Insight Into Autonomic Dysfunctions With Novel Interventions: Focusing On Vascular Tone And Breathing Regulations

The autonomic nervous system (ANS) controls most involuntary functions of the body. Dysfunctions of the ANS can be life-threatening. However, several critical questions related to cardiovascular and breathing regulations remain unclear. One of the open questions is how the system lose control of the vascular tones under certain circumstances. Using the septic shock model induced by lipopolysaccharide (LPS) in isolated and perfused mesenteric arterial rings, we found the vascular hyporeactivity is attributed to the decreased vasoconstriction to α-adrenoceptor agonists. The endotoxin-induced vasodilation can be intervened with endothelin-1 (ET-1), serotonin (5-HT) or vasopressin, which have never been used in clinical treatment. It is unclear how the excitability of endothelium affects vascular tones. Using optogenetics and transgenic mice with channelrhodopsin expression in endothelial cells (ECs), we found selective activation of the ECs induces a fast, robust, reproducible and long-lasting vasoconstriction in isolated and perfused hearts and kidneys. Breathing control by the ANS within the brain becomes abnormal in certain genetic diseases, such as Rett syndrome with defected norepinephrine (NE) system in locus coeruleus (LC). The LC neurons are hyperexcitable while NE release is deficient. Using optogenetics and double transgenic mice with Mecp2 null and channelrhodopsin expression in LC neurons, we found the NE-ergic modulation of hypoglossal neurons was impaired in transgenic mice, which cannot be improved with optostimulation, suggesting that LC neuronal hyperexcitability may not benefit the NE modulation in Rett syndrome. Collectively, our results provide insight into the autonomic dysfunctions using experimental interventions that have barely been used before

Serotonergic And Hypocretinergic Systems Modulate Ventilation And Hypercapnic Ventilatory Responses

Thesis (Ph.D.) University of Alaska Fairbanks, 2009Serotonergic (5-HT) cells of the medullary raphe are putative central chemoreceptors, one of multiple chemoreceptive sites in the brainstem that interact to produce the respiratory chemoreflex. This role is debated, and the importance of 5-HT neurons as chemoreceptors in relatively intact systems is unclear. The main focus of this dissertation is to provide further physiological evidence for the involvement and modulation of 5-HT neurons in CO2 chemosensitivity. This is of interest as a large number of Sudden Infant Death Syndrome (SIDS) cases report dysfunction in the 5-HT system, and CO2 may be an exogenous stressor leading to SIDS when in combination with this underlying vulnerability. Also, since SIDS occurs primarily during sleep, I also focus on the potential functional interaction between the 5-HT and hypocretinergic systems, as hypocretins play a role in arousal and also potentially in chemosensitivity. I confirm the hypothesis that the serotonergic and hypocretinergic systems modulate ventilation and hypercapnic ventilatory responses. Using the in situ preparation derived from juvenile rats and the in vitro medullary slice preparation from mice, I verify that 5-HT neurons are critical in generating a response to CO2, primarily via facilitation of the respiratory rhythm through 5-HT2 receptors. I also find evidence to support the hypothesis that hypocretins play a significant role in the neuroventilatory response to CO2 through activation of hypocretin receptors type 1. By comparing results from rhythmic medullary slice preparations from wildtype (normal 5-HT function) and Lmx1bf/f/p (lack central 5-HT neurons) neonatal mice, I attempt to identify whether changes in hypoglossal nerve output in response to acidosis are affected by hypocretin receptors, and whether this is dependent on the presence of 5-HT neurons. Frequency results from such studies are inconclusive; however, hypocretins do appear to mediate the burst duration response via serotonergic mechanisms. I also find that hypocretins facilitate baseline neural ventilatory output in part through 5-HT neurons. Thus, both the 5-HT and hypocretinergic systems are involved in modulating ventilation and hypercapnic ventilatory responses

