Leszek Kubin

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.

Pontomedullary distribution of 5-HT2A receptor-like protein in the rat

Serotonin (5‐HT) exerts excitatory effects in many brainstem regions involved in autonomic, somatic, motor, and sensory functions, and in control of vigilance. To determine the potential role of 5‐HT2A receptors in these effects, we immunohistochemically mapped the distribution of 5‐HT2A receptor‐like protein in the rat pontomedullary brainstem. Areas containing the densest labeling included the trigeminal, facial, hypoglossal, dorsal vagal motor nuclei, medullary linear nucleus, and the inferior olive. In the nucleus ambiguus, labeled cells were located in the areas containing pharyngeal and laryngeal motoneurons. Intensely labeled cells were loosely scattered in the reticular formation adjacent to the raphe magnus and obscurus nuclei, in the gigantocellular region, in the caudal pedunculopontine and laterodorsal tegmental nuclei, dorsomedial pontine reticular formation, and nucleus subcoeruleus. In the nucleus prepositus hypoglossi, all vestibular, abducens, cuneate, and lateral reticular nuclei, labeled neurons commingled with unlabeled ones. Few labeled neurons were located in the rostral and caudal ventrolateral medulla and parvicellular reticular formation. In the nucleus of the solitary tract, two patches of diffuse labeling not associated with cellular profiles were present: one in the medial, and the other in the interstitial subnucleus. Similar diffuse labeling was present in the lateral parabrachial region and the lateral rim of the caudal spinal trigeminal sensory nucleus. No labeled cells were found in the locus coeruleus, dorsal raphe, superior olive, or area postrema. The distinct pontomedullary distribution of 5‐HT2A receptors, combined with the known arousal‐dependent activity of serotonergic neurons, show that these receptors may mediate post‐ and presynaptic effects in the motor, selected somatic and visceral sensory, oculo‐vestibulo‐precerebellar, and sleep‐related regions. J. Comp. Neurol. 418:323–345, 2000.

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.

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.

Pontine cholinergic mechanisms and their impact on respiratory regulation

Activation of pontomedullary cholinergic neurons may directly and indirectly cause depression of respiratory motoneuronal activity, activation of respiratory premotor neurons and acceleration of the respiratory rate during REM sleep, as well as activation of breathing during active wakefulness. These effects may be mediated by distinct subpopulations of cholinergic neurons. The relative inactivity of cholinergic neurons during slow-wave sleep also may contribute to the depressant effects of this state on breathing. Cholinergic muscarinic and nicotinic receptors are expressed in central respiratory neurons and motoneurons, thus allowing cholinergic neurons to act on the respiratory system directly. Additional effects of cholinergic activation are mediated indirectly by noradrenergic, serotonergic and other neurons of the reticular formation. Excitatory and suppressant respiratory effects with features of natural states of REM sleep or active wakefulness can be elicited in urethane-anesthetized rats by pontine microinjections of the cholinergic agonist, carbachol. Carbachol models help elucidate the neural basis of respiratory disorders associated with central cholinergic activation.

Role of chloride‐mediated inhibition in respiratory rhythmogenesis in an in vitro brainstem of tadpole, Rana catesbeiana.

1. The isolated brainstem of larval Rana catesbeiana maintained in vitro generates neural bursts that correspond to the lung and gill ventilatory activity generated in the intact specimen. To investigate the role of chloride channel‐dependent inhibitory mechanisms mediated by GABA(A) and/or glycine receptors on fictive lung and gill ventilation, we superfused the isolated brainstems with agonists, antagonists (bicuculline and/or strychnine) or a chloride‐free solution while recording multi‐unit activity from the facial motor nucleus. 2. Superfusion with the agonists (GABA or glycine) produced differential effects on frequency, amplitude and duration of the neural bursts related to lung and gill ventilation. At a GABA or glycine concentration of 1.0 mM, fictive gill bursts were abolished while fictive lung bursts persisted, albeit with reduced amplitude and frequency. 3. At the lowest concentrations used (1.0‐2.5 microM), the GABA(A) receptor antagonist bicuculline produced an increase in the frequency of lung bursts. At higher concentrations (5.0‐2.0 microM) bicuculline produced non‐specific excitatory effects. The glycine antagonist strychnine, at concentrations lower than 5.0 microM, caused a progressive decrease in the frequency and amplitude of the gill bursts and eventually abolished the rhythmic activity. At higher concentrations (7.5 microM), non‐specific excitatory effects occurred. Superfusion with bicuculline (10 microM) and strychnine (5 microM) combined abolished the neural output for gill ventilation but increased the frequency, amplitude and duration of lung bursts. 4. Superfusion with Cl(‐)‐free solution also abolished the rhythmic neural bursts associated with gill ventilation, while it significantly increased the amplitude (228 +/‐ 51%; P < 0.05) (mean +/‐ S.E.M.) and duration of the lung bursts (3.5 +/‐ 0.1 to 35.3 +/‐ 3.7 s; P < 0.05) and improved the regularity of their occurrence. 5. We conclude that different neural systems generate rhythmic activity for lung and gill ventilation. Chloride‐mediated inhibition may be essential for generation of neural bursts associated with gill ventilation. In contrast, the burst associated with lung ventilation can be generated in the absence of Cl(‐)‐mediated inhibition although the latter plays a role in shaping the normal lung burst.

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.

Serotonin receptor mRNA expression in the hypoglossal motor nucleus.

Brainstem serotonin (5-HT)-containing cells are remarkable for their widespread axonal projections and having their highest activity during wakefulness and lowest during rapid eye movement sleep. One important site of action of 5-HT is on upper airway motoneurons. However, which of the 14 known 5-HT receptors mediate the effects is uncertain. We used the reverse transcriptase/polymerase chain reaction to detect mRNA for six distinct 5-HT receptors (1A, 1B, 2A, 2C, 3 and 7) in 50 nl micro-punches collected from the hypoglossal (XII) motor nucleus and, for comparison, from the viscerosensory nucleus of the solitary tract (NTS) in adult rats. The relative abundance of the distinct mRNAs was characterized by the minimal number of amplification cycles (25-40) necessary to detect a given mRNA. In the XII nucleus, mRNA for type 1B, 2A and 2C receptors was detectable after 29-31 cycles, detection of type 3 and 7 receptor mRNA required 33-35 cycles; and type 1A receptor mRNA was not detected. In the NTS, detection of mRNA for type 1B, 2C and 7 receptors required 31-33 cycles; type 1A receptor mRNA required 39 cycles; and type 2A receptor mRNA was not detected. The data from the XII nucleus demonstrate that not only the previously recognized type 1B, 2A and 2C receptors, but also type 3 and 7 receptors have the potential to mediate serotonergic effects in 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.