Irene C. Solomon - US grants

DESCRIPTION (Applicant's abstract): Severe brain hypoxia results in respiratory and sympathetic excitation. Respiratory excitation takes the form of gasping which is characterized by an abrupt onset, short duration, high amplitude burst of activity, associated exclusively with inspiratory discharge. Survival during hypoxia exposures appears to be critically dependent upon this integrated cardiorespiratory reflex which has been referred to as "autoresuscitation", and is associated with rapid reoxygenation of arterial blood and restoration of blood pressure. Failure to gasp has been proposed as a potential cause of sudden infant death syndrome. The principle hypothesis of this proposal is that the putative respiratory pacemaker is located in the pre-Botzinger complex (the proposed locus of respiratory rhythm generation; pre-BotC), is hypoxia chemosensitive, and when released from strong GABAergic inhibition, exhibits chemosensitivity to systemic hypoxia over the range associated with chemoreception of the carotid bodies. Additionally, we propose that both disinhibition of GABA" ?receptors and direct hypoxic excitation of neurons (i.e., hypoxic chemosensitivity) located in the pre-BotC play complimentary roles in the genesis of hypoxia related gasping. The goal of the experiments proposed in this application is to examine the roles of direct hypoxic excitation of pre-BotC neurons, GABAergic disinhibition of pre-BotC neurons, and ionotropic excitatory amino acid (EAA) receptor activation of pre-BotC neurons as potential mechanisms for the respiratory excitation seen during gasping in response to severe brain hypoxia. Microinjection of neurotransmitter agonists and antagonists in conjunction with whole nerve and medullary single unit extracellular recordings will be used. Experiments will be conducted in both decerebrate and chloralose-anesthetized, vagotomized, deafferented, paralyzed, and ventilated cats. The specific aims are: (1) test whether pre-inspiratory (I-driver) neurons located in the pre-BotC are activated by focal hypoxia, and whether focal hypoxia phase shifts and synchronizes other respiratory-modulated subtypes or respiratory neurons located in the pre-BotC to a gasp-synchronous discharge, (2) test whether pre-inspiratory (I-driver) neurons located in the pre-BotC are activated during severe systemic hypoxia phase shifts and synchronizes other inspiratory-modulated subtypes of respiratory neurons located in the pre-BotC to a gasp-synchronous discharge, (3) test whether GABA" -mediated disinhibition neurons located in the pre-BotC plays a facilitatory role in the production of respiratory excitation seen during hypoxia, and (4) test whether ionotropic EAA receptor activation of neurons located in the pre-BotC plays a modulatory role in the respiratory excitation seen during hypoxia-induced gasping.

DESCRIPTION (provided by applicant): Recent evidence indicates that gap junctions play a more prominent role in normal functioning of the mammalian central nervous system (CNS) than was once believed. Accumulating evidence from both neonatal and adult rodents indicates that gap junctions participate in multiple aspects of respiratory control, including central CO2 chemoreception. Central CO2 chemoreceptors have been demonstrated to be distributed at several sites in the mammalian brainstem, and respiratory neurophysiologists have gained tremendous insight as to how presumptive central CO2 chemoreceptor neurons work by studying the electrophysiological and anatomical properties of cells in in vitro preparations of the mammalian CNS. One feature that has been identified in CO2-chemosensitive neurons is cell-to-cell coupling which occurs via gap junctions. The presence of gap junctions between adjoining CO2-chemosensitive neurons and the demonstration of neuronal expression of the gap junction proteins (connexin; Cx) Cx26 and Cx32 in CO2-chemosensitive brainstem regions suggest that either electrical coupling and/or metabolic coupling is/are involved in respiratory control. The principle hypothesis of this proposal is that gap junctional communication plays an important role in mediating and maintaining the ventilatory response to elevated levels of CO2 (i.e., hypercapnia). The experiments proposed in this application will use both in vitro and in vivo models along with biochemical and immunohistochemical procedures to further define the role of gap junctions in CO2 chemoreception. The specific aims of the project are: (1) investigate the effects of pharmacological blockade (i.e., uncoupling) of brainstem gap junctions on CO2-chemoreception, (2) investigate the effects of genetic manipulation (deletion) of the neuronal gap junction proteins Cx32 and Cx 36 on CO2-chemoreception in vivo, (3) identify the repertoire of neuronal and glial gap junction proteins, including regional and postnatal developmental expression, in putative CO2-chemosensitive brainstem regions, (4) investigate the effects of hypercapnia on modulation of gap junction protein expression in putative CO2-chemosensitivie regions and (5) investigate the effects of a prior hypercapnia conditioning exposure on CO2 chemoreception.

