Dorsal medullary inspiratory neurons: effects of superior laryngeal afferent stimulation

In decerebrate paralyzed cats ventilated with a cycle-triggered pump, we examined the responses of inspiratory (I) neurons in the region of the ventrolateral nucleus tractus solitarius (NTS) to single electrical stimuli delivered to the ipsilateral superior laryngeal nerve (SLN). Sixty-five I neurons were classified as: I(-), I(0), I(+, early), I(+, late) or I(other) on the basis of responses to lung inflation, and as I(bulbophrenic) or I(non-bulbophrenic) on the basis of evidence of an excitatory projection to the contralateral phrenic motoneuron pool. The peristimulus histograms of contralateral phrenic activity showed an early peak of excitation with average latency of 4.9 +/- 0.1 ms (mean +/- S.E.M.), followed by depression at 7.3 +/- 0.2 ms, start of recovery from depression at 22.7 +/- 1.0 ms, and recovery to control levels at 28.4 +/- 1.1 ms. The peristimulus histograms of ipsilateral I unit activity showed an initial excitation (latency 2.9 +/- 0.3 ms), followed by spiking silence (latency 6.0 +/- 0.6 ms) and recovery to control discharge frequency at 38.8 +/- 3.6 ms. This time of inhibition was significantly longer than the time of phrenic depression, suggesting that other bulbophrenic excitatory projections are able to rapidly compensate for decreased NTS output. Subgroups of I neurons, as classified by lung inflation tests, did not differ significantly with respect to these timing variables. In contrast, latencies of excitation for I(bulbophrenic) neurons were significantly less than for I(non-bulbophrenic) neurons.     

Evidence for medullary projections of the intercostal nerve-elicited inspiratory termination

This study hypothesized that the ICN-elicited inspiratory termination reflex required synaptic activation in two distinct regions of the ventral respiratory group (VRG): (1) transitional (tVRG), and (2) pre-B?tzinger complex (pre-B?tC). Data from adult cats indicate that axons of passage associated with the ICN-elicited termination reflex traverse tVRG, but that relevant synaptic processing does not occur in this region. Furthermore, data indicate that neither synaptic nor axonal transmission within the pre-B?tC is required for the SLN- or ICN-elicited termination reflex.     

Central projections of the hamster superior laryngeal nerve

The superior laryngeal nerve (SLN) is known to innervate taste buds on the epiglottis of several mammalian species. Because of an increasing interest in the physiology of the gustatory system of hamsters, the brainstem projections of the SLN were investigated in this species. Crystallized HRP was applied to the proximal portion of the cut SLN or to one of its five distal branches. Anterograde transganglionic transport of HRP revealed afferent fibers of the SLN projecting into the ipsilateral solitary tract (ST) from 0.3 to 3.0 mm caudal to the dorsal cochlear nucleus (DCN), with the major area of termination in the nucleus of the solitary tract (NST) between 0.6 and 1.6 mm caudal to DCN. Some afferent fibers crossed the midline approximately 2.0 mm caudal to DCN to terminate contralaterally within the NST. Efferent cell bodies were retrogradely labeled within the nucleus ambiguus (NA) and in and around the more rostral portions of NST. There were five identifiable distal branches of SLN, termed A1, A2, M1, M2 and P, from anterior to posterior. Afferent fibers were carried in A2 and P, whereas efferent fibers were evident in all five branches. The heaviest projection from the NA occurred in the two middle branches (M1 and M2) and that from the NST in the posterior branch (P). Afferent projections of the Xth cranial nerve, along with those from the VIIth and IXth, into the NST provide a neural substrate for the integration of sensory inputs related to a number of oral and respiratory reflexes.     

