Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 167 pages. Includes Vita. Includes bibliographical references.

A clear definition of the mechanisms involved in synaptic transmission is of paramount importance for the understanding of the processes governing synaptic efficacy. Despite decades of intense study, these mechanisms remain poorly understood. The work contained in this thesis examines several such mechanisms using the hypothalamic-neurohypophysial system (HNS), a classical preparation for the study of Ca2+-dependent neuropeptide release. The first portion of this thesis is comprised of my efforts to define the cellular machinery essential for the exocytosis of secretory granules isolated from peptidergic neurohypophysial terminals of the HNS. Here, using the planar lipid bilayer model system, I have been able to show that syntaxin alone in the target membrane is sufficient to elicit fusion of modified neurohypophysial secretory granules. Surprisingly, SNAP-25 does not appear to be necessary for this process. This suggests that syntaxin may be able to substitute for SNAP-25 to form functional non-cognate fusion complexes. Additionally, the coupling of amperometric detection with the planar lipid bilayer system has allowed me to confirm these results using native, unmodified secretory granules, and also provides some insight into the kinetics of release in this reconstituted system. This model system should provide a convenient means for the study of additional regulatory factors believed to be involved in secretory vesicle exocytosis. The second and third sections of this thesis involve my examination of the role of presynaptic Ca2+ stores in neuropeptide secretion from isolated peptidergic neurohypophysial terminals (NHT). I initially examined the source of recently discovered ryanodine-sensitive Ca2+ stores in this system. Using Immuno-electron microscopy I have found that ryanodine receptor (RyR) labeling appears to co-localize with large dense core granules. Additionally, I have shown that a large conductance cation channel, with similarities to the RyR, found in the membrane of these granules has the same characteristic response to pharmacological agents specific for the RyR. Further, application of RyR agonists modulates basal neuropeptide release from NHT. These results suggest that the large dense core granules of NHT serve as the source of a functional ryanodine-sensitive Ca2+store. Recent work has revealed that spark-like Ca2+ transients, termed syntillas, can be observed in NHT. These syntillas arise from ryanodine-sensitive intracellular stores. In other neuronal preparations, similar Ca2+ transients have been suggested to affect spontaneous transmitter release. However, such a role for syntillas had yet to be examined. To assess if syntillas could directly trigger spontaneous release from NHT, I used simultaneous Ca2+imaging along with amperometric detection of release. Amperometry was adapted to this system via a novel method of false-transmitter loading. Using this approach I have found no apparent correlation between these two events, indicating that syntillas are unable to directly elicit spontaneous transmitter release. As this finding did not rule out an indirect modulatory role of syntillas on release, I additionally present some preliminary studies examining the ability of ryanodine-sensitive Ca2+ release to modulate vesicular priming. Using immunocytochemistry, I have shown that RyR agonist treatment shifts the distribution of neuropeptides toward the plasma membrane in oxytocinergic NHT, but not in vasopressinergic NHT. RyR antagonists have the opposite affect, again only in oxytocinergic NHT. Further, I have found that application of RyR agonists result in a facilitation of elicited release in NHT using membrane capacitance recording. This facilitation appears to be due primarily to an increase in recruitment of vesicles to the readily-releasable pool. These findings suggest that ryanodine-sensitive Ca2+stores may be involved in vesicular priming in NHTs. Taken together, the work presented in this thesis provides some new and interesting insights into the underlying mechanisms and modulation of transmitter release in both the HNS and other CNS terminals.

