Involvement of the motor trigeminal nucleus in respiratory phase-switching in the cat.

We investigated the hypothesis that the motor trigeminal nucleus, consisting of expiratory motoneurons, might be influential in termination of inspiration. We addressed the issue by comparing the effects on neural respiration of a reversible, unilateral, pharmacologic blockade of the motor trigeminal nucleus (5M), the medial parabrachial nucleus (PB), and of other nearby structures that are neutral for respiration in anesthetized, vagotomized, paralyzed, and ventilated cats. The blockade was achieved by microinjections of 2% xylocaine, laced with Pontamine Sky Blue to identify sites of injections, from the tip of a penetrating microelectrode. Integrated phrenic neurograms were recorded to quantify the time of neural inspiration (TI), expiration (TE), and the peak phrenic amplitude. We found that blockade of the 5M caused a pattern of apneustic respiration, consisting of a selective prolongation of inspiratory phases that were interrupted by short expiratory pauses. In contrast, blockade of the PB resulted in a prolongation of both TI and TE, which corresponds to a mere slowing of respiration. The results confirmed important functions of the rostral pons in ventilatory control but pointed to the 5M rather than PB as a structure underlying the inspiratory off-switch. We conclude that the motor trigeminal nucleus may have a part in the pontine pneumotaxic mechanism.

Changes in the central respiratory rhythm following pharmacological blockade of the nucleus parabrachialis medialis in the rabbit.

Experiments were performed in halothane-anesthetized, paralyzed, bilaterally vagotomized and artificially ventilated rabbits. Arterial blood pressure, expiratory carbon dioxide concentration, and electrical activity of the right phrenic nerve were recorded prior to and after xylocaine microinjection to the left nucleus parabrachialis medialis (NPBM). The location of the xylocaine blockade was verified histologically. It was found that blockade of the NPBM results in a decelerated respiratory rhythm due to lengthening of both phases of the respiratory cycle. These results do not corroborate the role of NPBM in the regulation of respiration as postulated by Bertrand and Hugelin (1971).

Horseradish peroxidase localization of the mylohyoid motoneurones in the rabbit.

Experiments were performed with anaesthetized (urethan and chloralose) spontaneously breathing rabbits. Horseradish peroxidase (HRP) was injected (1 mg) with a Hamilton syringe into the end of the sectioned mylohyoid nerve. The HRP-labelled neurones could be localized in the motor nucleus of the trigeminal nerve (N.V.mt.). It was found that the labelled neurones are present in the caudal and intermediate part of the nucleus. However, their largest agglomerations were noted in the caudal part, where they occupied the entire frontal section of the nucleus. It is assumed that the N.V.mt. directing their axons to the muscles of the upper respiratory airways, simultaneously send collaterals to the central respiratory neurones participating in the regulation of the respiratory rhythm. According to this hypothesis the neurones of the N.V.mt. may ensure synchronisation between the upper and lower respiratory system.

Motor nucleus of the V-th nerve and the control of breathing. Breuer-Hering reflexes and apneustic breathing.

Earlier studies from this department have demonstrated that neurones of the V-th nerve motor nucleus (NVmt) have oligosynaptic, inhibitory output to the inspiratory motoneurone themselves under the influence of a polysynaptic input from vagal afferents. To check the hypothesis that NVmt is a part of the pneumotaxic mechanism, we studied the effects of pharmacological microblockade of the NVmt on Breuer-Hering reflexes in halothane-anesthetized, paralyzed and artificially ventilated rabbits. Activities of NVmt neurones and phrenic nerve firing were recorded. Acid-base balance was controlled and histologic examinations were routinely performed. Expiratory activities were regulatory found in NVmt. Its blockade elicited a typical apneustic breathing. During the blockade the Breuer-Hering reflexes gave "paradoxical" effects: an increase in central respiratory frequency following inflation, inspiratory apneusis in response to deflation. We conclude that the NVmt is an important component of the pneumotaxic mechanism or even the anatomical substrate of the pneumotaxic "centre".

Motor nucleus of the V-th nerve and the control of breathing (Breuer-Hering reflexes and apneustic breathing).