Convergence of afferent inputs from the chorda tympani, lingual-tonsillar and pharyngeal branches of the glossopharyngeal nerve, and superior laryngeal nerve on the neurons in the insular cortex in rats

The responses of single neurons in the insular cortex to electrical stimulation of the chorda tympani (CT), lingual-tonsillar branch of the glossopharyngeal (LT-IXth) nerve, pharyngeal branch of the glossopharyngeal (PH-IXth) nerve, and superior laryngeal (SL) nerve were recorded in anaesthetized and paralyzed rats. Ninety-four neurons responding to stimulation of at least one of the four nerves were identified from the insular cortex. Most of the neurons were located in the posterior portion of the insular cortex; the mean location was 0.8 mm anterior to the anterior edge of the joining of the anterior commissure (AC) and was 1.4 mm dorsal to the rhinal fissure (RF). Of the 94 neurons, 84 (89%) received convergent inputs from two or more nerves, and the remaining 10 (11%) received inputs from one nerve. The neurons responding to the CT stimulation were distributed more anteriorly than those responding to other three nerves in the anterior-posterior dimension. Our results indicate that the neurons recorded mainly from the posterior portion of the insular cortex receive convergent inputs from the oropharyngolaryngeal regions.     

High-frequency and medium-frequency components of different inspiratory nerve discharges and their modification by various inputs

In decerebrate paralyzed cats, spectral analysis was performed on simultaneous recordings of efferent inspiratory nerves (phrenic, recurrent laryngeal, hypoglossal). Spectral peaks were present both in the high-frequency (HFO) range (50-100 Hz) and the medium-frequency (MFO) range (20-50 Hz). Different activities were coherent only in the HFO range, indicating that the HFOs arise in a common inspiratory pattern generator that drives the different motoneuron populations, whereas the MFOs are specific to different systems.     

Morphological study of long axonal projections of ventral medullary inspiratory neurons in the rat

The aim of this study was to examine medullary and spinal axonal projections of inspiratory bulbospinal neurons of the rostral ventral respiratory group (VRG) in the rat. A direct visualization of long (9.8–33 mm) axonal branches, including those projecting to the contralateral side of the medulla oblongata and the spinal cord, was possible due to intracellular labeling with neurobiotin and long survival times (up to 22 h) after injections. Seven of the nine labeled neurons had bilateral descending axons, which were located in discrete regions of the spinal white matter; ipsilateral axons in the lateral and dorsolateral funiculus, contralateral in the ventral and ventromedial funiculus. The collaterals issued by these axons at the mid-cervical level formed close appositions with dendrites of phrenic motoneurons, which had also been labeled with neurobiotin. None of these collaterals crossed the midline. The significance of this finding is discussed in relation to the crossed-phrenic phenomenon. Additional spinal collaterals were identified in the C₁ and T₁ segments. Within the medulla, collaterals with multiple varicosities were identified in the lateral tegmental field and in the dorsomedial medulla (in the hypoglossal nucleus and in the nucleus of the solitary tract). These results demonstrate that inspiratory VRG neurons in the rat have some features which have not been previously described in the cat, including frequent bilateral spinal projection and projection to the nucleus of the solitary tract. In addition, this study shows that intracellular labeling with neurobiotin offers an effective way of tracing long axonal projections, supplementing results previously obtainable only with antidromic mapping, and providing morphological details which could not be observed in previous studies using labeling with horseradish peroxidase.     

Effects of intravenous bicuculline and strychnine on inspiratory inhibitory responses in the cat

Single shock stimulation of the superior laryngeal nerve (SLN), intercostal nerve (ICN), phrenic nerve (PN) or within the medullary respiratory groups (DRG-VRG) produces a transient, short-latency attenuation of inspiratory motor activity. Trains of stimuli delivered to SLN and ICN cause premature termination of inspiration. This study examined involvement of glycine and GABAA receptors in these reflex inhibitions. Experiments were conducted in decerebrate, vagotomized, and paralyzed cats. Control responses of left PN activity to threshold single shock stimulation of SLN, PN, ICN and the DRG-VRG were recorded and the thresholds for SLN- and ICN-evoked inspiratory termination were determined. Five min after intravenous injection of bicuculline (1 mg/kg) or strychnine (50 micrograms/kg), the responses to stimulation were again recorded. This procedure was reiterated until the cumulative dose elicited marked convulsions. Neither drug affected the inspiratory terminating reflexes. Systemic bicuculline had no effect on transient inspiratory inhibition. However strychnine prolonged the onset latency and the duration of all four inhibitory responses. Since the degree of transient inhibition was not lessened (only delayed), it appears that these inspiratory inhibitory reflexes do not rely exclusively on actions of glycine or GABAA receptors.     

