Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor α2 (GFRα2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRα2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.

Our purpose was to identify and localize intrinsic cardiac ganglia innervating distinct regions of the heart using postmortem tracing of nerve projections with DiI, a method not previously used to study the intrinsic cardiac nervous system. We also investigated the possibility of collateral innervation of myocardium and intrinsic ganglia. In isolated paraformaldehyde-fixed guinea pig hearts, crystals of DiI (1,1′- dioctadecyl-3,3,3′,3′-tetramethylin-docarbocyanine perchlorate) were inserted into the posterior ventricular myocardium below the atrioventricular groove, the right atrium, or the left ventricular septum. Hearts were placed in the dark at 37°C for 2-14 weeks to allow DiI diffusion within neuronal membranes. Labeled neurons were observed in intracardiac ganglia after at least 4 weeks of dye exposure. Labeling was restricted to the inferior-most ganglia (those near the atrioventricular groove) when DiI was inserted into the posterior ventricular myocardium and to ganglia near the sinus node after right atrial DiI placement. Application of DiI to the left ventricular septum resulted in neuron labeling in ganglia primarily in the interatrial septum near the atrioventricular node. After 8 weeks, DiI-labeled nerve fibers and varicosities were seen surrounding unlabeled neurons in some ganglia, suggesting that axons terminating in or passing through the DiI application site in posterior ventricular tissue had collateral branches innervating these ganglia. These results indicate that intrinsic innervation of major cardiac subdivisions is accomplished by regionally segregated cardiac ganglia. Also, tracing with DiI has provided evidence for collateral nerve projections that could be the substrate for novel intracardiac regulatory circuits.

Immunohistochemistry is used widely to identify cholinergic neurons, but this approach has some limitations. To address these problems, investigators developed transgenic mice that express enhanced green fluorescent protein (GFP) directed by the promoter for choline acetyltransferase (ChAT), the acetylcholine synthetic enzyme. Although, it was reported that these mice express GFP in all cholinergic neurons and non-neuronal cholinergic cells, we could not detect GFP in cardiac cholinergic nerves in preliminary experiments. Our goals for this study were to confirm our initial observation and perform a qualitative screen of other representative autonomic structures for the presences of GFP in cholinergic innervation of effector tissues. We evaluated GFP fluorescence of intact, unfixed tissues and the cellular localization of GFP and vesicular acetylcholine transporter (VAChT), a specific cholinergic marker, in tissue sections and intestinal whole mounts. Our experiments identified two major tissues where cholinergic neurons and/or nerve fibers lacked GFP: 1) most cholinergic neurons of the intrinsic cardiac ganglia and all cholinergic nerve fibers in the heart and 2) most cholinergic nerve fibers innervating airway smooth muscle. Most cholinergic neurons in airway ganglia stained for GFP. Cholinergic systems in the bladder and intestines were fully delineated by GFP staining. GFP labeling of input to ganglia with long preganglionic projections (vagal) was sparse or weak, while that to ganglia with short preganglionic projections (spinal) was strong. Total absence of GFP might be due to splicing out of the GFP gene. Lack of GFP in nerve projections from GFP-positive cell bodies might reflect a transport deficiency.

The goal of this study was to build on the historic use of the avian model of development and also to further the knowledge of autonomic nervous system (ANS) regulation of cardiovascular function in vertebrates. Vasoactive drugs sodium nitroprusside, a vasodilator and phenylephrine, a vasoconstrictor were used to study the correlation of cardiovascular function relationship with nerve activity, both sympathetic and parasympathetic (vagal). Additionally, ANG II was used to assess its effects on vagal inhibition. The present study shows that pharmacologically-induced hypertension is associated with a fall in mSNA, indicating that the capacity for sympathetic autonomic cardiovascular regulation is established by late incubation however, late-stage embryonic chickens did not show a significant increase in mSNA during hypotension. The hypotensive response of the embryo was not accompanied by the expected inhibition of vagal discharge; however a slight but insignificant reduction in vagal discharge was noted. When vagal efferent output was isolated, a significant drop in vagal efferent activity was noted in response to hypotension. The present study showed late-stage embryonic chickens lack a vagal response to hypertension in both efferent and sensory limbs. In this study, vagal discharge was reduced from baseline levels in response to Ang II. Collectively, the present study indicates that the lack of a decreased heart rate, in response to increases in Pm caused by Ang II, is due to a central inhibitory action of Ang II on the vagus. Data from the present study suggests that although autonomic interaction with the cardiovascular system in present in late-stage chicken embryos, it is still underdeveloped and possesses a limited capacity.