Orientadores: Claudio Aparecido Casatti, Roelf Justino Cruz Rizzolo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T07:48:09Z (GMT). No. of bitstreams: 1 Andrade_JoaoCleberTheodorode_D.pdf: 4798610 bytes, checksum: b8d22ab273e3f97ec7f0b085497c5842 (MD5) Previous issue date: 2009 / Resumo: O hormônio concentrador de melanina (MCH) e a orexina A (OXA) são neuromediadores expressos em neurônios distribuídos no diencéfalo, principalmente na área hipotalâmica lateral, que emitem projeções para inúmeras regiões do neuroeixo. Esses neuropeptídeos exercem potente ação orexigênica, estimulando a ingestão alimentar e o controle do balanço energético. Em vista disso, propusemos analisar esses sistemas neuropeptidérgicos no núcleo motor dorsal do nervo vago, um importante grupamento neuronal pré-motor parassimpático responsável pelas modulações da motilidade e secreção do tubo gastrointestinal, que indiretamente colaboram na ingestão alimentar. Para esses propósitos, foram empregados os métodos de transporte neuronal retrógrado e imunoistoquímica. Os principais dados advindos desse estudo neuroanatômico permite concluir que o núcleo motor dorsal do nervo vago apresenta uma distribuição heterogênea da inervação imunorreativa a OXA (OXA-ir) e ao MCH (MCH-ir), com preponderância da inervação OXA-ir. Os neurônios desse núcleo parassimpático que enviam fibras nervosas para a parte abdominal do esôfago, estômago e segmento proximal do duodeno, recebem inúmeros contatos morfológicos dos terminais nervosos OXA-ir ou ao MCH-ir, sugestivos de contatos sinápticos. Finalmente, as aferências diencefálicas OXA-ir ou ao MCH-ir para o DMV, foram elucidadas, revelando que as partes dorsais e laterais da área hipotalâmica lateral são os principais locais de origem dessa inervação neuropeptidérgica. / Abstarct: Melanin-concentrating hormone (MCH) and orexin-A (OXA) are neuromediators expressed in neurons distributed in the diencephalum, mainly in the lateral hypothalamic area, projecting nerve fibers to several parts of the central nervous system. These neuropeptides exert potent orexigenic activity, increasing feeding and controlling the energetic balance. Consequently, these neuropeptidergic systems were evaluated in the dorsal motor nucleus of the vagus nerve, a fundamental premotor parasympathetic nucleus that modulates the motility and secretion of the gastrointestinal tract, indirectly collaborating in the feeding behaviour. For these purposes retrograde neuronal tracing and immunohistochemistry methods were used. The main results from this neuroanatomical study showed that the dorsal motor nucleus of the vagus nerve exhibits a heterogeneous rostro-caudal MCH- or OXA-innervation patterns, being the latter more significant. Parasympathetic neurons that send nerve fibers to subdiafragmatic esophagus, stomach and proximal duodenum receive MCH- or OXA-immunoreactive nerve endings like synapses. Finally, the MCH or OXA-immunoreactive diencephalic afferents to dorsal motor nucleus of the vagus nerve were elucidated. These afferents are mainly from dorsal and lateral parts of the lateral hypothalamic area. / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

L’espèce Astyanax mexicanus est composée de deux morphotypes de poissons radicalement différents : le très classique poisson de surface vivant dans des rivières et le poisson cavernicole (CF, cavefish) aveugle et dépigmenté. Ces deux morphotypes diffèrent sur de nombreux aspects, aussi bien en termes de modalités sensorielles, qu’en termes de physiologie ou de comportement. L’approche « Evo-Devo » consiste à tenter de relier des différences développementales précoces à des modifications phénotypiques plus tardives. Dans le cadre de ce travail, nous nous sommes concentrés sur les modifications précoces de l’hypothalamus et de l’œil du CF. Nous montrons que des modifications précoces de signalisation de morphogènes tels que Shh ou Fgf conduisent à une modification de la taille des groupes de neurones peptidergiques au sein de l’hypothalamus, via les facteurs de transcription Lhx, impliqués dans la spécification neuronale. Plus particulièrement, nous montrons l’augmentation de taille des groupes de neurones NPY ainsi qu’hypocretine, qui à son tour provoque une réduction du sommeil chez le CF.Nous nous sommes aussi intéressés à l’oeil du CF, qui commence par se développer avant de dégénérer. Une réduction du quadrant ventral de la rétine avait été précédemment décrit. Nous rafinons cette description grâce à une étude de la régionalisation de la coupe optique du CF qui suggère une réduction de la rétine temporale plus spécifiquement. Nous proposons également une première description de la morphogénèse de l’oeil du CF grace à l’imagerie live de lignées transgéniques fluorescentes. Cette étude révèle un défaut d’invagination de la coupe optique chez le CF. Globalement, ce travail ouvre la voie vers une meilleure compréhension de l’évolution de la tête du CF. / Astyanax mexicanus is a fish species comprising two strikingly different morphotypes : the classical river-dwelling surface fish and the blind depigmented cavefish. These two morphs differ in many aspects in terms of sensorial modalities, physiology and behaviour. In the Evo-Devo approach, we try to link early developmental differences to later phenotypic modifications. Here we focus on the early modification of the hypothalamus and the eye of the cavefish. We show that early signalling modification of morphogens such as Shh or Fgfs lead to the modification of neuropeptidergic clusters in the hypothalamus via the neuronal fate-specifying transcription factors Lhx. More particularly, we show an increase in NPY and hypocretin cluster size. In turn, this increased hypocretin cluster size triggers a reduction of sleep in the cavefish larva.We also examine the embryonic eye of the cavefish which first develops before degenerating. This eye was previously reported to have a reduced ventral retina. We refine this description by studying the regionalisation of the cavefish optic cup and suggest that this reduction concerns more specifically the temporal retina. We also attempt a first description of the cavefish eye morphogenesis by live imaging on fluorescent transgenic lines. This description reveals a defect in the optic cup invagination of the cavefish. Overall, this work started deciphering the developmental evolution of the cavefish head.

