Neural Mechanisms Controlling Breathing In Mammals

National Institute Of Neurological Disorders And Stroke

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Abstract

Research 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 developed novel methods for real-time structural and functional imaging of these neurons, as well as neurons in rhythm-transmission circuits, utilizing infrared and differential interference contrast (IR-DIC) imaging performed simultaneously with fluorescence imaging of activity patterns of the neurons labeled with calcium-sensitive dyes. This imaging approach has facilitated identification of respiratory circuit neurons for electrophysiological studies of biophysical and synaptic properties as well as molecular studies of neuron channel and receptor expression. With these approaches, we have imaged the activity and analyzed biophysical properties of respiratory neurons in the neonatal rodent pre-Botzinger complex and rhythm transmission circuits in vitro, providing the most direct experimental evidence to date that rhythm generation involves a network of neurons with specialized cellular properties that endow respiratory circuits with multiple mechanisms for producing respiratory oscillations. Methods for multi-photon imaging that will allow three-dimensional reconstruction of this network in the pre-Botzinger complex are currently under development. Studies of cellular membrane biophysical properties have provided additional evidence that persistent sodium and potassium leak 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, as well as immunohistochemical studies, show a profile of sodium, potassium, and neurotransmitter receptor-linked channels consistent with an important role of persistent sodium and leak conductances. Electrophysiological studies have also demonstrated that these conductance mechanisms are critically involved in the regulation of the breathing rhythm/pattern by a diverse set of neurochemicals that modulate these conductances, including serotonin and substance P, as well as physiological control signals including carbon dioxide and oxygen. Electrophysiological studies performed with more intact preparations of the brainstem-spinal cord in situ have confirmed the importance of these cellular and circuit mechanisms in both the neonatal and mature mammalian nervous systems. These results continue to support our hybrid pacemaker-network model that was formulated from previous work to explain the generation and control of respiratoy rhythm and pattern in the intact mammalian nervous system. Novel computational approaches including large-scale modeling of brainstem neural networks have been used in parallel to experimental studies. Our biophysically realistic computational models of respiratory neurons have been further developed and novel investigations were conducted on the dynamic behavior of synaptically coupled populations of these network cells. Computer simulations with these models mimicked many features of the single-cell and neuron population activity found experimentally in vitro and in situ, including instabilities of the rhythm produced by nonlinear dynamic phenomena such as quasiperiodicity arising in networks. Models of the respiratory neural control system that couple neural circuit dynamics with peripheral oxygen and carbon dioxide exchange, blood gas transport, and physiological feedback regulation of central respiratory circuits have also been developed. These models are currently being applied to further explore principles of operation of brainstem respiratory circuits at different stages of nervous system development and under various (patho)physiological conditions.