Afferents integration and neural adaptive control of breathing

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references.The respiratory regulatory system is one of the most extensively studied homeostatic systems in the body. Despite its deceptively mundane physiological function, the mechanism underlying the robust control of the motor act of breathing in the face of constantly changing internal and external challenges throughout one's life is still poorly understood. Traditionally, control of breathing has been studied with a highly reductionist approach, with specific stimulus-response relationships being taken to reflect distinct feedback/feedforward control laws. It is assumed that the overall respiratory response could be described as the linear sum of all unitary stimulus-response relationships under a Sherringtonian framework. Such a divide-and-conquer approach has proven useful in predicting the independent effects of specific chemical and mechanical inputs. However, it has limited predictive power for the respiratory response in realistic disease states when multiple factors come into play. Instead, vast amounts of evidence have revealed the existence of complex interactions of various afferent-efferent signals in defining the overall respiratory response. This thesis aims to explore the nonlinear interaction of afferents in respiratory control. In a series of computational simulations, it was shown that the respiratory response in humans during muscular exercise under a variety of pulmonary gas exchange defects is consistent with an optimal interaction of mechanical and chemical afferents. This provides a new understanding on the impacts of pulmonary gas exchange on the adaptive control of the exercise respiratory response. Furthermore, from a series of in-vivo neurophysiology experiments in rats, it was discovered that certain respiratory neurons in the dorsolateral pons in the rat brainstem integrate central and peripheral chemoreceptor afferent signals in a hypoadditive manner. Such nonlinear interaction evidences classical (Pavlovian) conditioning of chemoreceptor inputs that modulate the respiratory rhythm and motor output. These findings demonstrate a powerful gain modulation function for control of breathing by the lower brain. The computational and experimental studies in this thesis reveal a form of associative learning important for adaptive control of respiratory regulation, at both behavioral and neuronal levels. Our results shed new light for future experimental and theoretical elucidation of the mechanism of respiratory control from an integrative modeling perspective.by Chung Tin.Ph.D

Multi-Scale Modeling of the Neural Control of Respiration

The generation of respiration in mammals begins in the lower brainstem where groups of neurons, that together comprise the respiratory central pattern generator (CPG), interact to produce a motor output that controls breathing. The pre-Bötzinger complex (pre-BötC) in the ventrolateral respiratory column (VRC) is believed to be a major contributor to rhythmic inspiratory activity that interacts with other neural compartments within the VRC as well as with other brainstem areas, including the pons. Though there has been a substantial push to understand the exact cellular and network mechanisms operating within the pre-BötC, as well as the way it is incorporated into the larger respiratory network, there is still much to be resolved. The overarching goal of the work presented in this dissertation is to contribute to our understanding of the neural control of respiration at several hierarchical levels. It is my hope that better insight into the complexities of these multiscale neural control mechanisms will provide a more complete framework for understanding various respiratory pathologies, and ultimately guide the development of novel therapies that will improve patient outcomes. I applied techniques from the fields of mathematics and computer science to develop computational models that reproduced results from electrophysiological recordings (done by our collaborators) and generated verifiable predictions. The scale of my modeling work encompasses the interaction of neurons in a single population, several interconnected populations of neurons that encompass the core of the mammalian respiratory network, and an integration of the respiratory network into a larger control system that includes afferent feedback loops. At each level I address specific, but related, topics that add to the general understanding of the neural control of respiration. The aims of my thesis address specific issues at each of the scales mentioned above. These issues may be summarized as follows: (i) the characteristic rhythmic bursting behavior observed in the pre-BötC, which was studied at the cellular levels with a particular interest in how this behavior impacts respiratory rhythmogenesis; (ii) a respiratory network connectome that defines interactions between several populations of neurons that together form the VRC, which produces an alternating pattern of inspiration, post-inspiration and expiration, and, how such a pattern may be affected by changes in chemical environment, e.g. elevated carbon dioxide or diminished oxygen concentrations; and (iii) the role of afferent feedback to the VRC, from the pons and lungs, which was studied in the context of respiratory phase switching mechanisms.Ph.D., Biomedical Engineering -- Drexel University, 201