PACAP and VIP have prominent effects on cardiac function in several species, but little is known about their influence on the murine heart. Accordingly, we evaluated the expression of PACAP/VIP receptors in mouse heart and the response of isolated atria to peptide agonists. Quantitative PCR demonstrated that PAC1, VPAC1, and VPAC2 receptor mRNAs are present throughout the mouse heart. Expression of all three receptor transcripts was low, PAC1 being the lowest. No regional differences in expression were detected for individual receptor mRNAs after normalization to L32. Pharmacological effects of PACAP-27, VIP, and the selective PAC1 agonist maxadilan were evaluated in isolated, spontaneously beating atria from C57BL/6 mice of either sex. Incremental additions of PACAP-27 at 1 min intervals caused a concentration-dependent tachycardia with a log EC50=-9.08±0.15 M (n=7) and a maximum of 96.3±5.9% above baseline heart rate. VIP and maxadilan also caused tachycardia but their potencies were about two orders of magnitude less. Increasing the dosing interval to 5 min caused a leftward shift of the concentration-response curve to maxadilan but no changes in the curves for PACAP-27 or VIP. Under this condition, neither the potency nor the efficacy of maxadilan differed from those of PACAP-27. Neither PACAP-27 nor maxadilan caused tachyphylaxis, and maximal responses to maxadilan were maintained for at least 2 h. We conclude that all three VIP/PACAP family receptors are expressed by mouse cardiac tissue, but only PAC1 receptors mediate positive chronotropic responses to PACAP-27 and VIP.

PACAP and VIP have prominent effects on cardiac function in several species, but little is known about their influence on the murine heart. Accordingly, we evaluated the expression of PACAP/VIP receptors in mouse heart and the response of isolated atria to peptide agonists. Quantitative PCR demonstrated that PAC1, VPAC1, and VPAC2 receptor mRNAs are present throughout the mouse heart. Expression of all three receptor transcripts was low, PAC1 being the lowest. No regional differences in expression were detected for individual receptor mRNAs after normalization to L32. Pharmacological effects of PACAP-27, VIP, and the selective PAC1 agonist maxadilan were evaluated in isolated, spontaneously beating atria from C57BL/6 mice of either sex. Incremental additions of PACAP-27 at 1 min intervals caused a concentration-dependent tachycardia with a log EC50=-9.08±0.15 M (n=7) and a maximum of 96.3±5.9% above baseline heart rate. VIP and maxadilan also caused tachycardia but their potencies were about two orders of magnitude less. Increasing the dosing interval to 5 min caused a leftward shift of the concentration-response curve to maxadilan but no changes in the curves for PACAP-27 or VIP. Under this condition, neither the potency nor the efficacy of maxadilan differed from those of PACAP-27. Neither PACAP-27 nor maxadilan caused tachyphylaxis, and maximal responses to maxadilan were maintained for at least 2 h. We conclude that all three VIP/PACAP family receptors are expressed by mouse cardiac tissue, but only PAC1 receptors mediate positive chronotropic responses to PACAP-27 and VIP.

Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of atrial and ventricular arrhythmias. While this system has been studied extensively in animal models, far less is known about the intrinsic cardiac nervous system of humans. This study was initiated to anatomically identify neurotransmitters associated with the right atrial ganglionated plexus (RAGP) of the human heart. Biopsies of epicardial fat containing a portion of the RAGP were collected from eight patients during cardiothoracic surgery and processed for immunofluorescent detection of specific neuronal markers. Colocalization of markers was evaluated by confocal microscopy. Most intrinsic cardiac neuronal somata displayed immunoreactivity for the cholinergic marker choline acetyltransferase and the nitrergic marker neuronal nitric oxide synthase. A subpopulation of intrinsic cardiac neurons also stained for noradrenergic markers. While most intrinsic cardiac neurons received cholinergic innervation evident as punctate immunostaining for the high affinity choline transporter, some lacked cholinergic inputs. Moreover, peptidergic, nitrergic, and noradrenergic nerves provided substantial innervation of intrinsic cardiac ganglia. These findings demonstrate that the human RAGP has a complex neurochemical anatomy, which includes the presence of a dual cholinergic/nitrergic phenotype for most of its neurons, the presence of noradrenergic markers in a subpopulation of neurons, and innervation by a host of neurochemically distinct nerves. The putative role of multiple neurotransmitters in controlling intrinsic cardiac neurons and mediating efferent signaling to the heart indicates the possibility of novel therapeutic targets for arrhythmia prevention.

