Stephen Mark Johnson - US grants

DESCRIPTION (Applicant's abstract): The fundamental goal of this proposal is to test the hypothesis that bulbospinal respiratory synaptic transmission expresses activity-dependent synaptic plasticity. Using an in vitro brainstem-spinal cord preparation from adult turtles as an experimental model, electrical stimulation will be applied to the spinal cord (C5) during reversible blockade of axonal conduction between brainstem and spinal cord (C3-C4). Evoked short-latency responses and endogenous respiratory motor output (before and after blockade) will be assessed with extracellular recordings from the ventral roots (C8). Based on preliminary data, we propose to test three specific hypotheses: (1) Low frequency stimulation of descending synaptic inputs to spinal motoneurons induces long-term depression of both evoked and spontaneous respiratory synaptic inputs; (2) High frequency stimulation of descending inputs to spinal motoneurons induces long-term potentiation of evoked and spontaneous respiratory synaptic inputs, but only when preconditions are satisfied by the activation of spinal serotonin receptors; and (3) Spinal long-term potentiation and depression are opposing processes whose expression depends on the relative activity of postsynaptic protein kinases and phosphatases, respectively. Our findings will yield insights into a potentially important mechanism that adjust (enhance or depress) synaptic inputs to respiratory motoneurons, thereby maintaining an appropriate drive to respiratory muscles. Perhaps more importantly, this will be one of the first experimental preparations that allow an assessment of plasticity in evoked synaptic responses with the relevant behavior (respiratory motor output). An understanding of the role played by neuromodulators (e.g., serotonin) and intracellular signalling pathways (kinases and phosphatases) that control such activity-dependent synaptic plasticity may provide the rationale to develop effective therapeutic approaches in patients with compromised respiratory function of spinal injuries.

The goal of this project is to test hypotheses regarding vertebrate oscillatory motor networks with respect to their organization and cellular mechanisms underlying rhythmogenesis using turtle brainstems in vitro. The vertebrate respiratory control system was chosen because this system is necessary for life and it produces spontaneous quantifiable rhythmic motor output under in vitro conditions. This project examines the adult turtle respiratory control system because reptiles represent an important phylogenetic intermediate between amphibians and mammals, and turtles have unique physiological features that are advantageous for in vitro studies. Hypotheses to be tested include: (1) the adult turtle respiratory rhythm generator is composed of separate synaptically-coupled oscillatory networks, (2) expiratory neurons, such as novel pre-expiratory cells, initiate breathing cycles, (3) endogenous pacemaker properties in respiratory neurons are necessary for rhythm generation. To address these hypotheses, a multidisciplinary approach will be used whereby well-established multichannel recording techniques are applied for the first time to study respiratory neural control in vitro. The results of this proposal will help place the organizational principles and underlying mechanisms for respiratory rhythmogenesis within a broader phylogenetic and evolutionary context, thereby focusing attention on conserved mechanisms that are critical for respiratory rhythmogenesis and other rhythmic motor networks in vertebrates. The project will support a postdoctoral fellow and graduate student. Undergraduate students from underrepresented minorities will be recruited each summer from the University of Puerto Rico.

[unreadable] DESCRIPTION (provided by applicant): Neural networks in the brain, which generate fundamental motor behaviors such as walking and breathing, are hypothesized to consist of coupled rhythmic networks that can be reconfigured to produce different motor behaviors. The long-range goal is to determine how coupling and reconfiguration in rhythmic networks contribute to motor behavior in mammals. Highly versatile 'microfluidic chambers' will be constructed (of biocompatible polydimethylsiloxane [PDMS]) to study brain slices in vitro. These 'closed-top' and 'open-top' chambers will permit spatiotemporal control of slice extracellular space combined with a variety of neural recording configurations (e.g., 2-D and 3-D multichannel extracellular, intracellular, and suction electrodes). Transverse slices of neonatal rat medulla will be used because these slices spontaneously produce rhythmic respiratory-related motor output in these chambers for hours. Medullary slices are also ideal because the rhythmic motor networks that generate respiratory-related motor output (i.e., the pre-Botzinger Complex) are synaptically-coupled and located bilaterally in slices (i.e., spatially separate). In each type of chamber, multiple parallel fluid streams exhibiting laminar flow will pass over (and under) the slice. Since laminar flow prevents fluid streams from mixing, the composition of fluid bathing the slice in specific regions will be altered by controlling laminar stream composition and width. In both closed-top and open-top chambers, Aim 1 will establish how different factors affect laminar flow and diffusion in slices, and establish the microelectrode conformation that best captures network activity. Aim 2 will show that focused laminar streams can: (a) reversibly uncouple bilateral rhythmic motor networks (with sodium-free sucrose solution), and (b) reconfigure one rhythmic motor network (with hypoxic solution) while it is coupled to another rhythmic motor network. Microfluidic chambers will provide a novel approach to neural circuit analysis, which is a major goal of NINDS. Innovative studies on network coupling and reconfiguration may lead to therapeutic insights for treating spinal cord injury, stroke, cerebral palsy, and Parkinson's disease. Microfluidic chambers can also be modified to perform novel studies on synaptic and network plasticity, effects of local ischemia or hypoxia, intracellular signaling networks, and cell-based biosensing. [unreadable] [unreadable] [unreadable]