Richard O. Davies

Serotonergic excitatory drive to hypoglossal motoneurons in the decerebrate cat

In decerebrate, paralyzed, vagotomized and artificially ventilated cats, serotonin (5-HT) and its analogues, microinjected into the hypoglossal (XII) motor nucleus, altered the activity of the genioglossal branch of XII nerve. 5-HT, carboxamidotryptamine maleate (5-CT) and DOI (1-5 mM) increased the activity by over 200%. Methysergide reversed this increase. Methysergide, mianserin, or ketanserin (100-250 nl, 1 mM) reduced the spontaneous hypoglossal activity by 20-50%. Buspirone, 8-OH-DPAT and (-)-propranolol were without effect. Thus, 5-HT provides a substantial tonic excitatory drive to XII motoneurons. The 5-HT receptors involved are likely to be type 1C or 2, but uncertainty regarding the affinity profiles of the drugs used in in vivo conditions in the cat precludes a definite identification.

Suppression of hypoglossal motoneurons during the carbachol-induced atonia of REM sleep is not caused by fast synaptic inhibition

The depression of upper airway motor activity that develops during the rapid eye movement (REM) stage of sleep is a major factor allowing upper airway obstructions to occur in patients with sleep apnea syndrome. Microinjections of carbachol, a cholinergic agonist, into the dorsal pontine tegmentum of chronically instrumented cats produce REM sleep. In acutely decerebrate cats, carbachol induces postural atonia, eye movements and a depression of the motor output to respiratory pump and upper airway muscles. In lumbar motoneurons, the depression of activity is due to a glycinergic inhibition that has the same characteristics during natural REM sleep in chronic cats and carbachol-induced atonia in decerebrate cats (Neurophysiology, 57 (1987) 1118-1129). The mechanisms that lead to the suppression of upper airway motoneuronal activity during REM sleep are unknown. In this study, we assessed whether the depression of hypoglossal (XII) nerve activity induced by pontine carbachol injections is caused by inhibitory amino acids acting within the XII nucleus. In decerebrate, paralyzed and artificially ventilated cats, we recorded the activities of both XII nerves (genioglossal branches), one phrenic and a cervical motor branch (to monitor postural activity). Postural atonia and respiratory depression were induced by pontine carbachol injections. The inhibitory amino acid receptor antagonists, strychnine (glycine receptors) or bicuculline (GABAA receptors), were injected (100-250 nl; 1.0-2.5 mM) into one XII nucleus (the other served as control) in an attempt to reduce or abolish the depression subsequently induced by pontine carbachol. Prior to the carbachol injections, both antagonists caused similar elevations of XII nerve activity on the treated side (30-40%). However, following carbachol, the XII nerve activity on the treated side was depressed to about 25% of the (pre-antagonist and pre-carbachol) control level, whereas the depression on the untreated side was slightly greater, to 10-15% of the control. Additional injections of antagonists during the carbachol-induced depression produced no further increase in nerve activity. This minor effect of the antagonists on the carbachol-induced depression of XII nerve activity was in contrast to the marked disinhibitory effects that both antagonists had on the XII nerve response to electrical stimulation of the lingual nerve. The latter was used as a control for the ability of strychnine and bicuculline to exert disinhibitory effects within the XII nucleus. Thus, there is little, if any, contribution of these inhibitory amino acids to the depression of XII motoneurons during the carbachol-induced, REM sleep-like postural and respiratory depression; mechanisms other than fast synaptic inhibition must be involved.

Changes in serotonin level in the hypoglossal nucleus region during carbachol-induced atonia

