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O controle do sistema respiratório é realizado por neurônios localizados no bulbo e na ponte. Existem duas regiões localizadas próximas na coluna respiratória ventral do bulbo, mas que estão envolvidas com atividades respiratórias diferentes: núcleo retrotrapezóide (RTN) e região parafacial lateral (pFRG). O RTN contém neurônios quimiossensíveis que expressam o fator de transcrição Phox2b e respondem à hipercapnia e hipóxia; por este motivo, esta região é considerada uma forte candidata a conter os quimiorreceptores centrais. Além do seu envolvimento na quimiorrecepção central, experimentos descritos na literatura mostraram a importância do RTN também na inspiração. O pFRG está envolvido na geração da expiração ativa (E2), pois dados da literatura sugerem que existe uma inibição sináptica para essa região que, quando é inibida, gera a E2. Entretanto, até o presente momento, não se sabe a fonte desta inibição para os neurônios do pFRG, nem se os neurônios Phox2b do RTN contribuem para a geração da E2. Diante disso, o principal objetivo desta tese foi investigar, de forma anatômica e funcional, a localização da fonte de inibição para o pFRG. Foram utilizados ratos Wistar que receberam injeções de traçadores retrógrados no RTN e no pFRG, a fim de observarmos projeções de regiões que possuem neurônios inibitórios. Observamos projeções oriundas de diversas regiões envolvidas com a respiração que contêm neurônios inibitórios. Para o RTN, as projeções se originaram no núcleo do trato solitário intermediário (NTSi), núcleo tegmental pedunculopontino (PPT), complexo de Botzinger (BotC), rafe bulbar (RB) e Kolliker-Fuse (KF); e para o pFRG, as projeções se originaram dos mesmos núcleos, com exceção do PPT. É importante apontar que o pFRG também recebeu projeções do núcleo tegmental laterodorsal (LDT). Foi realizado um protocolo de hibridização in situ utilizando a metodologia de RNAscope, a qual nos confirmou que muitas das projeções observadas para o pFRG eram inibitórias. Adicionalmente, constatamos também projeções dos neurônios quimiossensíveis do RTN para o pFRG, sugerindo a participação do RTN na expiração ativa. Além disso, realizamos também experimentos funcionais para investigar a importância destas projeções na expiração ativa, por meio de inibições de algumas dessas regiões e registro das variáveis respiratórias. Os resultados mostraram que a inibição do NTSi gera E2 de forma tônica e inibição do PPT gera E2 fásica. Também constatamos que a inibição da RB promove aumento da amplitude abdominal durante hipercapnia, sugerindo que a RB pode modular a atividade do pFRG durante hipercapnia. Os resultados mostraram que as regiões do NTSi e PPT são fortes candidatas a serem fontes para o pFRG, inibindo a E2. / Respiratory system control is performed by neurons located in the medulla and the pons. There are two regions located close in the ventral column of the medulla, but are involved in different respiratory phases: retrotrapezoid nucleus (RTN) and lateral parafacial region (pFRG).The RTN contains chemosensitive neurons that express the transcription factor Phox2b and respond to hypercapnia and hypoxia; for this reason, this region is considered a candidate to contain the central chemoreceptors. In addition to its involvement in central chemoreception, experiments described in the literature showed the importance of RTN also in inspiration. The pFRG is involved in the generation of active expiration (E2); data from the literature suggest that there is synaptic inhibition for this region which, when inhibited, generates E2. However, the source of this inhibition to pFRG neurons and whether Phox2b neurons of the RTN contribute to the generation of E2 are still not known. Therefore, the main objective of this study was to investigate, anatomically and functionally, the location of the source of inhibition for pFRG. We used male Wistar rats that received injections of retrograde tracers into the RTN and pFRG, to investigate projections from regions that have inhibitory neurons. We observed projections from several regions involved on respiration containing inhibitory neurons. There were projections to the RTN from the intermediate nucleus of the solitary tract (NTSi), pedunculopontine tegmental nucleus (PPT), Botzinger complex (BotC), medullary raphe (RB), Kolliker-Fuse (KF). The projections to the pFRG are the same as observed to RTN, except PPT. There is also projection from tegmental dorsal laterodorsal nucleus (LDT) to pFRG. An in situ hybridization protocol was performed using the RNAscope methodology, which confirmed that many of the projections observed to pFRG were inhibitory. In addition, we observed projections of the chemosensitive neurons from the RTN to the pFRG, suggesting the participation of the RTN in E2. In addition, we performed functional tests to verify the involvement of some of those projections with E2. We performed inhibition of some of those regions and record the respiratory variables. The data showed that the inhibition of NTSi generates tonic E2 and inhibition of PPT generates phasic E2. We also found that inhibition of RB promotes increased abdominal amplitude during hypercapnia, suggesting that RB may modulate pFRG activity during hypercapnia. The results showed that the NTSi and PPT regions are candidates to be sources for pFRG, inhibiting E2.

