate (NaHS), when perfused onto PVN slices at various concentrations (0.1, 1, 10, and 50 mM), elicited a concentration-dependent response relationship from the majority of recorded neurons, with almost exclusively depolarizing effects. Input resistance differences from baseline, and during the NaHS-induced depolarization, uncovered a biphasic response, implicating both a potassium (K+) and non-selective cation conductance. In order to further investigate H2Ss effects on K+ conductances, we used both voltage- and current-clamp techniques to examine the effects of NaHS at either 1 or 10 mM on both the transient and sustained voltage-activated K+ currents in these neurons. We applied TEA+ (10 mM) to isolate the transient/rapidly inactivating current (IA) and 4-AP (5 mM) to isolate the sustained/delayed rectifier current (IK), and were able to show that both of these conductances were significantly reduced by H2S. Finally, we were able to demonstrate, using current-clamp, that when 4-AP and TEA+ were applied together with NaHS, they were able to completely eliminate the previously observed NaHS-induced depolarization, and the effects on membrane potential reversed to show a small hyperpolarization. These data highlight the potential role of H2S as a critical modulator of the voltage-gated repolarizing conductances, IA and IK, which in turn regulate neuronal excitability within the PVN. This can have a large impact on the way neurotransmitters and hormones such as vasopressin, oxytocin, corticotrophin-releasing hormone, and thyrotrophin-releasing hormone are released from the PVN, which influence a wide range of neuroendocrine and autonomic functions such as cardiovascular function, fluid balance, and food intake. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2013-09-13 10:51:34.585