DESCRIPTION (provided by applicant): Fast oscillatory neuronal activity is a prominent feature observed in many areas of the central nervous system (CNS), including CNS regions associated with respiratory motor control. In the respiratory neural control system, it has long been recognized that fast oscillatory rhythms are present in inspiratory-related muscles, nerves, and neurons, and that these fast rhythmic oscillations provide an index of short-time scale synchronization within the inspiratory burst. The neuronal mechanisms that underlie fast oscillations in the CNS are poorly understood. It has been hypothesized, however, that interneuronal gap junctions and fast inhibitory synaptic mechanisms play a critical role in the generation of fast network oscillations. Recent observations further indicate that gap junctions, composed of the neuron specific gap junction protein connexin36 (Cx36), among GABAergic inhibitory neurons may participate in the generation of fast oscillations in some areas of the CNS. The experiments proposed in this application will use biochemical and immunohistochemical analyses in conjunction with in vivo and in vitro experiments in wild type and Cx36- deficient mice to further define the roles of brainstem gap junctions (including gap junctions composed of Cx36) and GABAA-mediated synaptic inhibition in the generation of fast oscillations in the inspiratory neural control system. The specific aims of this research proposal are: (1) investigate the effects of genetic manipulation (deletion) of the neuronal gap junction protein Cx36 on fast oscillatory rhythms in inspiratory motor discharges, (2) investigate the effects of pharmacological blockade of GABAA receptors on fast oscillatory rhythms in inspiratory motor discharges in Cx36-deficient mice, (3) investigate the effects of pharmacological blockade of gap junctions and GABAA receptors in the dorsal respiratory group (DRG), rostral ventral respiratory group (rVRG), and phrenic motor nucleus (PMN) on fast oscillatory rhythms in inspiratory discharges, (4) identify the repertoire of gap junction proteins in GABAergic neurons in respiratory-related medullary regions, and (5) investigate whether the expression levels of other neuronal gap junction proteins in respiratory-related brainstem regions are altered in Cx36-deficient mice.

Project Summary Spinal cord injury (SCI) damages axonal connections between the spinal cord and brain resulting in reduction or loss of motor, sensory, and autonomic function below the level of the injury. While most SCIs are incomplete, spontaneous functional recovery resulting from neural plasticity occurs albeit this recovery is rarely complete. Thus, efforts aimed at enhancing the extent of functional recovery by augmenting neural plasticity following SCI are essential, and a variety of therapies focusing on this goal have been implemented. Amongst the therapies investigated, exposure to acute intermittent hypoxia (AIH) has been shown to elicit functional improvements in both respiratory and non-respiratory motor spinal systems following incomplete SCI in rodents and humans, with repetitive or daily AIH (dAIH) producing persistent functional improvements that may last one week or more. These observations suggest that respiratory and somatic motor systems are affected by AIH in similar ways. The effects of AIH on autonomic dysfunction, including lower urinary tract (LUT) dysfunction, following SCI are less well understood or altogether unknown. Moreover, the impact of AIH on recovery of respiratory function, including expiratory motor output, following mid-thoracic SCI have not been examined. This R21 application proposes an exploratory/developmental project that is designed to investigate the therapeutic potential of acute intermittent hypoxia (AIH) as a tool for improving lower urinary tract (LUT) and respiratory dysfunction following mid-thoracic SCI. In young adult female rats, LUT and respiratory motor function will be measured before and after SCI produced by mid-thoracic moderate contusion or complete spinal cord transection. In Specific Aim 1, at 1-week (immediate) versus 4-week (delayed) following SCI, LUT and respiratory-related motor activities will be measured and quantified in response to and for 2-hours following AIH or sham gas treatment. In Specific Aim 2, beginning four weeks after SCI, rats will receive dAIH or dSHAM gas treatment for one week and then monitored at 1-day versus 1-week post gas treatment; an additional terminal acute AIH gas exposure experiment will also be conducted at this final time point. Functional outcome measures will be acquired at regular time points before SCI, during the 4-week recovery period following SCI, and at specific time points post-AIH/SHAM gas treatment, and will include residual urine volume (manual bladder expression); frequency and volume of spontaneous voids (metabolic chamber); bladder pressure threshold, frequency of subthreshold bladder contractions, duration and frequency of external urethral sphincter (EUS) bursting, detrusor-sphincter dyssynergia (cystometry with EUS EMG recording); and rate and depth of breathing (plethysmography and respiratory-related motor activities). It is anticipated that AIH and dAIH treatments will lead to improved LUT function (e.g., reduced detrusor-sphincter dyssynergia and residual urine volume) and enhanced respiratory motor output following SCI. If successful, this approach could be an effective non-invasive intervention to improve bladder control and respiratory dysfunction in individuals with spinal cord injury.