O núcleo do trato solitário é uma área integrativa do sistema nervoso central (CNS) envolvida no controle autonômico e respiratório. Estudos da literatura sugerem que o óxido nítrico (NO) exerce um importante papel na modulação dos reflexos cardiovasculares e ventilatórios no NTS. Além disso, evidências da literatura indicam uma possível interação entre o NO e o ATP no SNC. Fundamentados nessas evidências, no presente estudo, avaliamos a possível participação do NO na modulação dos parâmetros cardiorespiratórios basais e no processamento das respostas cardiovasculares e respiratórias à ativação do quimiorreflexo no NTS caudal de ratos não-anestesiados. Além disso, o possível papel do NO produzido pela óxido nítrico sintase neuronal (nNOS) nas respostas cardiovasculares e respiratórias à microinjeção unilateral de ATP no NTS caudal também foi avaliado. Para tanto, os animais foram submetidos ao implante de cânulas guia em direção ao NTS caudal e à canulação da artéria e veia femoral. Os parâmetros ventilatórios foram avaliados pelo método de pletismografia de corpo inteiro. A análise dos resultados monstrou que as microinjeções bilaterais do L-NAME, um inibidor nãoseletivo da NOS, no NTS caudal, promoveram um aumento significativo na pressão arterial basal dos animais, sugerindo um papel modulatório do NO sobre os neurônios envolvidos com as vias neurais do barorreflexo. No entanto, as microinjeções bilaterais do N-PLA, um inibidor seletivo da nNOS, no NTS caudal, não promoveram alterações significativas na pressão arterial basal, sugerindo que a produção do NO envolvido no controle autonômico basal no NTS caudal não é dependente da atividade da nNOS. Com relação às respostas do quimiorreflexo, as microinjeções bilaterais do L-NAME ou do N-PLA, no NTS caudal de ratos nãoanestesiados, promoveram um atenuação significativa no aumento da freqüência respiratória (fR) à ativação do quimiorreflexo, sugerindo a participação do NO Resumo xii produzido pela nNOS na modulação do componente respiratório do quimiorreflexo no NTS caudal. No entanto, as respostas pressora e bradicárdicas decorrentes da ativação do quimiorreflexo não foram alteradas pelas microinjeções bilaterais do LNAME ou N-PLA no NTS caudal, sugerindo que o NO não está envolvido na modulação das respostas cardiovasculares decorrentes da ativação deste reflexo. No que diz respeito às respostas decorrentes da microinjeção de ATP no NTS caudal, a análise dos resultados demonstrou que as respostas de aumento na pressão arterial, fR e ventilação minuto produzidas pela microinjeção unilateral de ATP no NTS caudal de ratos não-anestesiados foram significativamente atenuadas após a microinjeção do N-PLA no mesmo sítio, sugerindo a participação do NO produzido pela nNOS na modulação de tais respostas. Neste contexto, os achados do presente trabalho sugerem que no NTS caudal: 1) o NO, provavelmente produzido pela NOS endotelial, exerce um importante papel modulatório nas vias neurais do barorreflexo; 2) a neurotransmissão do aumento da fR decorrente da ativação do quimiorreflexo envolve a formação de NO produzido pela nNOS; 3) a neurotransmissão das respostas cardiovasculares decorrentes da ativação do quimiorreflexo não envolve a formação de NO; 4) a neurotransmissão das respostas cardiovascualres e respiratórias decorrentes da microinjeção unilateral de ATP envolve a formação de NO produzido pela nNOS. / The nucleus tractus solitarius (NTS) is an integrative area in the central nervous system (CNS) involved with the ventilatory and autonomic control. Several studies suggest that nitric oxide (NO) in the NTS plays an important role in the modulation of the cardiovascular and ventilatory reflexes. In addition, there is evidence indicating a possible interaction of NO and ATP in the CNS. Considering these findings, in the present study, we evaluated the possible role of NO on the modulation of the basal cardiorespiratory parameters as well as on the processing of the cardiovascular and ventilatory responses elicited by chemoreflex activation in the caudal NTS of awake rats. In addition, the possible role of NO produced by neuronal nitric oxide sintase (nNOS) on the cardiovascular and respiratory responses produced by unilateral microinjection of ATP into the caudal NTS was also evaluated. For this purpose, rats received bilateral guide