La vasopressine (VP), hormone anti-diurétique, joue un rôle fondamental dans l’homéostasie des compartiments liquidiens de l’organisme. Ainsi, la concentration de VP libérée dans la circulation sanguine est proportionnelle à l’osmolalité plasmatique. Or, la libération de la VP est étroitement dépendante de l’activité électrique des neurones magnocellulaires hypothalamiques (MNCs) qui la synthétisent. L’activité électrique des MNCs est donc régulée lors de variations de l’osmolalité plasmatique. Cette régulation implique la modulation concertée de propriétés intrinsèques dépendant de l’expression de canaux ioniques mécanosensibles, mais aussi d’entrées synaptiques excitatrices provenant de structures cérébrales osmosensibles. Les Extracellular signal-regulated kinases (ERK 1 et 2) sont des protéines de la voie des MAPK (Mitogen-Activated Protein Kinases) qui convertissent des stimulus extracellulaires en réponses intracellulaires transcriptionnelles et post-traductionnelles. Elles contribuent en particulier à la plasticité des propriétés membranaires intrinsèques et des synapses excitatrices glutamatergiques dans le système nerveux central. Elles sont exprimées par les MNCs de l’hypothalamus. L’objet de ce travail de thèse a été l’étude du rôle de ERK dans l’activation des osmorécepteurs hypothalamiques. Les expériences d’immunohistochimie montrent qu’en réponse à une stimulation systémique hypertonique, la voie ERK 1/2 est activée rapidement, de façon transitoire et sélectivement dans des structures osmoréceptrices de l’encéphale ou directement impliquées dans l’osmorégulation : noyaux supraoptiques (NSO) et paraventriculaires (NPV), organe subfornical, noyau préoptique médian et OVLT. ERK est aussi activée dans la portion parvicellulaire du NPV, mais pas dans une structure témoin qui ne participe pas à l’osmorégulation, la strie médullaire thalamique. De plus, il existe un codage de l’intensité de la stimulation osmotique par le nombre de neurones exprimant phosphoERK dans l’encéphale. Les Western Blot indiquent qu’il existe également un codage de l’intensité du stimulus hypertonique par le degré d’expression de la protéine phosphoERK dans le NSO. Nous montrons aussi qu’il existe une activation plus importante de ERK dans les neurones à VP que dans les neurones à ocytocine. L’ensemble de ces résultats suggère que phosphoERK pourrait contribuer aux mécanismes de l’osmorégulation. Cette hypothèse a été testée en mesurant les effets de l’inhibition de la phosphorylation de ERK sur l’activation des centres osmorégulateurs. Effectivement, l’administration intracérébroventriculaire d’un inhibiteur sélectif de MEK 1/2, l’U 0126, inhibe la phosphorylation de ERK et l’activation neuronale (mesurée par l’expression de Fos) résultant de l’application d’un stimulus hypertonique. La phosphorylation de ERK joue donc un rôle majeur dans l’activation des centres osmorégulateurs de l’encéphale. En accord avec ces données, les enregistrements électrophysiologiques sur tranches d’hypothalamus montrent que ERK est impliqué dans la réponse cellulaire des neurones magnocellulaires à une stimulation osmotique : l’inhibition de la phosphorylation de ERK réduit la dépolarisation membranaire et diminue l’augmentation de la fréquence de décharge des potentiels d’action ainsi que l’augmentation de l’amplitude des potentiels synaptiques spontanés induits par un stimulus hypertonique. Les données obtenues sur les neurones supraoptiques isolés montrent qu’une partie au moins des effets de l’activation de ERK implique une modulation de la conductance membranaire qu’on sait responsable de l’osmotransduction dans ces neurones. En résumé, l’activation de ERK est nécessaire à la réponse normale des neurones magnocellulaires à une stimulation osmotique. / The release of the anti-diuretic hormone vasopressin into the general circulation varies as a function of plasma osmolality and therefore plays a major role in systemic osmoregulation. It is primarily determined by the firing rate of the hypothalamic magnocellular neurons (MNCs). The regulation of MNC firing rate during changes in systemic osmolality involves the concerted modulation of intrinsic mechanosensitive ion channels in these neurons, as well as excitatory glutamatergic inputs derived from osmosensitive forebrain regions. Extracellular signal-Regulated protein Kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, which include changes in intrinsic neuronal properties and synaptic function. In the present work, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of osmoregulatory networks. First, we found that within 10 minutes of intraperitoneal injections of hypertonic saline (3M, 6M), many phosphoERK-immunopositive neurons were observed within osmosensitive forebrain regions including suparoptic and paraventricular nuclei, subfornical organ, median preoptic nucleus and Organum vasculosum lamina terminalis (OVLT), but not in a control region such as the thalamic stria medullaris. This staining was intensity dependent and was reduced 30 minutes after the stimulus. In the supraoptic nucleus, it predominated in vasopressin neurons compared to oxytocin neurons. Western blotting experiments confirmed that ERK phosphorylation in the supraoptic nucleus was intensity dependent. Inhibition of ERK phosphorylation by a MEK inhibitor reduced both the numbers of phosphoERK-immunopositive neurons and Fos expressing neurons, a measure of neuronal activation after hypertonic stimuli. Inhibition of ERK phosphorylation also decreased the membrane depolarization, the increase in firing frequency and the increase in excitatory and inhibitory synaptic potential amplitude that were osmotically-induced in both oxytocin and vasopressin supraoptic neurons recorded from adult acute hypothalamic slices. It also reduced the increase in mechanically-gated membrane cation conductance evoked by hypertonic stimuli in isolated MNC neurons. Altogether, these findings strongly suggest that ERK phosphorylation is a key step in the transduction and integration of osmotic stimuli into activity changes in osmosensitive hypothalamic neurons.