The excitability of hypoglossal (XII) motoneurons innervating genioglossal muscles is markedly suppressed during the rapid-eye-movement (REM) stage of sleep. This may contribute to airway obstructions in sleep apnea patients. Based on our earlier studies in decerebrate cats using injections of carbachol into the pons to induce a REM sleep-like atonia and microinjections of serotonin (5HT) into the XII motor nucleus, we hypothesized that a sleep-related withdrawal of the serotonergic excitatory input to XII motoneurons may play a major role in these processes. To test one aspect of this hypothesis, we inserted microdialysis probes into the XII nucleus region of decerebrate, paralyzed, vagotomized and artificially ventilated cats. The probes were perfused without or with the addition of a 5HT reuptake blocker, clomipramine. The levels of 5HT and its metabolite, 5-hydroxyindoleacetic acid (5HIAA), were determined using HPLC and electrochemical detection in dialysate samples collected over successive 20 min periods under four successive experimental conditions: control (at least 2 h after probe insertion); during the postural atonia and respiratory depression produced by pontine microinjection of carbachol; recovery from the effects of carbachol produced by pontine microinjection of atropine; and, to verify that the presence of 5HT in the dialysate was related to the activity of serotonergic cells of the brainstem, following administration of 8-OH-DPAT, a 5HT 1A receptor agonist known to suppress activity in the serotonergic cells of the raphe system. After correcting for recovery rates of individual probes, the mean control 5HT level in the extracellular space of the XII nucleus region was 7.9 +/- 4.4 nM (S.D.) in eight experiments without reuptake blockers. During the carbachol-induced depression, it was reduced to 70 +/- 20% of the pre-carbachol level. It increased to the original control level 98 +/- 27% after pontine injection of atropine. 8-OH-DPAT reduced the 5HT level to 43 +/- 14% of the post-atropine level. Changes in the 5HIAA level were not as consistent as for 5HT and did not reach statistical significance under any of the experimental conditions. Thus, a functionally significant amount of 5HT is present in the extracellular space within the XII nucleus region, and its decrement during carbachol-induced, REM sleep-like atonia is likely to reflect that occurring during natural REM sleep; this may contribute to the decreased tone of upper airway muscles and airway patency.

Carotid sinus nerve projections to the brain stem in the cat

The distribution of carotid sinus nerve (CNS) afferent and efferent fibers in the brain stem was examined in eight cats using horseradish peroxidase (HRP) neurohistochemistry. The transganglionic transport of HRP yielded dense extraperikaryal labeling within the nucleus of the tractus solitarius (nTS). Labeling was also present in the area postrema (ap) and in the region of the nucleus ambiguus (nA). The nTS labeling was bilateral, the ipsilateral side being more intense. Within the nTS, the labeling was not uniform, being heaviest in the dorsal, dorsolateral and commissural subnuclei. Moderate labeling was seen in the ventrolateral nTS. In the region of the obex, HRP labeled fibers could be followed from the nTS to the region of the nA, where extraperikaryal labeling could be seen. HRP labeled perikarya were found in the rostral pole of nA. In two controls, the CSN was sectioned close to its junction to the glossopharyngeal nerve just prior to HRP injection. In both cases, no labeling was found in either the petrosal ganglion or brain stem.

BEHAVIOUR OF RAPHE CELLS PROJECTING TO THE DORSOMEDIAL MEDULLA DURING CARBACHOL-INDUCED ATONIA IN THE CAT

1. The activity of most brainstem serotonergic cells is suppressed during sleep, particularly the rapid eye movement (REM) phase. Thus, they may play a major role in state‐dependent changes in CNS functioning. Our main goal was to search for medullary raphe cells having axonal branches in the region of the hypoglossal (XII) motor nucleus and assess their behaviour during the atonia produced by microinjections of a cholinergic agonist, carbachol, into the dorsal pontine tegmentum. In chronic animals, such microinjections evoke a desynchronized sleep‐like state similar to natural REM sleep; in decerebrate animals, they produce eye movements and a motor suppression similar to the postural atonia of REM sleep. 2. In decerebrate, paralysed, vagotomized and artificially ventilated cats, we recorded extracellularly from medullary raphe cells antidromically activated from the XII nucleus region. Forty‐five cells recorded in the raphe obscurus and pallidus nuclei were antidromically activated with latencies characteristic of non‐myelinated fibres (4.4‐42.0 ms). For thirty‐three of the forty‐five cells, we found one or more axonal branches within or just below the XII nucleus. The remaining twelve cells, in addition to the XII nucleus, had axonal ramifications in the medial nucleus of the solitary tract (NTS) and/or the dorsal motor nucleus of the vagus (DMV). 3. A subset of fourteen spontaneously active cells with identified axonal projections were held long enough to be recorded during the carbachol‐induced atonia, and eight of these also during the subsequent recovery and a systemic administration of the serotonergic 1A receptor agonist (+/‐)8‐hydroxy‐2‐(di‐N‐propylamino)tetrealin hydrobromide (8‐OH‐DPAT). All but one were suppressed during the atonia in parallel to the suppression of XII, phrenic and postural nerve activities (firing rate, 1.3 +/‐ 0.7 Hz before and 0.1 +/‐ 0.2 Hz after carbachol (means +/‐ S.D.)). Following the recovery from the atonia, the firing rates of the eight cells increased to the pre‐carbachol level (1.6 +/‐ 1.0 Hz). Subsequently, all were silenced by 8‐OH‐DPAT. 4. These cells fulfil most physiological criteria for serotonergic cells and have the potential to modulate, in a state‐dependent manner, activities in the motor XII nucleus, visceral sensory NTS, and DMV. The decrements in serotonergic neuronal activity that occur during the carbachol‐induced atonia suggest that a similar withdrawal of serotonergic input may occur during REM sleep and contribute to the characteristic reductions in upper airway motor tone.