Towards an understanding of the role of Connexin26 in breathing

Connexin26 (Cx26) hemichannels expressed in glia at the ventral medullary surface (VMS) have been proposed to play a role in respiratory chemoreception, although this is disputed. At the VMS Cx26 hemichannels open in response to CO2 directly, causing ATP release that is capable of increasing respiratory drive. The main aim of this work was to establish a genetic strategy that can be used in vivo to elegantly remove Cx26 CO2- sensitivity from chemosensitive areas of the VMS, and to thereby investigate the role that Cx26 CO2 sensitivity plays in the chemoreception of awake mice. Using Forster resonance energy transfer and dye loading studies a Cx26 mutant (Cx26DN) was found to coassemble with Cx26WT subunits (forming heteromeric connexon hemichannels) and to remove CO2-induced hemichannel opening from cells stably expressing Cx26WT. In mice aged 12-18 weeks, bilateral lentivirus injections were used to express Cx26DN in GFAP+ cells at the VMS, as a means of removing CO2-induced Cx26 hemichannel opening and subsequent ATP release. As determined by whole-body plethysmography, expression of Cx26DN in the retrotrapezoid nucleus (RTN) had no effect on the hypercapnic ventilatory response in mice. Accidental Cx26DN expression in the caudal chemosensitive area resulted in reduced tidal volume 3 weeks post-transduction, however this was not well supported statistically. Such an auspicious result from suboptimal caudal expression warrants this to be repeated in order to validate these results. The work performed in this thesis outlines the first use of a highly novel genetic tool to remove the CO2-sensitivity property of Cx26 from specific cells, without removing the protein from the system. The results shed light on our understanding of central respiratory chemoreception, suggesting that Cx26 plays no role in chemoreception in the RTN but is likely to play a role in caudal areas of the VMS. Such a tool could aid research into the virtually unexplored role that Cx26 CO2 gating has in the body

The breathing brainstem : development of inspiration

Breathing is essential for life, and yet we do not fully understand the mechanisms that control it. The main central pattern generators for respiration include the inspiratory generating region called the preBötzinger Complex (preBötC), and the chemosensitive region called the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN). These are located in the ventrolateral medulla oblongata of the brainstem. To study these centers in detail, organotypic cultures of the respiratory brainstem were developed to keep the structural integrity of the neural tissue essentially intact while still allowing careful control of the microenvironment. The cultures generated synchronized respiratory network activity and motor output for up to three weeks. Characterization revealed a network organization of the respiratory regions with a so called small-world structure. These respiratory networks consists of both neurons and astrocytes. Detailed examination identified two subgroups of astrocytes. Most appeared dormant, but a subset of the astrocytes displayed persistent, rhythmic oscillating calcium activity. These active astrocytes formed an individual network, interacting with a distinct neuronal network. Furthermore, stimulation of the astrocytes increased their calcium oscillation frequency in both the preBötC and pFRG/RTN. However, while neuronal calcium oscillations in the preBötC were unaffected, they were significantly increased in frequency in the pFRG/RTN. The organotypic culture also preserved the respiratory center reactivity towards exogenous stimulus, such as opioids and hypercapnia. Hypercapnic challenge elicited a gapjunction dependent release of the inflammatory molecule prostaglandin E2 (PGE2) in the pFRG/RTN, increasing the overall network activity of this region. A release of PGE2 was also triggered by stimulation of astrocytes, which blunted a subsequent hypercapnic challenge. Thus, a new respiratory signaling pathway where PGE2 release from astrocytes following hypercapnic challenge modifies respiratory behavior to meet physiological demands was identified. In a clinical setting this might be beneficial at birth, where high PGE2 levels set the respiratory system to perform deep breaths. However, increased levels of PGE2 would also blunt the hypercapnic response, inducing a vulnerable period for infants. The first week after birth, cardiorespiratory dysfunction may lead to sudden unexpected postnatal collapse (SUPC), where seemingly healthy infants collapse and require resuscitation. In a retrospective study, we identified 115 SUPC cases among 313 351 live births during a 15.5-year period. Thus, the incidence of 36.7 SUPC events per 100 000 live births makes SUPC events more common than sepsis caused by group B streptococci. Seven percent of the affected children died, and about one fourth developed hypoxic ischemic encephalopathy. The majority of SUPCs occurred during the first hours after birth and where related to co-bedding, emphasizing the importance of these risk factors. Urinary PGE2 metabolite levels were high during the first days after birth when most (97 %) of SUPC events occur. These findings are important for understanding how respiratory behavior is affected during inflammatory states, such as immediately after birth and during infections, and have an impact on our ability to detect and protect against respiratory dysfunction