Estrogen and glucocorticoid receptors (ER and GR) are both members of the same subfamily of steroid nuclear receptors, and can both signal classically as ligand-activated transcription factors. However, many responses to estrogen and glucocorticoid exposure occur through the non-classical pathways, which include rapid activation of kinase cascades, activation of membrane-associated receptors, gene regulation through transcription by non-classical transcription factors, and protein regulation by translation and post-translational modification. Male territorial aggression is a hypothalamically-mediated steroid hormone-dependent adaptive behavior in mice. The hypothalamus, which expresses multiple ERs and GRs, is also responsive to estrogen and glucocorticoid treatment at a cellular level. Experiments were conducted to test the effects of estrogen and glucocorticoid interactions on spinogenesis in the ventromedial hypothalamus (VMH) and on male territorial aggression through the resident-intruder paradigm. Studies in male postnatal primary hypothalamic cell cultures demonstrate the expression of classic ERα, the variant ERα-36, and GPR30. PSD-95 protein, a marker for dendritic spines, is increased in response to 12 hours of treatment with the GPR30 agonist G-1 in an ERK/MAPK-dependent manner. Further work in immortalized embryonic hypothalamic cell lines (mHypoE-11 and mHypoE-42) demonstrate non-classical effects of a membrane-limited glucocorticoid on rapid nuclear translocation of the intracellular GR. Additionally, pharmacological inhibition of the ERK/MAPK pathway results in similar GR translocation in the absence of a ligand. Male postnatal primary hypothalamic cell cultures also respond to glucocorticoid exposure with increased 17Î_-E synthesis, suggesting crosstalk between GR signaling and estrogen signaling. Spine density in the gonadally intact adult male VMH decreases following suppression of estrogen synthesis with the aromatase inhibitor letrozole, suggesting estrogen is necessary to maintain spine density. In vivo studies in adult male mice demonstrate that estrogen is necessary to maintain basal peripheral CORT synthesis. Behavior testing using the resident-intruder paradigm showed that dexamethasone-suppression of adrenal CORT synthesis increases the amount of time resident mice spent engaged in aggressive bouts, and CORT treatment 20 minutes prior to aggression testing abolished this effect. The findings presented here provide support for the importance of the interactions between classical and non-classical estrogen and glucocorticoid signaling pathways on hypothalamic spinogenesis and male territorial aggression. / 1 / Jennifer Rainville

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Gonadotropin-Releasing Hormone (GnRH), the master regulator of the reproductive axis, is hypothesized to play an essential role in directly sensing changes in energy levels to maintain fertility. There are a number of GnRH cell lines that have been utilized to study reproductive function; however these have been embryonic in origin and clonally derived. To investigate the cellular mechanisms underlying glucose responsiveness in GnRH neurons, we generated a novel GnRH cell line through immortalization, and fluorescent activated cell (FAC)-sorting of hypothalamic primary culture taken from a GnRH-GFP transgenic mouse. The mHypoA-GnRH/GFP neurons express a complement of markers of fully differentiated GnRH neurons. Glucose induces neuronal activation of mHypoA-GnRH/GFP neurons and glucose metabolism initiates an AMP-Protein Kinase (AMPK)-dependent mechanism to exert transcriptional and secretory control of GnRH. These findings support the use of this novel GnRH cell model for defining components involved in cellular and molecular action of glucose in GnRH neurons.

Gonadotropin-Releasing Hormone (GnRH), the master regulator of the reproductive axis, is hypothesized to play an essential role in directly sensing changes in energy levels to maintain fertility. There are a number of GnRH cell lines that have been utilized to study reproductive function; however these have been embryonic in origin and clonally derived. To investigate the cellular mechanisms underlying glucose responsiveness in GnRH neurons, we generated a novel GnRH cell line through immortalization, and fluorescent activated cell (FAC)-sorting of hypothalamic primary culture taken from a GnRH-GFP transgenic mouse. The mHypoA-GnRH/GFP neurons express a complement of markers of fully differentiated GnRH neurons. Glucose induces neuronal activation of mHypoA-GnRH/GFP neurons and glucose metabolism initiates an AMP-Protein Kinase (AMPK)-dependent mechanism to exert transcriptional and secretory control of GnRH. These findings support the use of this novel GnRH cell model for defining components involved in cellular and molecular action of glucose in GnRH neurons.

Zugl.: Giessen, Universiẗat, Diss., 2009.

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