We visualized respiratory neuron activity covering the entire ventral medulla using optical recordings in a newborn rat brainstem–spinal cord preparation stained with voltage-sensitive dye. We measured optical signals from several seconds before to several seconds after the inspiratory phase using the inspiratory motor nerve discharge as the trigger signal; we averaged the optical signals of 50–150 respiratory cycles to obtain an optical image correlating particularly to inspiratory activity. The optical images we obtained from the ventral approach indicated that neuron activity first appeared during the respiratory cycle in the limited region of the rostral ventrolateral medulla (RVLM), preceding the onset of inspiratory activity by ∼500 msec. During the inspiratory phase, plateau activity appeared in the more caudal ventrolateral medulla at the level of the most rostral roots of the XIIth nerve. Comparison with electrophysiological recordings from respiratory neurons in the RVLM suggested that the optical signals preceding the inspiratory burst reflect preinspiratory neuron activity in this area. This RVLM area was determined to be ventrolateral to the facial nucleus and close to the ventral surface. We referred to this functional neuron group as the para-facial respiratory group (pFRG). Partial, bilateral electrical lesioning of the pFRG significantly reduced the respiratory frequency, together with changes in the spatiotemporal pattern of respiratory neuron activity. Our findings suggest that the pFRG comprises a neuronal population that is involved in the primary respiratory rhythm generation in the rostrocaudally extending respiratory neuron network of the medulla.

Phox2b protein is a specific marker for neurons in the parafacial region of the ventral medulla, which are proposed to play a role in central chemoreception and postnatal survival. Mutations ofPHOX2Bcause congenital central hypoventilation syndrome. However, there have been no reports concerning electrophysiological characteristics of these Phox2b-expressing neurons in the parafacial region of the neonate immediately after birth. This region overlaps with the parafacial respiratory group (pFRG) composed predominantly of preinspiratory (Pre-I) neurons that are involved in respiratory rhythm generation. We studied (1) whether pFRG neurons are Phox2b immunoreactive or not and (2) whether they show intrinsic CO₂chemosensitivity. We found that most pFRG/Pre-I neurons were Phox2b immunoreactive and depolarized upon increase in CO₂concentration under condition of action potential-dependent synaptic transmission blockade by tetrodotoxin. We also confirmed that these pFRG neurons expressed neurokinin-1 receptor. They were tyrosine hydroxylase negative and presumed to be glutamatergic. Our findings suggest that Phox2b-expressing parafacial neurons play a role in respiratory rhythm generation as well as central chemoreception and thus are essential for postnatal survival.

Respiratory activities are produced by medullary respiratory rhythm generators and are modulated from various sites in the lower brainstem, and which are then output as motor activities through premotor efferent networks in the brainstem and spinal cord. Over the past few decades, new knowledge has been accumulated on the anatomical and physiological mechanisms underlying the generation and regulation of respiratory rhythm. In this review, we focus on the recent findings and attempt to elucidate the anatomical and functional mechanisms underlying respiratory control in the lower brainstem and spinal cord.

There are at least two respiration-related rhythm generators in the medulla: the pre-Bötzinger complex, which produces inspiratory (Insp) neuron bursts, and the parafacial respiratory group (pFRG), which produces predominantly preinspiratory (Pre-I) neuron bursts. The pFRG Pre-I neuron activity has not been correlated with motor neuron activity in slice or block preparations of rostral medulla. In this study, we attempted to detect pFRG Pre-I activity as motor output in the rostral medulla. We recorded respiratory activity of the facial nerve in the brain stem–spinal cord preparation of 0- to 2-day-old rats. Facial nerve activity consisted of preinspiratory, Insp, and postinspiratory activity. Pre- and postinspiratory activity corresponded well with membrane potential trajectories of Pre-I neurons in the rostral ventrolateral medulla. In response to perfusion of 1 μM DAMGO (a μ-opiate agonist), fourth cervical ventral root (C4) Insp activity was depressed and facial nerve activity continued to synchronize with Pre-I neuron bursts. After transverse sectioning between the levels of the pre-Bötzinger complex and the pFRG, C4 Insp activity recovered within 15 min, but facial nerve activity was inhibited. When DAMGO was applied, C4 Insp activity was inhibited, and rhythmic facial nerve activity recovered. Subsequent elevation of K⁺ concentration reinduced C4 activity, but facial nerve activity was inhibited. Whole cell recordings in the rostral block revealed the presence of putative Pre-I neurons, the activity of which was synchronized with facial nerve activity. These results show that the rostral medulla, not including the pre-Bötzinger complex, produces Pre-I–like rhythmic activity that can be monitored as facial nerve motor output in newborn rat in vitro preparations.