Earlier studies from this department have demonstrated that neurones of the V-th nerve motor nucleus (NVmt) have oligosynaptic, inhibitory output to the inspiratory motoneurone pool being themselves under the influence of a polisynaptic input from vagal afferents. To check the hypothesis that NVmt is a part of the pneumotaxic mechanism, we studied the effects of pharmacological mictroblockade of the NVmt on Breuer-Hering reflexes in halothane-anaesthetized, paralyzed and artificially ventilated rabbits. Activities of NVmt neurones and phrenic nerve firing were recorded. Acid-base balance was controlled and histologic examinations were routinely performed. Expiratory activities were regularly found in NVmt. Its blockade elicited a typical apneustic breathing. During the blockade the Breuer-Hering reflexes gave "paradoxical" effects: an increase in central respiratory frequency following inflation, inspiratory apneusis in response to deflation. We conclude that the NVmt is an important component of the pneumotaxic mechanism or even the anatomical substrate of the pneumotaxic "centre".

Breuer-Hering reflexes in ketamine-induced apneustic breathing in the rabbit.

The effects of ketamine on the activities of the mylohyoid nerve (a branch of the Vth nerve) and of both phrenic nerves were investigated in rabbits anaesthetized with halothane, paralyzed and artificially ventilated. Intravenous administration of ketamine elicited a marked prolongation of the phrenic inspiratory discharge (without significantly affecting its amplitude) and a depression of the mylohyoid expiratory activity. An elimination of the volume-related input from the lungs ("no-inflation manoeuvre") or deflation elicited under these conditions typical apneustic pattern of breathing. The response to tracheal occlusion at peak-inspiration was "classical". We conclude that ketamine inhibits the Vth nerve motor nucleus which is not only an important component of the central inspiratory-inhibitory neurones but also a "relay station" between the vagal and the central inspiratory "off-switch" mechanisms.

Studies on the mechanism of obstructive sleep apnea.

Several observations indicate that the mylohyoid nerve (NV) may play a crucial part in the mechanisms of obstructive sleep apnea (OSA). The activity of this nerve normally counteracts the collapse of the upper airways during inspiration. Any reduction in this activity may thus facilitate the occurrence of apnoeic spells. We have studied the effects of ethanol and lung inflations on the activity of NV recorded along with the activities of phrenic and facial nerve in rabbits anaesthetised with chloralose-urethan, paralyzed with curare and artificially ventilated. Under the control conditions the NV exhibited phasic expiratory activity; after vagotomy and additional, inspiratory component was observed. Lung inflation strongly enhanced the expiratory activity of NV whereas both the phrenic and facial nerve activities (both phasic-inspiratory) were typically inhibited. An injection of 5 ml of 20% ethanol very strongly inhibited the NV activity. The results may confirm the importance of NV in the mechanism of OSA. The well-known fact that OSA patients are particularly sensitive to alcohol finds support in the response of NV activity to ethanol injection. The analysis of the patterns of discharges of the three outputs from the respiratory controller may additionally suggest that the Vth nerve nucleus is involved in the control of respiratory pattern.

Respiratory activity of the pons and its influence on breathing in the guinea pig.

The localization of respiratory activity in the pons of the anaesthetized guinea pigs has been studied. The respiration related units were localized in/or close to the following anatomical structures: nucleus parabrachialis, nucleus trigemini motorius and nucleus reticularis pontis caudalis. The influence of the pontine respiratory activity on the pattern of breathing was studied by the use of a reversible pharmacological block (local anaesthetic) of these structures. The application of lignocaine to all investigated sites of the pons caused the prolongation of both respiratory phases, then an effect comparable to vagotomy. In the vagotomized animal the focal block of nucleus parabrachialis has never produced apneusis (pronounced prolongation of inspiration and very short lasting expiration). Since the removal of some pontine structures involved in the breathing control caused the depression of respiration, we concluded that the respiratory activity of the pons in the guinea pigs exerts an excitatory influence on the rhythm generating mechanism in this animal.

The split-respiratory centre in the cat: responses to hypercapnia.