The origin of cardiac preganglionic neurons in the rat was investigated using the retrograde transport of horseradish peroxidase (HRP). A single injection of HRP was made into the right myocardium in either a sinoatrial or mid-ventricular location. Labeled cells were found in the mid- and lower medulla primarily in and around the nucleus ambiguus (NA) 600-1800 μm above the obex. The dorsal motor nucleus of X (DMN) was sparsely labeled and a few cells were found in an intermediate zone near the level of the obex. Labeling was bilateral with slightly heavier labeling found ipsilateral to the injection site than contralateral to it. Following a unilateral vagotomy, labeled cells were only found ipsilateral to the intact vagus. Atrial and midventricular injections yielded similar results. Occasionally only 1- cells in the NA were labeled per section. Inspection of serial sections revealed that in these sparsely labeled rats, the HRP was often in the same location within the NA forming a column of cells within the nucleus. The columns sometimes extended at least 240 μm in the rostral-caudal direction. The columnar organization was most apparent in rats with few labeled cells presumably because it was obscured in nuclei that were heavily labeled. In a second group of rats, the right vagus was cut at the cervical level and dipped in HRP to determine the extent of the NA and DMN in rats. In these animals, heavier labeling was found in the DMN than in the NA. Cells in the DMN were filled from the upper spinal cord to its most rostral extent 1200 μm above the obex. Thus, although the DMN and NA send projections in the vagus nerve, those axons terminating in the myocardium primarily originate in the NA.

Murine models have become increasingly popular to study various aspects of cardiovascular diseases due to their ease of genetic manipulation. Unfortunately, there has been little effort put into describing the distribution of autonomic nerves in the mouse heart, making it difficult to compare current findings from clinical and experimental models related to cardiovascular diseases. Furthermore, determination of the requirements for the development of this system and its maintenance in adult mice remains largely unexplored. This study represents the first detailed mapping of cholinergic neuroanatomy of the mouse heart based on immunohistochemical staining using true cholinergic markers. We found cholinergic innervation of the mouse heart to be largely focused in the atrium and conducting system. We investigated the involvement of the neurotrophic factor neurturin (NRTN) in the development of cholinergic innervation, because there was indirect evidence that implicated it as a crucial factor. Results from our work definitively demonstrate that NRTN plays a major role in the development of cardiac parasympathetic ganglia and cholinergic innervation of the mouse heart. Adult NRTN knockout mice exhibited a drastic reduction in the number of intracardiac neurons with decreased atrial acetylcholine, cholinergic nerve density at the sinoatrial node and negative chronotropic responses to vagal stimulation. The presence of NRTN and its receptors in hearts from adult wild-type mice suggests that this neurotrophic factor might also be required for maintenance of cardiac cholinergic innervation. Finally, we wanted to determine how intracardiac neurons and their processes change during diseased states, specifically type 1 diabetes. This work has shown that the cardiac cholinergic nervous system in the mouse undergoes structural and functional remodeling when challenged with streptozotocin-induced diabetes. Cholinergic nerves in diabetic hearts undergo extensive sprouting at the sinoatrial node with no change in the number of intracardiac neurons. Cholinergic function appears to be enhanced in diabetic mice, based on pharmacological testing, despite decreased response to direct vagal nerve stimulation. Evidence also suggests that diabetic mice have an imbalance in autonomic control of heart rate. The latter findings suggest that disruption of central input into intrinsic cardiac ganglia also contributes to the neuropathology of type 1 diabetes.