Noradrenergic, serotonergic and GABAergic antagonists injected together into the XII nucleus abolish the REM sleep-like depression of hypoglossal motoneuronal activity.

Recently, we reported that the suppression of hypoglossal (XII) motoneuronal activity that occurs during the carbachol‐induced, rapid eye movement (REM) sleep‐like state is abolished by the microinjection into the XII nucleus of a drug mix that antagonizes aminergic excitation and amino acid‐mediated inhibition (prazosin, methysergide, bicuculline and strychnine). We now assess the role of glycinergic inhibition in the depression of XII motoneuronal activity and estimate the distribution of the antagonists around the XII nucleus at the time when they are effective. Towards the first goal, REM sleep‐like episodes were elicited in urethane‐anesthetized rats by 10 nl carbachol microinjections into the dorsomedial pons prior to, and at different times after, combined microinjections into the XII nucleus of only three antagonists (strychnine omitted). As in our previous study, the carbachol‐induced depression of XII activity was abolished during tests performed 42–88 min after the antagonists, whereas other characteristic effects of carbachol (appearance of hippocampal theta, cortical activation, decreased respiratory rate) remained intact. The depressant effect of carbachol on XII motoneurons partially recovered after 2.5 h. Towards the second goal, using a drug diffusion model, we determined that the tissue concentrations of the antagonists at the time when they were effective were within the range of their selective actions, and the drugs acted within 0.9–1.4 mm from the injection sites, thus within a space containing XII motoneurons and their dendrites. We conclude that antagonism of α‐adrenergic, serotonergic, and GABAA receptors are sufficient to abolish the REM sleep‐like atonia of XII motoneurons.

The medullary projections of afferent bronchopulmonary C fibres in the cat as shown by antidromic mapping.

1. The activity of eighty‐seven bronchopulmonary vagal afferent neurones with unmyelinated axons (C fibres) was recorded extracellularly in the nodose ganglia of decerebrate, paralysed and artificially ventilated cats. On the basis of their response latencies following the right atrial injection of capsaicin or phenyldiguanide, the cells were classified as having their receptor endings within the reach of pulmonary (latency less than 3.5 s) or bronchial (latency above 3.5 s) circulation. 2. Pulmonary and bronchial receptor cells differed only slightly in their response characteristics (firing rate, burst duration) and the conduction velocity of their peripheral axons. Bronchial C fibres represented about 70% of the population studied. 3. The medullary distributions of the central branches of six pulmonary and six bronchial C fibres were determined by means of the antidromic mapping technique. The two receptor subtypes did not differ in their central projection patterns. 4. Rostral to the obex, the central branches of the bronchopulmonary C fibres were localized within the medial portions of the nucleus tractus solitarii (NTS) and area postrema, and were most densely distributed along the borders of the parvicellular subnucleus of the NTS. Caudal to the obex, the most dense branching was found in the dorsal portion of the commissural subnucleus. Projections to the contralateral NTS were found, but these were of a much lower density. 5. The central distribution of bronchopulmonary C fibres is compared to the projection patterns of vagal and glossopharyngeal afferents of other modalities that are involved in respiratory and cardiovascular control. This is discussed in relation to the concept of a modality‐specific organization of the NTS.

Pulmonary stretch receptor relay neurones of the cat: location and contralateral medullary projections.