In 22 cats anaesthetized with halothane, vagotomised, paralyzed with d-tubocurarine and artificially ventilated, the medulla was split in the midline and the response of phrenic motoneurones, efferent vagus nerve and medullary respiratory neurones to hypercapnia was studied. In 18 successful experiments the mid-sagittal incision abolished all electrical activities but an inhalation of a 5% CO2-oxygen mixture promptly restored rhythmic firing in both the medullary units and efferent nerves when PaCO2 reached 55 mm Hg on the average (mean pH = 7.20). Switching the ventilation back to normocapnia was followed by a gradual disappearance of the activities usually when PaCO2 and pH returned to control values. We conclude that splitting the brainstem in cat affects the respiratory rhythmogenesis, presumably by increasing the CO2-threshold of medullary respiratory neurones.

The effects of pontine or bulbar transection on the respiratory pattern in the rabbit.

Rabbits were anesthetized with halothane, paralyzed with d-tubocurarine and mechanically ventilated at eucapnic level. The activity of both phrenic nerves was recorded before and after brainstem transection in animals with vagi intact and then cut. The effect of transection depended upon its level: midpontine transections elicited an apneustic pattern of firing in phrenic nerves prior to vagotomy and a considerable prolongation of expiratory time after vagotomy. Transections through the rostral part of the n. facialis in animals with vagi intact increased the duration of both inspiratory time and - even more - expiratory time. Vagotomy abolished the activities of phrenic nerves. Transection through the n. retrofacialis resulted in fast, irregular, low-amplitude volleys of phrenic nerves; vagotomy elicited inspiratory apneusis. Low frequency electrical stimulation of the vagus nerve enhanced this tonic firing, whereas high-frequency stimulation interrupted it. The results indicate that medullary neurons are capable of generating the basic respiratory pattern, and that the vagal input is integrated at the bulbar level in the rabbit.

Phrenic motoneurone activity in split-brainstem cats and monkeys.

A mid-line incision of the lower brainstem (from 2 mm caudal to 14 mm rostral to the obex) was performed in 14 cats and (from 2 mm caudal to 7 mm rostral to the obex) in three green monkeys and its effects on the respiratory activity of both phrenic nerves were studied under similar experimental conditions (halothane anaesthesia, artificial ventilation). Splitting the medulla in cats abolished the phrenic nerve activity under eupnoeic conditions, but hypercapnia or (with more rostral cuts) hypercapnia with hypoxia restored synchronous, rhythmic firing in both phrenic nerves. The response to splitting in eupnoeic monkeys was qualitatively different and consisted in a 'desynchronisation', i.e. independent firing of both phrenic nerves at different rhythms and patterns. It is concluded that cats and monkeys have basically different functional organisation of the respiratory controller.

The effects of brainstem transection on respiratory activity in the rabbit.

Mid-sagittal incisions and transverse hemisections of the medulla were performed in rabbits anaesthetized with halothane and artificially ventilated at eupnoeic level. A midsagittal incision extending from the obex to (at least) 4 mm rostral elicited asynchronous (independent) firing in both phrenic nerves. A subsequent transverse hemisection interrupted the activity of the ipsilateral phrenic nerve. An electrical stimulation of the vagal input temporarily restored this activity. A midsagittal incision, which had not completely separate both halves of the medulla, did not affect the synchrony of firing of both phrenic nerves; in these cases also the transverse hemisection did not arrest the phrenic nerve activity. It is concluded that synchronous activity of the inspiratory output depends upon intact connnections crossing in the lower medulla and that proper generation of respiratory pattern depends upon a critical level of excitation created by neurones at rostral pontine and suprapontine levels of the CNS, neighboring structures (presumably including the medial reticular formation) and/or vagal input. A concept of a "critical mass" of active neurones is introduced.

Respiratory activity generated by a split brainstem preparation of the rabbit.

Mid-sagittal incisions of the brainstem were performed in anaesthetized, vagotomised, paralysed and artificially ventilated rabbits. The activity of both phrenic nerves, blood pressure and end-tidal CO2 percent were continuously monitored. The results fell into two groups: in one of them a relatively small separation of both halves of the medulla extending from the obex to at least 4 mm rostrally elicited asynchronous firing in both phrenic nerves ("split respiratory centre"). In the other group, in which the incisions were placed either more caudally or more rostrally, or when small strands of the nervous tissue were left to provide connections between both sides of the brainstem, this. phenomenon did not appear. It is concluded that there are two symmetrical respiratory networks in both halves of. the brainstem and that their synchronous firing depends upon intact connections extending from the obex to the caudal end of nucleus of VII nerve.

