Jeffrey C. Smith - US grants

This research program seeks to explain the neurogenesis of respiratory pattern in mammals in terms of the biophysical, synaptic, and network properties of neurons in the central nervous system (CNS). During the award period, research will be conducted to further develop and exploit a novel system for the study of these aspects of respiratory pattern generation. This system consists of the mammalian brainstem and spinal cord in vitro and has unique properties that facilitate experimental analysis of neural mechanisms. Unlike conventional experimental approaches in vivo, the in vitro system allows routine application of most relevant neurobiological techniques to study neural pattern generation, including direct utilization of pharmacological and membrane channel probes in the CNS, and intracellular recording to determine neuronal properties. Information to be obtained from these studies is fundamental for defining CNS mechanisms responsible for respiratory homeostasis and for understanding pathologies where ventilatory failure results from dysfunction of CNS mechanisms controlling breathing.

This proposal is to further develop and exploit a novel in vitro mammalian brainstem-spinal cord preparation to study neural mechanisms controlling breathing. The preparation consists of the brainstem and spinal cord isolated from neonatal rat and maintained under controlled in vitro conditions. This preparation retains major components of the central nervous system (CNS) respiratory pattern generating circuitry and spontaneously generates a respiratory motor output pattern of brainstem origin resembling the pattern generated in the neonate in vivo. Unlike the situation in vivo, the in vitro system allows routine application of most current neurobiological techniques to study mechanisms of neural pattern generation, including direct utilization of pharmacological and membrane channel probes in the CNS, and intracellular recording from different brainstem- spinal cord regions to determine neuron electrophysiological properties. Preliminary studies have shown the in vitro brainstem-spinal cord to be a versatile and potentially powerful experimental system for investigation of motor control systems in the mammalian CNS. The in vitro system will be used to investigate the following aspects of respiratory pattern generation: (1) Functional organization of the pattern generation system. Information will be obtained on how brainstem respiratory networks are organized for the generation of the basic components of the respiratory pattern including respiratory cycle timing (rhythm) and the detailed spatiotemporal patterns of motoneuronal activity. (2) Synaptic mechanisms in pattern generation, including the roles of excitatory and inhibitory synaptic interactions and neurotransmitter systems in rhythm generation and generation of brainstem neuronal discharge patterns. (3) Membrane biophysical properties of respiratory neurons. Intracellular recording will be used to examine electrophysiological behavior of identified brainstem respiratory neurons and to infer the nature of their synaptic inputs and the ionic basis of neuronal excitability. The long range goal of this work is to explain the neurogenesis of respiratory pattern in mammals in terms of the biophysical, synaptic and network properties of CNS respiratory neurons. Information to be obtained from this proposal is fundamental for defining CNS mechanisms responsible for respiratory homeostasis and for understanding pathologies where ventilatory failure results from dysfunction of neural mechanisms controlling breathing.

Research addressing the main specific aims of this project focused on cellular and circuit mechanisms generating the respiratory rhythm and neural activity patterns in the brainstem of rodents. Experimental studies were performed with isolated in situ perfused brainstem-spinal cord and in vitro brainstem slice preparations from neonatal and mature rats. Previously we have identified the brainstem locus (called the pre-Botzinger complex) containing populations of neurons participating in rhythm generation. We have further exploited methods for real-time structural and functional imaging of these neurons, as well as neurons in rhythm-transmission circuits, utilizing structural imaging performed simultaneously with functional activity imaging by multi-photon laser scanning microscopy of the neurons labeled with fluorescent calcium-sensitive dyes and/or fluorescent proteins. This imaging approach has facilitated identification of respiratory circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of expression of neuron channels, receptors, and neurotransmitter-related proteins. With these approaches, we have performed high-resolution spatiotemporal imaging of neuron activity and analyzed biophysical properties of respiratory neurons in the neonatal rodent pre-Botzinger complex and rhythm transmission circuits in vitro. These studies have provided the most direct experimental evidence to date that rhythm generation involves an excitatory network of neurons with specialized cellular properties that endow respiratory circuits with multiple mechanisms for producing respiratory oscillations. Studies of neuronal synaptic interactions and cellular membrane biophysical properties in the pre-Botzinger complex, including with intracellular recording techniques in situ and advanced electrophysiolgical approaches such as the dynamic clamp applied in vitro, continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of respiratory rhythm and pattern in the intact mammalian nervous system. Studies in progress based on intracellular recording approaches applied in situ are analyzing in detail how distinct populations of excitatory and inhibitory neurons interact to generate the respiratory rhythm and pattern as well as to test predictions of our network models. Other studies have provided additional evidence that neuronal persistent sodium currents and several types of leak or background conductances represent critical ionic conductance mechanisms for generation and control of respiratory oscillations. Molecular profiling with RT-PCR of messenger RNA expressed in single functionally identified neurons in vitro, as well as immunohistochemical studies, have identified a specialized set of transient receptor potential (TRP) cationic channels that may also represent important regulators of neuron excitability and current studies are directed toward understanding how these channels may contribute to electrophysiological behavior of respiratory circuit neurons. Other electrophysiological studies have demonstrated that leak conductance mechanisms are critically involved in the regulation of rhythmic breathing patterns by a diverse set of endogenous neurochemicals that modulate these conductances as well as by physiological control signals including carbon dioxide and oxygen. A particular focus of these latter studies was elucidating neuromodulatory control of respiratory circuit activity by neurons of the brainstem raphe nucleus that constitute the brainstem serotonin (5-HT) system, which is postulated to have a critical function in brain state-dependent control of breathing in vivo and is associated with pathophysiological disturbances of breathing such as thosse underlying sudden infant death syndrome (SIDS). Our continuing electrophysiological studies performed in vitro and in situ have established critical functional interactions between raphe and respiratory circuit neurons and have determined the essential modulatory actions of raphe 5-HT neurons in both the neonatal and mature mammalian nervous systems. Previously we have shown that raphe 5-HT neurons have slow pacemaking properties dependent in part on the kinetic properties of sodium and leak channels, and these pacemaking properties were demonstrated to be essential for continuous modulation of respiratory network excitability and respiratory rhythm generation. We have now established that the activity of 5-HT neurons is regulated by carbon dioxide/hydrogen ion for homeostatic regulation of respiratory circuit activity in vitro and in situ. We have also continued to analyze how the pharmacological properties of various types of 5-HT receptors on different populations of respiratory circuit neurons can be exploited to reverse opioid-induced depression of breathing with potential translational therapeutic applications. In previous studies employing novel pharmacogenetic approaches applied in situ and in vivo, neurons of the retrotrapezoid nucleus (RTN) that also have slow pacemaking and chemosensory properties were also shown to provide a critical excitatory modulatory input to core components of the respiratory network for generation and coordination of inspiratory and expiratory neural activity. Accordingly new models for the operation of brainstem respiratory circuits that incorporate multiple neuromodulatory input control mechanisms have been formulated to explain how specific brainstem circuit components are controlled and regulate patterns of respiratory oscillatory activity. We are currently employing optogenetic approaches for manipulation of activity of specific neuronal populations to further investigate how different populations of network neurons contribute to respiratory pattern generation in various (patho)physiological states.

