Qi-Jian Sun

The pre-Bötzinger complex and phase-spanning neurons in the adult rat.

To characterise respiratory neurons in the pre-Bötzinger complex of adult rats, extracellular recordings were made from 302 respiratory neurons in the ventral respiratory group of sodium pentobarbitone anaesthetised adult rats. Neurons were located 0 to 1.6 mm caudal to the facial nucleus, and ventral to the nucleus ambiguus. The pre-Bötzinger complex comprised expiratory neurons (22%, 22/100), inspiratory neurons (37%, 37/100) and phase-spanning neurons (41%, 41/100). In contrast, 80% (125/157) of Bötzinger neurons were expiratory, and 80% (36/45) of rostral ventral respiratory group neurons were inspiratory. Rostrocaudally, the pre-Bötzinger complex extended about 400 microns, starting at the caudal pole of the nucleus ambiguus compact formation. The pre-Bötzinger complex was also characterised by a predominance of propriobulbar neurons (81%, 13/16). Furthermore, 68% (33/48) of expiratory-inspiratory neurons found were located within the pre-Bötzinger complex. The variety of neuronal subtypes in the pre-Bötzinger complex, including many firing during the expiratory-inspiratory transition is consistent with the hypothesis that this nucleus plays a key role in respiratory rhythm generation in the adult rat.

Bötzinger neurons project towards bulbospinal neurons in the rostral ventrolateral medulla of the rat.

Sympathetic nerve activity often fluctuates with the respiratory cycle, but the central neurons that impose this respiratory modulation have not been conclusively identified. In the present study, we used intracellular recording and dye‐filling to identify expiratory neurons in the Bötzinger complex. Our aim was to see if Bötzinger neurons project towards putative cardiovascular neurons in the rostral ventrolateral medulla. In the first series of experiments, histochemistry and immunohistochemistry were used to reveal the labelled Bötzinger neurons and neurons immunoreactive for tyrosine hydroxylase. Two out of four Bötzinger neurons had axon varicosities that were closely apposed to tyrosine hydroxylase‐immunoreactive neurons with cell bodies located within 0.6 mm caudal to the facial nucleus (three and five close appositions, respectively). In a second series of studies, rats were injected with cholera toxin B into the intermediolateral cell column of the spinal cord 4–7 days before the electrophysiological recording. Eight of the fourteen labelled Bötzinger neurons had a direct projection towards cholera toxin B‐labelled neurons in the rostral ventrolateral medulla. Close appositions were found on both somata and proximal dendrites (5 ± 2 close appositions/neuron, n=8). The present study supports the idea that a direct projection from Bötzinger neurons to presympathetic neurons in the rostral medulla plays a role in the respiratory modulation of sympathetic nerve activity. J. Comp. Neurol. 388:23–31, 1997.

Calbindin-immunoreactive neurons in the reticular formation of the rat brainstem: catecholamine content and spinal projections.

Calbindin‐D28k (calbindin) is a calcium‐binding protein that is distributed widely in the rat brain. The localisation of calbindin immunoreactivity in the medulla oblongata and its colocalisation with adrenaline‐synthesising neurons [phenylethanolamine‐N‐methyltransferase‐immunoreactive (PNMT‐IR)] was examined (Granata and Chang [1994] Brain Res. 645:265–277). However, detailed information about the distribution of calbindin‐IR neurons in the reticular formation of the medulla oblongata in particular is lacking. In this report, the authors address this issue with an emphasis on the quantitation of calbindin‐IR neurons, catecholamine neurons [tyrosine hydroxylase (TH)‐IR, or PNMT‐IR], and spinally projecting neurons in the ventral brainstem. Rats received injections of the retrograde tracing agent cholera toxin B (CTB) into the thoracic spinal cord or into the superior cervical ganglion. Immunocytochemistry was used to reveal calbindin, TH, PNMT, and CTB immunoreactivity. Ten calbindin‐IR cell groups were identified within the pontomedullary reticular formation. Seven previously undescribed but distinct clusters of calbindin‐IR neurons were found. Within the ventral pons, a population of calbindin‐IR neurons occurred dorsal but adjacent to the A5 cell group. These calbindin‐IR neurons did not contain either TH or PNMT immunoreactivity, and few if any of these neurons projected to the spinal cord. A distinct group of calbindin‐IR neurons was present in the ventral medulla. Seventy‐five percent of these calbindin‐IR neurons contained TH immunoreactivity, 45% contained PNMT immunoreactivity, and 21% were spinally projecting neurons. Spinally projecting, calbindin‐IR neurons were a subpopulation of PNMT‐IR cells. In the caudal ventral medulla, no TH‐IR or PNMT‐IR cells were calbindin‐IR. In the intermediolateral cell column, close appositions of calbindin‐IR terminals on identified sympathetic preganglionic neurons as well as calbindin‐IR synapses indicated that these neurons may affect directly the sympathetic outflow. The results demonstrate for the first time the existence of a new subpopulation of spinally projecting, PNMT‐IR neurons in the rostral ventrolateral medulla. J. Comp. Neurol. 424:547–562, 2000.