1. The activity of pump (p.) cells, second‐order neurones in the pulmonary stretch receptor pathway, was recorded extracellularly in the nucleus of the tractus solitarius (n.t.s.) of the decerebrate cat. Their firing was proportional to changes in lung volume but unrelated to the centrally determined respiratory rhythm. A systematic search of the n.t.s. for the location of p. cells was made and an assessment of their efferent projection to the contralateral n.t.s. was determined electrophysiologically by the antidromic mapping technique. 2. P. cells were located around, and in close proximity to, the solitary tract. The two sites of greatest density were ventromedial and dorsolateral to the tract, with lower concentrations found laterally and ventrolaterally. 3. For twelve of the thirty p. cells tested, evidence of a projection to the contralateral n.t.s. was obtained; in seven of these cells, axonal arborizations within the projection area were identified. Almost all the cells that sent axons to the contralateral n.t.s. were located dorsolateral to the tract; there was no evidence that cells in the ventromedial region had contralateral projections. 4. No evidence that R beta neurones project to the contralateral commissural and ventrolateral subnuclei was found. 5. No p. cells projected to the contralateral ventrolateral n.t.s. The site of projection and branching was consistently localized just caudal to the obex and medial to the solitary tract, in the caudal medial, and commissural subnuclei of the n.t.s. This same region has been shown to receive a dense, direct projection from pulmonary rapidly adapting receptors.

Differential sensitivity of laryngeal and pharyngeal motoneurons to iontophoretic application of serotonin.

Serotonergic neurons decrease their activity during sleep, especially rapid eye movement sleep, thereby reducing their facilitatory effect on upper airway motoneurons. The magnitude of teh sleep-related loss of tone varies among upper airway muscles (e.g., pharyngeal dilator motoneurons are more suppressed than laryngeal motoneurons). We hypothesized that these differences may be related to the sensitivity of different groups of upper airway motoneurons to serotonin. Experiments were done on decerebrate, vagotomized, paralysed and artificially-ventilated cats. Hypoglossal and laryngeal motoneurons were recorded extracellularly using five-barrel pipettes filled with: serotonin, glutamate and methysergide (serotonergic antagonist) for iontophoresis, and NaCl for recording and current balancing. All but two of the 65 hypoglossal motoneurons (45 inspiratory, 10 expiratory, 10 tonic) and 27 out of 32 laryngeal motoneurons (14 inspiratory, 18 expiratory) were excited by serotonin, and the excitation was abolished by methysergide. To compare the magnitude of the excitatory effect among distinct motoneuronal groups, we applied small ejection currents in a standardized manner (+15 nA for 3 min; 10 mM serotonin in 150 NaCl) onto spontaneously active motoneurons (13 inspiratory hypoglossal, 11 inspiratory laryngeal and 11 expiratory laryngeal). Serotonin increased the number of spikes per respiratory burst of inspiratory hypoglossal motoneurons from 19 +/- 4.0 (S.E.M.) to 35 +/- 4.8, of inspiratory laryngeal motoneurons from 44 +/- 8.3 to 55 +/- 8.8, and of expiratory laryngeal motoneurons from 23 +/- 4.8 to 33 +/- 6.2. The relative increases in activity (to 220% +/- 24, 147% +/- 23 and 148% +/- 9 of control, respectively) were significantly higher in hypoglossal than in laryngeal motoneurons. In addition, the excitatory effect developed significantly faster in hypoglossal than in laryngeal motoneurons. Methysergide reduced the spontaneous activity of about half the hypoglossal and laryngeal motoneurons to 66% +/- 5 of control. Thus, the sensitivity to the excitatory effects of serotonin varies among different pools of upper airway motoneurons. These differences correlate with the pattern of airway muscle hypotonia seen during sleep.

Absence of carotid chemoreceptor response during hypoxic exercise in the cat

Abstract The ventilatory response to hypoxia is augmented by muscular exercise. In order to determine the role of peripheral chemoreceptors in the ventilatory response during hypoxic exercise we measured carotid chemoreceptor activity in few-fiber and multi-fiber preparations in 15 anesthetized and 6 decerebrate cats simultaneously with ventilation and systemic blood pressure. These determinations were made at three levels of P co 2 (about 460, 80 and 36 torr) during hind limb exercise elicited by spinal cord stimulation and also during passive hind limb movement. The ventilatory response to each exercise maneuver was prompt and persistent. Although the carotid chemoreceptor preparation responded normally to chemical stimuli, there was no modification of the response by the simulated exercise. On the other hand, in 6 separate experiments on anesthetized cats, exercise increased ventilation in proportion to the increase in oxygen uptake and enhanced the hypoxic effect on ventilation. Therefore these results demonstrated, at each level of inspired 0; tested, that the hyperpnea of exercise occurred without an increase in mean activity of the carotid chemoreceptors. We conclude that the effect of exercise is not mediated by the peripheral chemoreceptors and that the augmenting and interacting effects of hypoxia on the ventilation during exercise occur centrally.