The brain beating and heart breathing: a unifying theory of the neuro- cardiac- respiratory control in infant and adult sudden unexpected deaths

Background: Sudden Infant Death Syndrome (SIDS) is characterized by the death of an infant that cannot be explained, despite a systematic case examination, including death scene investigation, autopsy and review of the clinical history. Nowadays, Sudden Unexpected Infant Death (SUID) is a wide-ranging concept used to describe any sudden and unexpected death, whether explained or unexplained, including SIDS, which occurs during the first year of life. Several differing and sometimes contradictory hypotheses of the underlying mechanisms of SIDS have been proposed. The most reliable seems to be the “triple risk hypothesis”. Based on this theory, unexpected infant deaths might arise as a consequence of the combination of three factors coming together: a vulnerable infant, a vulnerable phase of development and a final insult occurring in this window of vulnerability. Recently, a unified neuropathological theory contributes to describing SIDS. According to this, serotonergic neurons play a crucial homeostatic function in the cardiorespiratory brainstem centres. A high incidence of morphological abnormalities and biochemical defects of serotoninergic neurotransmission have been reported in the brainstem of SIDS victims. This brain region includes the main nuclei and structures that coordinate the vital activities, such as cardiovascular function and breathing, perinatal and after birth. Nevertheless, evidence suggests likely genomic complexity and a degree of overlap among SIDS, Sudden Intrauterine Death (SIUD), Sudden Cardiac Death (SCD) and Sudden Unexpected Death in Epilepsy (SUDEP). In SUDEP, which has clinical parallels with SIDS, alterations to medullary serotoninergic neural populations and autonomic dysregulation have been shown too. Molecular profiling of SUDEP cases and the investigation of genetic models have directed to the identification of putative SUDEP genes of which most are ion channel active along the neurocardiac, neuroautonomic, and neurorespiratory pathway. Concurrently, anomalous time- activation, transcription or regional expression of candidate neuro-cardiac-respiratory genes implicated for SUDEP, could be similarly involved in other unexpected sudden deaths. A small but significant proportion of infants who die suddenly and unexpectedly have been shown on postmortem genetic testing to have Developmental Serotonopathies, Cardiac Channelopathies and Autonomic Nervous System Dysregulation, with considerable implications for surviving and future family members. This has led to the demonstration that neuro-cardiac genes are expressed in both tissues (brain and heart) and recently in the respiratory system. Aim: Despite their decreasing incidence, SIDS and SUDEP are still important causes of death. There are many nuclei in the cardio and respiratory centres of the brain involved in unexpected and sudden deaths. Cardiac, sympathetic, and respiratory motor activities can be viewed as a unified rhythm controlled by brainstem neural circuits for effective and efficient gas exchange. We aim to describe abnormalities in these nuclei, in part because robust molecular or functional examination of these nuclei has not been carefully performed. We intend to perform detailed functional mapping of these brainstem nuclei. Specifically, the cardiorespiratory and cardioventilatory coupling can be understood as a unified vital rhythm controlled by brainstem neural circuits. By cardiorespiratory coupling, we mean the Respiratory Sinus Arrhythmia (RSA) that is characterized by a heart rate (HR) increasing during inspiration and an HR decrease during expiration. Conversely, Cardioventilatory coupling (CVC) is considered the influence of heartbeats and arterial pulse pressure on respiration with the tendency for the next inspiration to start at a preferred latency after the last heartbeat in expiration. We hypothesized that these two reflex systems are not separate, but constitute an integrated network. We defined this last concept as "unifying theory". By studying all the maps of the cardiorespiratory nuclei of the Literature, we integrated this concept into a reworking map of brainstem nuclei that could also explain the gasping and blocking cardiorespiratory of sudden deaths. The theory of a unique, unifying cardiorespiratory network, it has been recently demonstrated in some cases of arrhythmia, in some cases of SUDEP with striking systolic hypotensive changes and in some cases of SIDS too. Material and Methods: We investigated articles, reviews indexed in PubMed describing putative neuro-cardiac-respiratory genes and cardiorespiratory, and cardioventilatory coupling theories. Specifically, we evaluated cardiorespiratory brainstem nuclei and whole brains of fetal, infant and adult autopsies respectively to detect congenital errors in the cerebral development or malformations, but also to identify the “normal” or “dysplastic” brainstem centres. Results: Based on the Literature, we identified a brain-heart gene mapping and a scheme of cardiorespiratory brainstem nuclei network. Contemporary, we collected a large pool of fetal brain malformations and cardiorespiratory nuclei dysgenesis both in infants both in adult sudden deaths. We found dysgenesia, agenesia and hypoplasia of brainstem nuclei associated with SIDS cases, compared with post-mortem infant control cases. However, the arcuate nucleus showed insignificant inter-variations regarding adults autoptic cases. Discussion: Many intrinsic and extrinsic factors increase fetal, perinatal, infant, and adult sudden death susceptibility. The final common pathway for SIDS and SUDEP involves a failure to arouse and autoresuscitate in response to environmental challenge. The different risk factors, among these a prone position, can directly alter the function of cardiorespiratory nuclei and impair the ability of this network to coordinate cardiorespiratory–cardioventilatory coupling. Conclusions: Neuropathological analysis of the infant brainstem and neuro-cardiac-respiratory gene mapping represents a good tool to infer on the final events of SIDS and SUDEP, although nothing it is clear regarding the role of adult cardiorespiratory centres. An integrated study of postmortem neuropathology and molecular autopsies could help to understand the network of this beating-breathing-thinking unit