Serotonin inputs to inspiratory laryngeal motoneurons in the rat

Serotonergic neurons are distributed widely throughout the central nervous system and exert a tonic influence on a range of activities in relation to the sleep–wake cycle. Previous morphologic and functional studies have indicated a role for serotonin in control of laryngeal motoneurons. In the present study, we used a combination of intracellular recording, dye‐filling, and immunocytochemistry in rats to demonstrate close appositions between serotonin immunoreactive boutons and posterior cricoarytenoid (PCA) and cricothyroid (CT) motoneurons, both of which are located in the nucleus ambiguus and exhibit phasic inspiratory activity. PCA motoneurons received 29 ± 5 close appositions/neuron (mean ± SD, n = 6), with the close appositions distributed more frequently on the distal dendrites, less frequently on the proximal dendrites, and sparsely on the axons and somata. CT motoneurons received 56 ± 15 (n = 6), with close appositions found on both the somata and dendrites, especially proximal dendrites. Close appositions on the axons were only seen on one CT motoneuron. These results demonstrate a significant serotonin input to inspiratory laryngeal motoneurons, which is more prominent on CT compared with PCA motoneurons, and may reflect the different functional role of the muscles that they innervate during the sleep–wake cycle. J. Comp. Neurol. 451:91–98, 2002.

Bulbospinal sympatho-excitatory neurons in the rat caudal raphe

Objectives To explore the rat caudal raphe nuclei for neurons that respond to activation of baroreceptor nerves and that have a spinal axon, and to compare the behavioural properties of barosensitive bulbospinal neurons in the rat caudal raphe with the properties of barosensitive bulbospinal neurons in the rostral ventrolateral medulla. Design Extracellular unit recordings were obtained from an area extending up to 1.0 mm caudally from the caudal edge of the facial nucleus. Two sites were explored: the rostral ventrolateral medulla and the midline. Materials and methods Single-unit recordings were made in anaesthetized (75 mg/kg chloral hydrate and 30 mg/kg sodium pentobarbitone then 3–6 mg intravenously as required) immobilized (2 mg pancuronium as required) Sprague-Dawley rats. Central respiratory drive was recorded from phrenic nerve discharge. The barosensitivity of single units was assessed by R-wave triggered histograms and by histograms of their responses to aortic nerve stimulation or to intravenous injection of phenylephrine. Nociceptors were activated by a brief pinch of the tail. Results Eleven spontaneously active units in the midline that were inhibited by baroreceptor stimulation and had a spinal axon were studied. Respiratory modulation was present and was predominantly inspiratory. Barosensitive neurons in the rostral ventrolateral medulla were activated by nociceptive inputs; midline barosensitive neurons were not. Conclusions The behavioural characteristics of midline neurons differ from those of the bulbospinal barosensitive neurons in the rostral ventrolateral medulla, indicating that raphe spinal neurons have different sets of afferent inputs and may subserve to a distinct physiological role. The present paper is the first report of bulbospinal neurons in the rat caudal raphe that are inhibited by activation of arterial baroreceptors.

The temporal relationship between non‐respiratory burst activity of expiratory laryngeal motoneurons and phrenic apnoea during stimulation of the superior laryngeal nerve in rat

Non‐technical summary  Nerve fibres in the larynx detect foreign substances and elicit a stereotypical airway protective response that can be simulated by electrical stimulation of the superior laryngeal nerve (SLN). In humans the response includes cough, swallowing and a cessation of breathing (apnoea). It is still unknown precisely how the central nervous system coordinates swallowing and breathing, and at which point the two vital systems converge and diverge in the brain. Here we report a temporal, sequential relationship between excitation of expiratory laryngeal motoneurons that close the larynx during swallowing, and inhibition of breathing, during stimulation of the SLN in rat. The two phenomena can be dissociated by inactivating different brain areas. This work therefore has implications for diseases such as sudden infant death syndrome and Parkinsons disease, in which incoordination of breathing and protective behaviours may result in aspiration of irritants and subsequent death or aspiration pneumonia.