Effects of reversible elimination of some bulbar structures on the generation of respiratory pattern in rabbits.

The regions of (i) nucl. n. VII (N. VII), (ii) nucl. retrofacialis (N. r. VII), (iii) nucl. Ambiguus (A.c) and (iv) nucl. ret. parvocellularis (n.rp) were explored with microelectrodes and then the active sites were injected with 5 microliters of lignocaine. The activity of both phrenic nerves, blood pressure and end-tidal CO2 percent were recorded throughout the experiment. Blockade of the n.VII region elicited apneustic discharge in both phrenic nerves; blockade of N.r.VII gave variable frequency responses but always reduced phrenic amplitude of discharges; microinjection of lignocaine into the region of A.c. invariably accelerated central respiratory rhythm, the amplitude of discharges being reduced predominantly contralaterally to the injection site; blockade of the R.pc elicited a dramatic prolongation of expiratory pause, decrease in amplitude and rate of rise of phrenic activity. All effects disappeared within 30 min after injection and histological examinations revealed only minor lesions at the sites of injections. It is concluded that the respiratory neurons of the medulla form a complex, strongly interconnected network.

The role of timing and magnitude of the vagal input in controlling the phrenic output in rabbits and baboons.

The effects of a short train of electrical impulses applied to the central stump of a cut vagus nerve at various moments of the central respiratory cycle were studied in 28 rabbits and 3 baboons. The animals were anaesthetized (halothane), vagotomized, paralyzed and artificially ventilated. Stimulation in inspiration elicited always an inhibitory effect (latency 8-9 ms) the magnitude of which increased towards the end of inspiration. The amplitude and duration of inhibition increased also with the frequency of impulses and/or the duration of the volley. Stimulation in expiration shortened this phase after latency being shorter towards the end of expiratory pause. It is suggested that excitation of thin myelinated vagal fibres has a facilitatory effect on the inhibitory response to information being conducted along thick myelinated fibres during inspiration.

Generation of respiratory pattern in the rabbit -- brainstem transections revisited.

The effects of unilateral transection of the lower brainstem on the generation of central respiratory rhythm and its amplitude were studied in halothane anesthetized, vagotomized, paralyzed and artificially ventilated rabbits. Transections involving N. VII or rostral part of N. r VII elicited apneustic pattern of discharge in both phrenic nerves. Lesions made at more caudal levels (1.5-3.5 mm rostral to obex) restored a more normal pattern of discharge as far as frequency is concerned, but reduced the amplitude, particularly in the ipsilateral phrenic nerve. Sections below the obex abolished the activity of the ipsilateral phrenic n. Since classical midpontine sections did not, elicit apneustic discharge, it is possible that this pattern is produced by modifications in the number and functional connections of inspiratory-inhibitory neurons.

Pentobarbitone effects on respiration related units; selective depression of bulbopontine reticular neurones.

Respiration related units (RRUs) were recorded in cats, locally anesthetized, vagotomized and immobilized, under two different experimental conditions: (1) receiving pentobarbitone intravenously at a dose inducing slight but highly significant changes in phrenic discharge; (2) unanesthetized, with a spinal section at C6. RRU characteristics were analysed in unitary volumes constituted by cubes of a matrix including the brain stem. Four parameters of RRU discharges were compared in cubes = RRU density (RRUD), RRU proportion and two indices of respiratory modulation. Cubes were sampled in: (1) dorsal and ventral bulbar respiratory nuclei, (2) pneumotaxic centre (PC), and (3) seven fields of the bulbopontine and mesencephalic reticular formation. Using the paired sample method for comparing data cube by cube in the two conditions it was shown that under pentobarbitone, RRU activity was profoundly depressed in the reticular formation surrounding dorsal and ventral bulbar respiratory nuclei, in the region bridging the gap between these and the pneumotaxic centre and extending from the pneumotaxic level to the decussation of the brachium conjunctivum. In contrast RRU activity was unchanged at the level of dorsal and ventral bulbar respiratory nuclei and in the nucleus parabrachiallis medialis (NPBM) and was increased in the Kölliker-Fuse nucleus (KF). In the mesencephalic reticular formation, increased activity was observed for non-modulated units and to a larger extent for units driven by the bulbopontine respiratory system.