No description available.

No / The nervous system communicates with peripheral tissues through nerve fibres and the systemic release of hypothalamic and pituitary neurohormones. Communication between the nervous system and the largest human organ, skin, has traditionally received little attention. In particular, the neuro-regulation of sebaceous glands (SGs), a major skin appendage, is rarely considered. Yet, it is clear that the SG is under stringent pituitary control, and forms a fascinating, clinically relevant peripheral target organ in which to study the neuroendocrine and neural regulation of epithelia. Sebum, the major secretory product of the SG, is composed of a complex mixture of lipids resulting from the holocrine secretion of specialised epithelial cells (sebocytes). It is indicative of a role of the neuroendocrine system in SG function that excess circulating levels of growth hormone, thyroxine or prolactin result in increased sebum production (seborrhoea). Conversely, growth hormone deficiency, hypothyroidism, and adrenal insufficiency result in reduced sebum production and dry skin. Furthermore, the androgen sensitivity of SGs appears to be under neuroendocrine control, as hypophysectomy (removal of the pituitary) renders SGs largely insensitive to stimulation by testosterone, which is crucial for maintaining SG homeostasis. However, several neurohormones, such as adrenocorticotropic hormone and α-melanocyte-stimulating hormone, can stimulate sebum production independently of either the testes or the adrenal glands, further underscoring the importance of neuroendocrine control in SG biology. Moreover, sebocytes synthesise several neurohormones and express their receptors, suggestive of the presence of neuro-autocrine mechanisms of sebocyte modulation. Aside from the neuroendocrine system, it is conceivable that secretion of neuropeptides and neurotransmitters from cutaneous nerve endings may also act on sebocytes or their progenitors, given that the skin is richly innervated. However, to date, the neural controls of SG development and function remain poorly investigated and incompletely understood. Botulinum toxin-mediated or facial paresis-associated reduction of human sebum secretion suggests that cutaneous nerve-derived substances modulate lipid and inflammatory cytokine synthesis by sebocytes, possibly implicating the nervous system in acne pathogenesis. Additionally, evidence suggests that cutaneous denervation in mice alters the expression of key regulators of SG homeostasis. In this review, we examine the current evidence regarding neuroendocrine and neurobiological regulation of human SG function in physiology and pathology. We further call attention to this line of research as an instructive model for probing and therapeutically manipulating the mechanistic links between the nervous system and mammalian skin. / Agency for Science, Technology and Research. Grant Numbers: A*STAR Research Attachment Programme (ARAP), IAF‐PP H17/01/a0/004, IAF‐PP H17/01/a0/008; NIHR Manchester Biomedical Research Centre, Inflammatory Hair Disease Programme; University of Manchester; University of Miami