Serotonin Inputs to Laryngeal Constrictor Motoneurons in the Rat

Objectives/Hypothesis: The objective was to demonstrate close appositions between serotonin‐immunoreactive boutons and laryngeal constrictor (LCon) motoneurons in Sprague‐Dawley rats.

Firing patterns of pre-Bötzinger and Bötzinger neurons during hypocapnia in the adult rat

Controversy exists about how a coordinated respiratory rhythm is generated in the brainstem. Some authors suggest that neurons in the pre-Bötzinger complex are key to initiation of all types of breathing. While, on the other hand, it has been reported that some pre-Bötzinger neurons fail to maintain a rhythmic discharge in phase with phrenic nerve discharge during mechanical hyperventilation. Extracellular recordings were made from respiratory units in the pre-Bötzinger and Bötzinger complexes of 13 anaesthetised, paralysed and vagotomised rats. Central respiratory activity was monitored from the C5 phrenic nerve. During mechanical hyperventilation, several changes were observed in the phrenic neurogram. Firstly, the frequency and amplitude of integrated phrenic nerve discharge were reduced and reversibly stopped. Secondly, the patterned discharges changed from an augmenting to a variety of non-augmenting patterns in 53 of 60 cases. In some cases (n=9) we observed that the pattern appeared to have two components, an early short duration discharge followed by a longer duration discharge. Respiratory units also started to show different firing patterns during mechanical hyperventilation. In general, they were divided into those units that fired tonically (n=28) and units that became silent (n=32), before phrenic nerve discharge ceased coincidently with complete apnoea. Of particular interest were those expiratory-inspiratory units in the pre-Bötzinger complex (n=8) that narrowed their firing period towards late expiration and early inspiration during mechanical hyperventilation. Given their firing features, it is possible that these expiratory-inspiratory units may participate in generation of the early inspiratory component of phrenic nerve discharge.

Thyrotropin-releasing hormone immunoreactive boutons form close appositions with medullary expiratory neurons in the rat

The aim of the present study was to assess the size of the input from TRH immunoreactive varicosities to medullary respiratory neurons in the Bötzinger complex and caudal ventral respiratory group. Neurobiotin was intracellularly injected into seven neurons in the Bötzinger complex, between 0.4 and 0.9 mm caudal to the facial nucleus. Five of the seven Bötzinger neurons had extensive local axonal projections, with bouton-like varicosities clustered predominantly between their somata and the nucleus ambiguus. Seven neurons in the caudal ventral respiratory group, located between 1.6 and 2.4 mm caudal to the facial nucleus, were also labelled. All but one caudal respiratory neurons had no, or very few, medullary collaterals. TRH immunoreactive fibres were seen in many medullary nuclei, including the ventral reticular formation. Bötzinger neurons were closely apposed by an average of 29 +/- 8 TRH immunoreactive boutons/neuron (mean +/- S.D., n = 7). In contrast, caudal ventral respiratory group neurons were apposed by only 5 +/- 3 TRH immunoreactive boutons/neuron (n = 7). Bötzinger neurons form many intramedullary and bulbospinal inhibitory connections with premotoneurons and motoneurons that are important in the timing, amplitude and shape, of respiratory activity. Our findings suggest a role for endogenous TRH-containing neurons in modulating the activity of inhibitory Bötzinger neurons and neurons in the caudal ventral respiratory group. The significance of the apparent difference in size of this input remains to be determined.

GABAA mediated inhibition and post-inspiratory pattern of laryngeal constrictor motoneurons in rat

Laryngeal constrictor motoneurons (LCMN) are activated during post-inspiration and act to slow expiratory airflow. However, little is known about how this phasic activity is generated. Here, we investigated the electrophysiological responses of identified LCMN to local application of GABA and bicuculline methiodide (BIC) in 14 anaesthetised Sprague-Dawley rats. During extracellular recordings, GABA iontophoresis (0.5M) strongly inhibited LCMN (n=6). Interestingly, BIC iontophoresis (5 mM) reduced, rather than increased, LCMN post-inspiratory activity (5 out of 6). Furthermore, intracellular recording revealed that BIC reduced not only the hyperpolarisation of the LCMN during inspiration (2.5+/-1.4 mV before and 1.5+/-0.4 mV after the BIC, P=0.05, n=5), but also the depolarisation during post-inspiration (3.0+/-1.3 mV before and 1.6+/-0.4 mV after the BIC, P=0.02, n=5). Our results demonstrate for the first time that the inspiratory inhibition of LCMN is primarily mediated by GABA(A) receptors. A possible involvement of a post-inhibitory rebound mechanism is discussed to explain how blockade of an inspiratory inhibition would affect LCMN excitability during post-inspiration.