ALTERATIONS IN GABAERGIC NTS NEURON FUNCTION IN ASSOCIATION WITH TLE AND SUDEP

Epilepsy is a neurological disorder that is characterized by aberrant electrical activity in the brain resulting in at least two unprovoked seizures over a period longer than 24 hours. Approximately 60% of individuals with epilepsy are diagnosed with temporal lobe epilepsy (TLE) and about one third of those individuals do not respond well to anti-seizure medications. This places those individuals at high risk for sudden unexpected death in epilepsy (SUDEP). SUDEP is defined as when an individual with epilepsy, who is otherwise healthy, dies suddenly and unexpectedly for unknown reasons. SUDEP is one of the leading causes of death in individuals with acquired epilepsies (i.e. not due to genetic mutations), such as TLE. Previous studies utilizing genetic models of epilepsy have suggested that circuitry within the vagal complex of the brainstem may play a role in SUDEP risk. Gamma-aminobutyric acid (GABA) neurons of the nucleus tractus solitarius (NTS) within the vagal complex receive, filter, and modulate cardiorespiratory information from the vagus nerve. GABAergic NTS neurons then project to cardiac vagal motor neurons, eventually effecting parasympathetic output to the periphery. In this study, a mouse model of TLE was used to assess the effect of epileptogenesis on GABAergic NTS neuron function and determine if functional alterations in these neurons impact SUDEP risk. It was discovered that mice with TLE (i.e. TLE mice) have significantly increased mortality rates compared to control animals, suggesting that SUDEP occurs in this model. Using whole cell electrophysiology synaptic and intrinsic properties of GABAergic NTS neurons were investigated in TLE and control mice. Results suggest that during epileptogenesis, GABAergic NTS neurons become hyperexcitable, potentially due to a reduction in A-type potassium channel current and increased excitatory synaptic input. Increases in hyperexcitability have been shown to be associated with an increased risk of spreading depolarization and action potential inactivation leading to neuronal quiescence. This may lead to a decreased inhibition of parasympathetic tone, causing cardiorespiratory collapse and SUDEP in TLE