Research involved the further development of novel neurodynamical models of neurons and networks comprising the respiratory neural control system as studied experimentally in parallel in the rodent brain. Data-based models developed included: (1) biophysically realistic cellular-level computational models of brainstem respiratory neurons incorporating current information on cellular architecture and biophysical properties such as ionic conductance mechanisms underlying neuronal activity;and (2) large-scale models of brainstem respiratory neural networks incorporating available information on network functional and structural architecture. The overall objective of these modeling studies was to gain mechanistic insights into the manner in which cellular- and circuit-level properties are integrated into microcircuits as well as large-scale respiratory networks for dynamical operation of the mammalian respiratory neural control system. A new model of respiratory central pattern generation (CPG) networks in the rodent brainstem was further developed consisting of interacting excitatory and inhibitory subnetworks distributed in serially arranged brainstem structural compartments, each with distinct functional roles in generation and control of the respiratory neural activity patterns that evolve during the normal breathing cycle of inspiration followed by expiration. The basic network architecture and cellular properties used in this CPG model were derived from electrophysiological and neuroanatomical reconstruction studies conducted in the rat brainstem-spinal cord in situ and on subnetworks isolated in living brainstem slice preparations in vitro with active circuits. These models also incorporated for the first time regulation of different circuit components by modeled afferent input signals, including rhythmically active inputs from critical neuromodulatory control systems that are known to be involved in regulation of respiratory pattern generation. For dynamical analysis of CPG network operation, methods from dynamical systems theory were also applied to identify critical dynamical variables and parameters of circuit operation that underlie respiratory rhythm and pattern generation and control the orderly transitions between the functionally distinct phases of inspiratory and expiratory neural activity. Computer simulations with the microcircuit and large-scale models mimicked many features of the single-cell and neuron population activity patterns found experimentally under different in vitro and in situ conditions. A major new hypothesis derived from experimental studies and further tested with these models was that the capability to generate oscillatory activity exists within the respiratory CPG at multiple levels of cellular and network organization, forming a dynamical system of coupled oscillatory mechanisms. Thus different mechanisms of respiratory rhythm generation can be functionally expressed in a brain state-dependent manner and underlie multiple respiratory motor behaviors, some of which occur under normal physiological conditions and others of which emerge under pathophysiological conductions such as during severe brain hypoxia (conditions of abnormally low oxygen). Simulations with models of different levels of cellular and network complexity further confirmed the plausibility of this new concept and have provided insights into the essential cellular and network mechanisms involved. We have also initated implementation of simulation approaches involving cluster computing on large distributed parallel processing systems including the NIH Biowulf cluster as well as desktop supercomputing systems utilizing graphics processing units (GPUs) that allow real-time simulation of large-scale network models. At the system level, models of the respiratory neural control system have been further developed that couple essential neural circuit dynamics with peripheral oxygen and carbon dioxide exchange, blood gas transport, and physiological feedback regulation off central respiratory circuits by signals such as blood/brain levels of oxygen and carbon dioxide. These latter models represent the first generation of system-level control models that integrate essential elements of nervous system structural-functional properties and realistic features of the respiratory gas exchange and transport system. All of these models are currently being applied to further explore principles of operation of brainstem respiratory circuits and control of respiratory activity including under various (patho)physiological conditions associated with disturbances of brain and body oxygen/carbon dioxide homeostasis.