Research has indicated that the frontal and temporal lobes are involved in the mediation of heart rate and blood pressure. However, whereas these regions of the brain have been identified in the mediation of heart rate and blood pressure, the specific cerebral processes involved in determining the direction and magnitude of change in heart rate and blood pressure has not been adequately addressed. The present paper proposes that changes in the magnitude of cerebral activation between the left and right frontal and temporal lobes is partly that which determines the direction and magnitude of changes in heart rate and blood pressure. The present investigation sought to test part of this proposition, namely, that increasing magnitude of cerebral activity within the right anterior temporal region generates increasing levels of sympathetic control of heart rate and blood pressure and that the right lateral frontal region acts to inhibit sympathetic activity. A total of 45 right handed men, with no history of significant head injury, were exposed to 55 dB, 75 dB, and 90 dB white noise presentations. Right frontal lobe functioning was assessed by performance on the Ruff Figural Fluency Test (RFFT), with the participants scoring in the lower one-third classified as Low Fluency. Those scoring in the upper one-third were classified as High Fluency. Quantitative electroencephalography, measured at 19 electrodes sites arranged according to the International 10/20 System, as well as heart rate and blood pressure responses to white noise presentation were measured. Although the results failed to support any of the hypotheses concerning the effects of varying intensity of white noise on cardiovascular activity, partial support was found for the hypotheses that varying intensity of white noise would generate differential changes in high beta magnitude between the Low and High Fluency groups. The results are discussed in terms of support for the model being tested. Alternative explanations of the findings are also provided that demonstrate correspondence between the QEEG and cardiovascular data. Finally, limitations of the model and the methods of the present investigation are discussed and suggestions for improvement are provided. / Ph. D.

É hipótese que (treinamento) T iniciado na fase pré-hipertensiva possa reduzir a atividade simpática e aumentar a vagal ao coração, melhorando a regulação autonômica nos SHR. Investigamos em SHR e WKY jovens (29 dias) os efeitos do T sobre parâmetros hemodinâmicos, funcionamento dos barorreceptores arteriais, tônus simpático e vagal ao coração e variabilidades, e expressão de neurônios pré-ganglionares vagais (Colina Acetil Transferase, ChAT) nos núcleos dorsal motor do vago (DMV) e ambíguo (NA). Ratos T ou sedentários (S) foram canulados nas semanas 0, 1, 2, 4 e 8, para registros funcionais e remoção dos encéfalos para a quantificação (peroxidade e estereologia). A participação dos aferentes periféricos na modulação dos efeitos do T foi avaliada com a desnervação sinoaórtica (SAD). Os efeitos do T são modulados por baro- e quimiorreceptores. O T precoce corrige a disfunção baroreflexa, reduz a hipertonia simpática, mantém parcialmente a tonicidade de neurônios colinérgicos, aumentando a modulação vagal ao coração e melhorando controle autonômico da circulação. / We hypothesized that aerobic training (T) starting at the pre-hypertensive phase reduces not only the sympathetic activity, but increases vagal outflow to the heart, thus improving autonomic cardiovascular control. We investigated in young SHR and WKY the T-induced effects on hemodynamic parameters, activation of arterial baroreceptor, sympathetic and vagal tone to the heart, and the expression of pre-ganglionic parasympathetic neurons (Choline acetyl transferase, ChAT) in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguous (NA). Rats (29 days) were T or sedentary (S) and cannulated at weeks 0, 1, 2, 4 and 8. After functional recordings, brains were harvested for quantification of neurons (peroxidase and stereology). The peripheral afferents was evaluated in sinoaortic denervation (SAD). SAD abrogated the improvement of baroreflex control and resting bradycardia in intact SHR-T. T-induced effects were modulated by arterial baro- and chemoreceptors. When started at the pre-hypertensive phase, T corrects baroreflex dysfunction, reduces sympathetic hyperactivity, maintain the tonicity of pre-ganglionic cholinergic neurons thus increasing vagal outflow to the heart and allowing a better autonomic control of the circulation.