cannulae in direction of the caudal NTS and femoral artery and vein were cannulated. The ventilatory measurements were obtained by whole-body pletismograph method. Our data showed that bilateral microinjections of L-NAME, a non-selective NOS inhibitor, into the caudal NTS, produced a significant increase in basaline mean arterial pressure, suggesting a modulatory role of NO in the neural pathways of the baroreflex. However, bilateral microinjections of N-PLA, a selective nNOS inhibitor, into the caudal NTS, produced no significant changes in the baseline mean arterial pressure, suggesting that NO produced by nNOS is not involved in the basal autonomic control in the caudal NTS. With respect to chemoreflex responses, bilateral microinjections of L-NAME or NPLA, into the caudal, produced a significant attenuation in the increase in respiratory frequency (fR) produced by chemoreflex activation, suggesting that NO produced by nNOS is involved in the modulation of the respiratory component of the chemoreflex. However, the pressor and bradicardic responses elicited by chemoreflex actiovation Abstract xv were not affected by microinjections of L-NAME or N-PLA, suggesting that NO is not involved in the modulation of the cardiovascular responses. With respect to ATP microinjection responses, the data showed that unilateral microinjection of ATP into the caudal NTS produced increase in arterial pressure, fR and minute ventilation, which were significantly attenuated by N-PLA, suggesting that NO produced by nNOS is involved in the modulation of the cardiovascular and ventilatory responses to ATP microinjection into the caudal NTS. In conclusion, the data of present study indicate that in the caudal NTS: 1) NO, produced probably by endothelial NOS, plays an important modulatory role on the neural pathways of the baroreflex; 2) the neurotransmission of the increase in respiratory frequency to chemoreflex activation involve NO production by nNOS. 3) NO is not involved in modulation of the autonomic components of chemoreflex; 4) the cardiovascular and ventillatory responses produced by ATP micronjection are, at least in part, mediated by NO produced by nNOS.

O núcleo do trato solitário é uma área integrativa do sistema nervoso central (CNS) envolvida no controle autonômico e respiratório. Estudos da literatura sugerem que o óxido nítrico (NO) exerce um importante papel na modulação dos reflexos cardiovasculares e ventilatórios no NTS. Além disso, evidências da literatura indicam uma possível interação entre o NO e o ATP no SNC. Fundamentados nessas evidências, no presente estudo, avaliamos a possível participação do NO na modulação dos parâmetros cardiorespiratórios basais e no processamento das respostas cardiovasculares e respiratórias à ativação do quimiorreflexo no NTS caudal de ratos não-anestesiados. Além disso, o possível papel do NO produzido pela óxido nítrico sintase neuronal (nNOS) nas respostas cardiovasculares e respiratórias à microinjeção unilateral de ATP no NTS caudal também foi avaliado. Para tanto, os animais foram submetidos ao implante de cânulas guia em direção ao NTS caudal e à canulação da artéria e veia femoral. Os parâmetros ventilatórios foram avaliados pelo método de pletismografia de corpo inteiro. A análise dos resultados monstrou que as microinjeções bilaterais do L-NAME, um inibidor nãoseletivo da NOS, no NTS caudal, promoveram um aumento significativo na pressão arterial basal dos animais, sugerindo um papel modulatório do NO sobre os neurônios envolvidos com as vias neurais do barorreflexo. No entanto, as microinjeções bilaterais do N-PLA, um inibidor seletivo da nNOS, no NTS caudal, não promoveram alterações significativas na pressão arterial basal, sugerindo que a produção do NO envolvido no controle autonômico basal no NTS caudal não é dependente da atividade da nNOS. Com relação às respostas do quimiorreflexo, as microinjeções bilaterais do L-NAME ou do N-PLA, no NTS caudal de ratos nãoanestesiados, promoveram um atenuação significativa no aumento da freqüência respiratória (fR) à ativação do quimiorreflexo, sugerindo a participação do NO Resumo xii produzido pela nNOS na modulação do componente respiratório do quimiorreflexo no NTS caudal. No entanto, as respostas pressora e bradicárdicas decorrentes da ativação do quimiorreflexo não foram alteradas pelas