Carotid afferent and parafacial neuronal excitatory effects on breathing

Clement J. Zablocki Va Medical Center, Milwaukee WI

Investigators

Abstract

DESCRIPTION (provided by applicant): Neurons within the respiratory network regulate and coordinate respiratory pump and airway muscle activation. As such, the level of pulmonary ventilation is determined by the balance of excitatory and inhibitory inputs to these neurons. It has long been assumed that the primary excitatory drive to breathe is from intracranial CO2- H+ chemoreceptors. However, severe hypoventilation and reduced central CO2-H+ chemosensitivity after carotid body denervation (CBD) suggest a major excitatory drive is provided by carotid chemoreceptor afferents. It is unknown how, despite the presence of the intact and highly-sensitive intracranial chemoreceptors, CBD leads to hypoventilation, and furthermore what mechanisms of plasticity govern the normalization of breathing two or more weeks after CBD. One proposed pathway (Res. Plan, Figure 1, page 1) for these excitatory carotid chemoreceptor effects is through second order solitary tract (NTS) neuronal projections to the parafacial respiratory group/retrotrapezoid nucleus (pFRG/RTN). Phox2b-expressing (Phox2b+) pFRG/RTN neurons are hypothesized to: 1) receive and integrate multiple excitatory inputs, including those from the carotid bodies, and 2) provide the critical excitatory drive to pre-Bvtzinger complex (preBvtzC) respiratory rhythmogenic neurons during sleep;hence, dysfunction of Phox2b+ neurons underlies central and obstructive sleep apnea, and abnormal chemoreceptor and exerciseventilatory responses. An alternative hypothesis is that the carotid excitatory effect is through changes in neuromodulator inputs to brainstem respiratory neurons. We will test these and other hypotheses with the following Specific Aims: 1) Determine the effects on breathing in awake and asleep goats of destruction of Phox2b+ pFRG/RTN neurons. Hypothesis: Bilateral destruction of Phox2b+ pFRG/RTN neurons will cause: a) hypoventilation while awake which will be accentuated during NREM sleep by prolonged apneas, and b) attenuated ventilatory responses to hypercapnia, hypoxia, and exercise. Validation of these hypotheses will support the concept that the pFRG/RTN neurons provide critical excitatory input for breathing, particularly during sleep. 2) Determine whether CBD in goats induces central shifts in the balance between excitatory and inhibitory neuromodulation of the brainstem respiratory network. Hypothesis: After CBD when goats hypoventilate and CO2 sensitivity is reduced, the concentration of excitatory and inhibitory neuromodulators in effluent mock cerebrospinal fluid (mCSF) dialyzed through the preBvtzC will be decreased and increased respectively from baseline. Also after CBD, the ventilatory response to an injection of a glutamate receptor agonist into the preBvtzC will be reduced. Validation of these hypotheses will support the concept that tonic excitatory carotid activity affects neuromodulator-mediated excitability of respiratory rhythmogenic neurons. 3) Determine whether the observed time-dependent plasticity (recovery) after CBD is through upregulation of excitatory neuromodulatory mechanisms. Hypothesis: Two weeks after CBD in goats when they are no longer hypoventilating, the concentrations of excitatory and inhibitory neuromodulators in effluent mCSF dialyzed through the preBvtzC and the ventilatory response to a glutamate receptor agonist injection into the preBvtzC will be at or above normal. Furthermore, post-mortem immunohistochemistry will show increased percentage of neurons with receptors for excitatory neuromodulators in the preBvtzC. Validation of these hypotheses will be consistent with the concept that plasticity after CBD is due to upregulation of brainstem excitatory neuromodulatory mechanisms within the respiratory network. 4) Determine the effects on breathing in awake and sleeping goats of CBD after pFRG/RTN lesions. Hypothesis: When goats undergo CBD a month after lesioning the pFRG/RTN, the effects on breathing will be less than what normally occurs after CBD. Validation of this hypothesis will indicate the major pathway (Res. Plan, Figure 1, page 1) for central mediation of carotid afferent excitatory effect is through the pFRG/RTN.