Made available in DSpace on 2017-07-10T14:17:02Z (GMT). No. of bitstreams: 1 kelly Jaque.pdf: 1788989 bytes, checksum: 65d07041b0d9003d6666dd84d7fee873 (MD5) Previous issue date: 2013-08-01 / The growing number of overweight and obesity has led to an increase in the number of patients with insulin resistance and diabetes mellitus type 2. MSG obese rats were glucose intolerant, insulin resistant and theirs pancreatic islets secrete more insulin in response to glucose. Subdiafragmatic vagotomy changes the response of islets to glucose and improves glucose homeostasis, supporting the hypothesis that an unbalance of autonomic nervous system with increased parasympathetic nervous system (PNS) action but a decreased sympathetic nervous system function. Studies showed that the PNS is also involved in β-cell proliferation. Therefore, we investigated of PNS participation, using a subdiafragmatic vagal denervation, upon pancreatic β-cell function and mass regulation, and the body glucose control disruption in MSG-obese rats. For this, Male Wistar rats received during the first five days of life monosodium glutamate (MSG) or saline. Subdiaphragmatic vagotomy was performed at 30 days of life. At 90 days of age, we verified static insulin secretion, pancreas morphometric, ERK expression in islets, glucose homeostasis and lipidis. The MSG treatment caused obesity at 90 days of life. MSG rats presented lower body weight and nasoanal length, increased Lee index and fat depots, normoglycemia, hyperinsulinemia, dyslipidemia, glucose intolerance and insulin resistance when compared to CTL. Vagotomy performed at 30-days of age prevented obesity, fat deposition in the liver and ameliorated glucose tolerance and insulin sensitivity in adult MVAG rats in relation to MSG rats. Islets from MSG rats secreted more insulin at stimulatory glucose concentrations than CTL islets. Histological analysis showed that pancreatic islets from MSG rats were lower with a reduction in β-cell area without modification in α-cell content when compared with CTL. Also, MSG group presented an increased number of pancreatic islets per mm2, with higher number of islets, which may contributes to the higher islet and β-cell relative mass in the MSG pancreas. These effects were associated with enhanced proliferation in MSG group. The number of MVAG pancreatic islet were less than MSG. Vagotomoy performed at 30-days of age, reduced islet and β-cell area in the pancreas from 90-days old CVAG rats. Finally, the relative islet and β-cell mass in MVAG and CVAG rats was similar to CTL. Here we verified if ERK was involved in β-cell replication in MSG rats, but presented no alteration. We demonstrate for the first time that adult MSG rats showed enhanced pancreatic β-cell proliferation which contributes to the higher islet insulin secretion in response to glucose. The vagus nerve is the main factor involved in such a process, since vagotomy performed at 30 days of age prevented islet morphological alterations in adult MVAG rats. Possibly this increase PNS activity in MSG endocrine pancreas is responsible to hyperinsulinemia that enhanced fat storage, damaged glucose homeostasis and insulin action in MSG obesity / O crescente número de pessoas com sobrepeso e obesidade tem levado ao aumento no número de pacientes com resistência à insulina (RI) e portadores do Diabetes mellitus tipo 2. Ratos obesos MSG são intolerantes à glicose (Gli), RI e suas ilhotas pancreáticas secretam mais insulina em resposta à concentrações de Gli. A vagotomia subdiafragamática altera a responsividade das ilhotas à Gli e melhora a homeostase glicêmica nestes animais, sugerindo um desbalanço do sistema nervoso autonômico, com aumento do tônus parassimpático e redução do simpático. Estudos demonstram que o sistema nervoso parassimpático (SNP) possui efeito na proliferação das células β-pancreáticas. Desta forma, investigamos a participação do SNP, através da vagotomia subdiafragmática, no metabolismo energético e na proliferação das ilhotas e de células β-pancreáticas de ratos obesos-MSG. Para isto, ratos Wistar machos receberem durante os cinco primeiros dias de vida glutamato monossódico (grupo MSG) ou salina (grupo CTL). A vagotomia subdiafragmática foi realizada aos 30 dias de vida formando os grupos MVAG e CVAG. Aos 90 dias, verificamos a secreção estática de insulina, homeostase glicêmica e lipídica, morfometria do pâncreas e conteúdo proteico da ERK nas ilhotas. Ratos MSG apresentaram redução do peso corporal e comprimento nasoanal, aumento do índice de Lee e acúmulo de gordura, normoglicêmia, hiperinsulinemia, dislipidemia, intolerância à Gli e RI comparados aos CTL. A vagotomia realizada aos 30 dias de vida preveniu obesidade, acúmulo de gordura no fígado e melhorou a tolerância à Gli e a sensibilidade à insulina em ratos MVAG adultos em relação aos ratos MSG. As ilhotas dos animais MSG secretaram mais insulina quando estimulada pela Gli, em relação aos animais CTL. As análises histológicas mostram que as ilhotas pancreáticas dos animais MSG são menores com redução da área das células β sem alteração nas células α em relação aos CTL. O grupo MSG apresenta um aumento do número das ilhotas por mm2, que pode estar contribuindo com o aumento da massa relativa das ilhotas e das células β. Esse efeito está associado ao aumento da proliferação no grupo MSG. O número de ilhotas foi menor nos MVAG em relação aos MSG. A vagotomia realizada aos 30 dias de vida reduziu a área das ilhotas e das células β aos 90 dias de vida nos animais CVAG. Finalmente, a massa relativa das ilhotas e da células β no MVAG e CVAG foram similares ao CTL. Verificamos se a ERK estava envolvida na proliferação das células β nos ratos MSG, porém não apresentaram alterações desta proteína. Pela primeira vez demonstramos que ratos MSG apresentam aumento da proliferação das células β que contribui com o aumento da secreção de insulina em resposta à Gli. O nervo vago é o principal fator envolvido neste processo, visto que a vagotomia realizada aos 30 dias de vida preveniu as alterações morfológicas das ilhotas nos ratos MVAG adultos