microinjeções bilaterais do LNAME ou N-PLA no NTS caudal, sugerindo que o NO não está envolvido na modulação das respostas cardiovasculares decorrentes da ativação deste reflexo. No que diz respeito às respostas decorrentes da microinjeção de ATP no NTS caudal, a análise dos resultados demonstrou que as respostas de aumento na pressão arterial, fR e ventilação minuto produzidas pela microinjeção unilateral de ATP no NTS caudal de ratos não-anestesiados foram significativamente atenuadas após a microinjeção do N-PLA no mesmo sítio, sugerindo a participação do NO produzido pela nNOS na modulação de tais respostas. Neste contexto, os achados do presente trabalho sugerem que no NTS caudal: 1) o NO, provavelmente produzido pela NOS endotelial, exerce um importante papel modulatório nas vias neurais do barorreflexo; 2) a neurotransmissão do aumento da fR decorrente da ativação do quimiorreflexo envolve a formação de NO produzido pela nNOS; 3) a neurotransmissão das respostas cardiovasculares decorrentes da ativação do quimiorreflexo não envolve a formação de NO; 4) a neurotransmissão das respostas cardiovascualres e respiratórias decorrentes da microinjeção unilateral de ATP envolve a formação de NO produzido pela nNOS. / The nucleus tractus solitarius (NTS) is an integrative area in the central nervous system (CNS) involved with the ventilatory and autonomic control. Several studies suggest that nitric oxide (NO) in the NTS plays an important role in the modulation of the cardiovascular and ventilatory reflexes. In addition, there is evidence indicating a possible interaction of NO and ATP in the CNS. Considering these findings, in the present study, we evaluated the possible role of NO on the modulation of the basal cardiorespiratory parameters as well as on the processing of the cardiovascular and ventilatory responses elicited by chemoreflex activation in the caudal NTS of awake rats. In addition, the possible role of NO produced by neuronal nitric oxide sintase (nNOS) on the cardiovascular and respiratory responses produced by unilateral microinjection of ATP into the caudal NTS was also evaluated. For this purpose, rats received bilateral guide cannulae in direction of the caudal NTS and femoral artery and vein were cannulated. The ventilatory measurements were obtained by whole-body pletismograph method. Our data showed that bilateral microinjections of L-NAME, a non-selective NOS inhibitor, into the caudal NTS, produced a significant increase in basaline mean arterial pressure, suggesting a modulatory role of NO in the neural pathways of the baroreflex. However, bilateral microinjections of N-PLA, a selective nNOS inhibitor, into the caudal NTS, produced no significant changes in the baseline mean arterial pressure, suggesting that NO produced by nNOS is not involved in the basal autonomic control in the caudal NTS. With respect to chemoreflex responses, bilateral microinjections of L-NAME or NPLA, into the caudal, produced a significant attenuation in the increase in respiratory frequency (fR) produced by chemoreflex activation, suggesting that NO produced by nNOS is involved in the modulation of the respiratory component of the chemoreflex. However, the pressor and bradicardic responses elicited by chemoreflex actiovation Abstract xv were not affected by microinjections of L-NAME or N-PLA, suggesting that NO is not involved in the modulation of the cardiovascular responses. With respect to ATP microinjection responses, the data showed that unilateral microinjection of ATP into the caudal NTS produced increase in arterial pressure, fR and minute ventilation, which were significantly attenuated by N-PLA, suggesting that NO produced by nNOS is involved in the modulation of the cardiovascular and ventilatory responses to ATP microinjection into the caudal NTS. In conclusion, the data of present study indicate that in the caudal NTS: 1) NO, produced probably by endothelial NOS, plays an important modulatory role on the neural pathways of the baroreflex; 2) the neurotransmission of the increase in respiratory frequency to chemoreflex activation involve NO production by nNOS. 3) NO is not involved in modulation of the autonomic components of chemoreflex; 4) the cardiovascular and ventillatory responses produced by ATP micronjection are, at least in part, mediated by NO produced by nNOS.

