Functional Imaging, Spatial Reconstruction, and Biophysical Analysis of a Respiratory Motor Circuit Isolated In Vitro

We combined real-time calcium-based neural activity imaging with whole-cell patch-clamp recording techniques to map the spatial organization and analyze electrophysiological properties of respiratory neurons forming the circuit transmitting rhythmic drive from the pre-Bötzinger complex (pre-BötC) through premotoneurons to hypoglossal (XII) motoneurons. Inspiratory pre-BötC neurons, XII premotoneurons (preMNs), and XII motoneurons (MNs) were retrogradely labeled with Ca2+-sensitive dye in neonatal rat in vitro brainstem slices. PreMN cell bodies were arrayed dorsomedially to pre-BötC neurons with little spatial overlap; axonal projections to MNs were ipsilateral. Inspiratory MNs were distributed in dorsal and ventral subnuclei of XII. Voltage-clamp recordings revealed that two currents, persistent sodium current (NaP) and K+-dominated leak current (Leak), primarily contribute to preMN/MN subthreshold current–voltage relationships. NaP or Leak conductance densities in preMNs and MNs were not significantly different. We quantified preMN and MN action potential time course and spike frequency–current (f–I) relationships and found no significant differences in repetitive spiking dynamics, steady-state f–I gains, and afterpolarizing potentials. Rhythmic synaptic drive current densities were similar in preMNs and MNs. Our results indicate that, despite topographic and morphological differences, preMNs and MNs have some common intrinsic membrane, synaptic integration, and spiking properties that we postulate ensure fidelity of inspiratory drive transmission and conversion of synaptic drive into (pre)motor output. There also appears to be a common architectonic organization for some respiratory drive transmission circuits whereby many preMNs are spatially segregated from pre-BötC rhythm-generating neurons, which we hypothesize may facilitate downstream integration of convergent inputs for premotor pattern formation.

[1]  J. C. Smith,et al.  Models of respiratory rhythm generation in the pre-Bötzinger complex. I. Bursting pacemaker neurons. , 1999, Journal of neurophysiology.

[2]  R. Stornetta,et al.  Inspiratory augmenting bulbospinal neurons express both glutamatergic and enkephalinergic phenotypes , 2003, The Journal of comparative neurology.

[3]  R. Fregosi,et al.  Coordination of intrinsic and extrinsic tongue muscles during spontaneous breathing in the rat. , 2004, Journal of applied physiology.

[4]  E. Marder,et al.  Variable channel expression in identified single and electrically coupled neurons in different animals , 2006, Nature Neuroscience.

[5]  J. Feldman,et al.  Differential innervation of protruder and retractor muscles of the tongue in rat , 1995, The Journal of comparative neurology.

[6]  N. Mellen,et al.  Normal breathing requires preBötzinger complex neurokinin-1 receptor-expressing neurons , 2001, Nature Neuroscience.

[7]  Hong Wang,et al.  A group of glutamatergic interneurons expressing high levels of both neurokinin‐1 receptors and somatostatin identifies the region of the pre‐Bötzinger complex , 2003, The Journal of comparative neurology.

[8]  J. Ramirez,et al.  Reconfiguration of the neural network controlling multiple breathing patterns: eupnea, sighs and gasps , 2000, Nature Neuroscience.

[9]  J C Smith,et al.  Models of respiratory rhythm generation in the pre-Bötzinger complex. III. Experimental tests of model predictions. , 2001, Journal of neurophysiology.

[10]  J. Feldman,et al.  PreBötzinger complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. , 1998, Annual review of physiology.

[11]  Lyle J. Graham,et al.  Contrasting Effects of the Persistent Na+ Current on Neuronal Excitability and Spike Timing , 2006, Neuron.

[12]  Robert J. Butera,et al.  Ionic Current Model of a Hypoglossal Motoneuron , 2005 .

[13]  Robert J Butera,et al.  Persistent sodium current, membrane properties and bursting behavior of pre-bötzinger complex inspiratory neurons in vitro. , 2002, Journal of neurophysiology.