Deux des trois divisions du système nerveux viscéral – le système nerveuxparasympathique et le système nerveux entérique – sont associés aux nerfscrâniens (le troisième, le système nerveux sympathique, est associé aux nerfsspinaux). Cette étude est centrée sur les nerfs crâniens et sur les ganglionsqui leur sont associés ; plus précisément sur les stratégies cellulaires ayantlieu lors de leur ontogenèse.Je propose des principes unificateurs concernant les interactions neuronalesmises en jeu lors de la formation des nerfs crâniens branchiomériques ainsiqu’un nouveau mode de migration des précurseurs des ganglionsparasympathiques couplé à la migration de leurs partenairespréganglionnaires jusqu’au site de formation du ganglion. Enfin, je présentecertaines observations préliminaires suggérant que les précurseurs dusystème nerveux entériques utilisent ce même modus operandi pour envahirl’oesophage. / Two of the three divisions of the visceral nervous system —theparasympathetic and the enteric nervous systems— are associated withcranial nerves (the third one, the sympathetic division, being associatedwith spinal nerves). This work is focused on cranial nerves and associatedganglia and more particularly on the cellular strategies presiding over theirontogeny and wiring.I propose unifying principles of neuronal interactions that govern theformation of branchiomeric cranial nerves, as well as a novel migrationpathway followed by parasympathetic precursors, which use theirpreganglionic nerves to migrate to the site of ganglion formation. Finally, Ipresent preliminary observations suggesting that the enteric neuronalprecursors use the same trick to populate the esophagus.