Project Summary Parkinson's Disease (PD) is the second most common neurodegenerative disease, affecting as many as 1 million Americans and ~7-10 million people worldwide. While PD is characterized by motor symptoms of resting tremor, rigidity, bradykinesia, and postural instability, a number of non-motor symptoms (NMS) are also observed. Amongst the NMS, breathing abnormalities have been noted in PD patients since the disease was first described in 1817, and respiratory complications remain the leading cause of death in PD. Respiratory dysfunction in PD manifests as a variety of altered breathing patterns as well as deficits in chemical control of breathing, which may further contribute to PD-related breathing abnormalities. Experimental (animal) models of PD have been developed and implemented in order to gain insight into causes and symptoms of PD as well as the impact of potential therapeutic interventions on ameliorating PD symptoms. One of the common and extensively used approaches involves administration of the neurotoxin 6-hydroxydopamine (6-OHDA) to selectively destroy dopaminergic (DA) neurons when injected directly into the nigrostriatal pathway, specifically into the substantia nigra pars compacta (SNpc), medial forebrain bundle (MFB), or caudate- putamen (CPu). While each site-targeted model has advantages and disadvantages, each seems to capture some, but not all, aspects of the clinical motor and non-motor PD phenotype albeit little is known about the respiratory phenotype in any of these models. Thus, there is a need to develop a better understanding of respiratory abnormalities in these experimental models of PD in order (1) to provide insights into the possible pathological mechanisms responsible for producing and exacerbating respiratory dysfunction in PD and (2) to aid in the development of new and effective therapeutic approaches to ameliorate this common NMS. This R21 application proposes an exploratory/developmental project designed to characterize and quantify the respiratory phenotype in these three commonly used 6-OHDA-induced PD rat models. In specific aim 1, we will evaluate the extent and progression (time course) of respiratory dysfunction in each PD model using serial plethysmography to measure basal ventilatory activity and the responses to acute changes in chemical respiratory drive in conscious spontaneously breathing adult rats. In specific aim 2, we will evaluate the contributions of neural respiratory-related upper airway (genioglossus), pump muscle (diaphragm), and expiratory (external oblique) motor dysfunction in each PD model using acute/terminal electrophysiology experiments to measure basal respiratory-related neural (EMG/ENG) activities and the responses to acute changes in chemical respiratory drive in anesthetized spontaneously breathing adult rats. Thus, this R21 application will (1) identify whether each of the 6-OHDA-lesioned PD rat models is a viable model for studying respiratory abnormalities in PD, (2) provide insight into prospective mechanisms underlying respiratory dysfunction, and (3) provide preliminary data that could serve as the basis of a future R01 application.