[14]  Edmund M Talley,et al.  The TASK family: two-pore domain background K+ channels. , 2003, Molecular interventions.

[15]  W. Crill,et al.  Persistent sodium current in mammalian central neurons. , 1996, Annual review of physiology.

[16]  S. J. Goldberg,et al.  Organization of the hypoglossal motoneurons that innervate the horizontal and oblique components of the genioglossus muscle in the rat , 2002, Brain Research.

[17]  Michael J. O'Donovan,et al.  Calcium imaging of rhythmic network activity in the developing spinal cord of the chick embryo , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[18]  Jeffrey C. Smith,et al.  Neuronal pacemaker for breathing visualized in vitro , 1999, Nature.

[19]  Edmund M Talley,et al.  TASK-1, a Two–Pore Domain K+ Channel, Is Modulated by Multiple Neurotransmitters in Motoneurons , 2000, Neuron.

[20]  J C Smith,et al.  Intrinsic bursters increase the robustness of rhythm generation in an excitatory network. , 2007, Journal of neurophysiology.

[21]  R. A. Davidoff.  Neural Control of Rhythmic Movements in Vertebrates , 1988, Neurology.

[22]  J C Smith,et al.  Pre-Bötzinger complex in the cat. , 1995, Journal of neurophysiology.

[23]  J. C. Smith,et al.  Neural control of respiratory pattern in mammals: an overview , 1995 .

[24]  K. Horikawa,et al.  A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates , 1988, Journal of Neuroscience Methods.

[25]  J. Leiter,et al.  Control and actions of the genioglossus muscle. , 1990, Progress in clinical and biological research.

[26]  J. C. Smith,et al.  Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals. , 1991, Science.

[27]  Marc D Binder,et al.  Persistent sodium and calcium currents in rat hypoglossal motoneurons. , 2003, Journal of neurophysiology.

[28]  D A Bayliss,et al.  Repetitive firing properties of developing rat brainstem motoneurones. , 1995, The Journal of physiology.

[29]  R. Stornetta,et al.  Neurokinin-1 Receptor-Expressing Cells of the Ventral Respiratory Group Are Functionally Heterogeneous and Predominantly Glutamatergic , 2002, The Journal of Neuroscience.

[30]  J. C. Smith,et al.  Models of respiratory rhythm generation in the pre-Bötzinger complex. II. Populations Of coupled pacemaker neurons. , 1999, Journal of neurophysiology.

[31]  H. Ellenberger.  Nucleus ambiguus and bulbospinal ventral respiratory group neurons in the neonatal rat , 1999, Brain Research Bulletin.

[32]  J. Duffin,et al.  Respiratory pre-motor control of hypoglossal motoneurons in the rat , 2002, Neuroscience.

[33]  J. Fetcho,et al.  Visualization of active neural circuitry in the spinal cord of intact zebrafish. , 1995, Journal of neurophysiology.

[34]  R K Powers,et al.  Contribution of outward currents to spike-frequency adaptation in hypoglossal motoneurons of the rat. , 1997, Journal of neurophysiology.

[35]  P. Guyenet,et al.  Tonic sympathetic chemoreflex after blockade of respiratory rhythmogenesis in the rat. , 1996, The Journal of physiology.

[36]  L. K. Purvis,et al.  Ionic current model of a hypoglossal motoneuron , 2002, Proceedings of the Second Joint 24th Annual Conference and the Annual Fall Meeting of the Biomedical Engineering Society] [Engineering in Medicine and Biology.

[37]  Hans-Ulrich Dodt,et al.  Infrared videomicroscopy: a new look at neuronal structure and function , 1994, Trends in Neurosciences.

[38]  J C Smith,et al.  Generation and transmission of respiratory oscillations in medullary slices: role of excitatory amino acids. , 1993, Journal of neurophysiology.

[39]  Fiona E. N. LeBeau,et al.  Microcircuits in action – from CPGs to neocortex , 2005, Trends in Neurosciences.

[40]  J. Foskett,et al.  Simultaneous Nomarski and fluorescence imaging during video microscopy of cells. , 1988, The American journal of physiology.