A brainstem-related abnormality in respiratory control appears to be one of the most compelling mechanisms for sudden infant death syndrome (SIDS). The elements calcium, copper, iron, potassium, magnesium, sodium, phosphorus, sulfur, and zinc were analyzed by inductively coupled plasma atomic emission spectroscopy in the brainstem of 30 infants who died from SIDS and 10 infants who died from other causes (control). No differences were found between SIDS and control for any element except for more calcium in the SIDS group. A multivariate analysis of the data failed to group the majority of SIDS and control subjects in different clusters. Further research is required to determine the biological significance of the higher calcium found in the SIDS group.,

By exploiting the concept of emergent network properties and the hierarchical nature of networks, we have constructed several levels of models facilitating the investigations of issues in the area of respiratory neural control. The first of such models is an intracellular second messenger pathway model, which has been shown to be an important contributor to intracellular calcium metabolism and mediate responses to neuromodulators such as serotonin. At the next level, we have constructed new single neuron models of respiratory-related neurons (e.g. the pre-Btzinger complex neuron and the Hypoglossal motoneuron), where the electrical activities of the neurons are linked to intracellular mechanisms responsible for chemical homeostasis. Beyond the level of individual neurons, we have constructed models of neuron populations where the effects of different component neurons, varying strengths and types of inter-neuron couplings, as well as network topology are investigated. Our results from these simulation studies at different structural levels are in line with experiment observations. The small-world topology, as observed in previous anatomical studies, has been shown here to support rhythm generation along with a variety of other network-level phenomena. The interactions between different inter-neuron coupling types simultaneously manifesting at time-scales orders of magnitude apart suggest possible explanations for variations in the outputs measured from the XII rootlet in experiments. In addition, we have demonstrated the significance of pacemakers, along with the importance of considering neuromodulations and second-messenger pathways in an attempt to understand important physiological functions such as breathing activities.

Oxygen and nitric oxide reductases are enzymes found in aerobic and anaerobic respiration, respectively. Both enzyme groups belong to the superfamily of Heme-Copper Oxidases, which is further divided into several subgroups: oxygen-reducing enzymes into A-, B- and C-type and nitric oxide reductases into qNORs and cNORs. Oxygen reducing enzymes use the energy released from oxygen reduction to take up electrons and protons from different sides of the membrane. Additionally, protons are pumped. These processes produce a membrane potential, which is used by the ATP-synthase to produce ATP, the universal energy currency of the cell. Nitric oxide reductases are not known to conserve the energy from nitric oxide reduction, although the reaction is highly exergonic. Here, the detailed mechanism of a B-type oxidase is studied with special interest in an element involved in proton pumping (proton loading site, PLS). The study supports the hypothesis that the PLS is protonated in one and deprotonated in the consecutive step of the oxidative catalytic cycle, and that a proton is pumped during the final oxidation phase. It further strengthens the previous suggestion that the PLS is a cluster instead of a single residue or heme propionate. Additionally, it is proposed that the residue Asp372, which is in vicinity of the heme a3 propionates previously suggested as PLS, is part of this cluster. In another study, we show that the Glu15II at the entry of the proton pathway in the B-type oxidase is the only crucial residue for proton uptake, while Tyr248 is or is close to the internal proton donor responsible for coupling proton pumping to oxygen reduction. The thesis also includes studies on the mechanism and electrogenicity of qNOR. We show that there is a difference in the proton-uptake reaction between qNOR and the non-electrogenic homolog cNOR, hinting at a different reaction mechanism. Further, studies on a qNOR from a different host showed that qNOR is indeed electrogenic. This surprising result opens up new discussions on the evolution of oxygen and nitric oxide reductases, and about how energy conservation can be achieved. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>