La douleur articulaire associée à l’arthrose est un problème clinique majeur, spécialement chez les personnes âgées. L’intensité de la douleur est souvent amplifiée lors de mouvement de l’articulation et principalement lors du soutien de la charge corporelle sur le membre lésé. Malheureusement, les traitements pharmacologiques proposés sont trop souvent associés à des effets secondaires néfastes et à une inefficacité pour le soulagement de la douleur à long terme. Divers modèles murins sont utilisés en laboratoire de recherche pour des études précliniques de molécules aux propriétés analgésiques. Une évaluation comparative de la réponse comportementale douloureuse des animaux d’un modèle d’instabilité articulaire induit par le sectionnement du ligament croisé antérieur accompagné d’une méniscectomie partielle (le modèle ACLT+pMMx) et d’un modèle de dégénérescence articulaire induite par le monoiodoacetate (le modèle MIA) a permis de sélectionner un modèle approprié pour la continuité du projet. Les deux modèles ont démontré des lésions tissulaires, mais le modèle MIA a démontré une réponse douloureuse plus prononcée que le modèle ACLT+pMMx. Par l’analyse de la démarche, le modèle MIA a démontré une boiterie claire dans le patron de la démarche des animaux qui est associée à une lésion unilatérale. Le modèle MIA a donc été choisi pour la suite du projet. La problématique principale dans la recherche sur la douleur associée à l’arthrose est une compréhension incomplète des mécanismes de douleur responsables de l’induction et du maintien de l’état de douleur. Il devient donc nécessaire d’améliorer nos connaissances de ces mécanismes en effectuant une caractérisation plus approfondie des modèles animaux employés pour l’évaluation de stratégies pharmacologiques analgésiantes. Afin de bien comprendre le modèle MIA, une caractérisation des événements moléculaires centraux lors de la progression du processus dégénératif des structures articulaires de ce modèle s’est effectuée aux jours 3, 7, 14, 21 et 28 post injection. Des mécanismes hétérogènes qui modulent l’information nociceptive en fonction de la progression temporelle de la pathologie ont été observés. Les changements du contenu i spinal des neuropeptides sélectionnés (substance P, CGRP, dynorphine A et Big dynorphine) ont débuté sept jours suivant l’injection de MIA. L’observation histologique a démontré que les dommages structuraux les plus importants surviennent entre les jours 14 et 21. C’est entre les jours 7 et 21 que les lésions démontrent le plus de similarités à la pathologie humaine. Cela suggère que lors d’une évaluation préclinique d’un traitement pharmacologique pour pallier la douleur articulaire utilisant le modèle MIA, l’étude doit tenir compte de ces événements afin de maximiser l’évaluation de son efficacité. Puisque les traitements pharmacologiques conventionnels proposés pour le soulagement de la douleur ne font pas l’unanimité en terme d’efficacité, d’effets non désirés et de coûts monétaires parfois onéreux, les molécules de dérivés de plante deviennent une alternative intéressante. L’eugénol, le principal constituant de l’huile de clou de girofle, a été administré oralement pour une période de 28 jours chez des rats ayant reçu l’injection intra-articulaire de MIA afin d’évaluer son efficacité pour le traitement de la douleur articulaire. L’eugénol à une dose de 40 mg/kg s’est révélé efficace pour l’amélioration du patron de la démarche des animaux ainsi que pour la diminution de l’allodynie mécanique secondaire. De plus, les concentrations spinales de neuropeptides pronocicepteurs ont diminué chez les animaux traités. Par une évaluation histopathologique, l’eugénol n’a démontré aucune évidence d’effets toxiques suite à une administration per os quotidienne pour une période prolongée. Ces résultats suggèrent le potentiel thérapeutique complémentaire de la molécule d’eugénol pour le traitement de la douleur articulaire. / Pain is the most predominant clinical symptom associated with osteoarthritis (OA), mostly among older people. Joint movement and weight bearing often increase the pain intensity. Unfortunately, the proposed pharmacological treatments are frequently associated with side effects and ineffective for pain alleviation for long time periods. Many murine models are used in laboratories for preclinical studies evaluating analgesic compounds. A comparative evaluation of the behavioral pain responses of animals with a joint instability model induced by the transection of the anterior cruciate ligament followed by a partial menisectomy (the ACLT+pMMx model) and of an articular degenerative model induced by an intra-articular injection of monoiodoacetate (the MIA model) was conducted to select an appropriate model for the continuation of the project. Both models demonstrated articular lésions, however the MIA model demonstrated a clearer behavioral pain response over the ACLT+pMMx model. The gait pattern of the MIA model revealed a clear limping gait similar to that observed with unilateral OA in humans. The MIA model was chosen for the subsequent studies. An unresolved issue in pain OA research is the lack of understanding of the pain mechanisms responsible for the induction and maintenance of the pain. Therefore, there is an urgent clinical need to improve the characterization of animal models to effectively discover novel pain relief pharmacological treatment stratégies for OA patients. A characterization of the spinal pain molecular events during the progression of the joint degenerative process in the MIA model was performed on days 3, 7, 14, 21 and 28 post injection. Heterogeneous nociceptive central molecular events were observed in respect to the time course of the pathology’s progression. Changes in selected spinal neuropeptide content (substance P, CGRP, dynorphin A, Big dynorphin) began 7 days following the MIA injection. Most severe joint structural damage on histology occured between days 14 and 21 post injection. These results suggest that preclinical drug evaluation employing this model should be conducted between 7 and 21 days post injection when the lesions resemble most those of human OA. iii As current pharmacological therapy for the alleviation of joint pain does not achieve the unanimity in respect to efficacy, side effects and cost, plant derivate compounds are now interesting alternatives to improve the situation. Eugenol, the main constituent of clove oil, was evaluated for its efficacy for alleviation of joint pain in rats who previously received an intra-articular injection of mono-iodoacetate to induce the MIA model. Eugenol, administered orally for 28 consecutive days at a dose of 40 mg/kg, improved gait pattern and reduced secondary mechanical allodynia. Furthermore, spinal concentrations of pronociceptive neuropeptides were also decreased in the treated animals. No toxic effects of the compoud were identified on histopathological assessment of the various tissues. These results suggest that eugenol could be a potential therapeutic option for alleviating OA joint pain.