Effects of ethmoidal nerve stimulation on respiration-related neurones in the dorsal medulla of the cat

Stimulation of the nasal mucosa produces a number of respiratory reflexes the afferent limb of which is provided by the ethmoidal nerve, a branch of the trigeminal nerve. In the cat this nerve terminates within the trigeminal nucleus. It has no direct projection to brainstem respiratory centres. This study examines the response of respiratory-related neurones in the nucleus of the solitary tract (NTS) to ethmoidal stimulation. It demonstrates that these neurones show both excitatory and inhibitory responses to ethmoidal stimulation. Thus, the NTS appears to be involved in respiratory reflexes initiated by stimulation of the nasal mucosa.     

Medium-frequency oscillations dominate the inspiratory nerve discharge of anesthetized newborn rats

In this study we examined the synchronization of the discharge of phrenic and recurrent laryngeal motoneurons in anesthetized rat pups 14 to 36 days of age and kittens, 14–15 days old. We found that the inspiratory nerve activity consisted of synchronized bursts separated by 20–35 ms, corresponding to medium-frequency oscillations (MFO). Accordingly, the autospectra of the neurograms had two peaks, one at the respiratory rate and the other between 22.8–43.0 Hz. No significant coherence was found between MFOs in the discharges of different nerves. High-frequency oscillations (HFO) characteristic for the adult inspiratory nerve activity were not present in the newborn rats. These findings demonstrate that phrenic nerve discharge of rat pups, like that of kittens and piglets, is in the MFO range, and suggest that MFO activity is an index of an early developmental stage of the respiratory system.     

Evidence for a tonic GABA-ergic inhibition of excitatory respiratory-related afferents to presympathetic neurons in the rostral ventrolateral medulla

The effect of blockade of ionotropic GABA and glutamate receptors in the rostral ventrolateral medulla (RVLM) on the relationship between phrenic nerve, splanchnic sympathetic nerve and lumbar sympathetic nerve activities was examined in urethane anesthetized, paralyzed and vagotomized Sprague-Dawley rats. Bilateral microinjection of the GABA-A receptor antagonist, bicuculline (4 mM, 100 nl), into the RVLM dramatically, and almost exclusively, increased the post-inspiratory related discharge in both splanchnic sympathetic nerve and lumbar sympathetic nerve activities and elicited hypertension with fluctuations of arterial pressure phase locked to the discharge of the phrenic nerve. Subsequent bilateral microinjection of kynurenate, a non-selective ionotropic excitatory amino acid receptor antagonist (50 mM, 100 nl), into the RVLM significantly attenuated the sympathoexcitation and hypertension evoked by injection of bicuculline. This was accompanied by an abolition of the post-inspiratory related burst discharge of splanchnic sympathetic nerve and lumbar sympathetic nerve activities. These data suggest that the GABAergic inputs to RVLM tonically inhibit glutamatergic inputs from central respiratory neurons that normally act to increase the firing of presympathetic neurons in the RVLM. Inputs from post-inspiratory neurons appear to be an especially potent excitatory synaptic drive to the presympathetic neurons in the absence of the GABAergic inhibition.     

Contribution of free nerve endings in the laryngeal epithelium to CO2 reception in rats

The superior laryngeal nerve (SLN) contains CO2-sensitive fibers. In the laryngeal epithelium, two candidates for CO2 reception have been identified, namely the intraepithelial free nerve endings and the taste buds. To elucidate the contribution of free nerve endings to CO2 reception, electrophysiological activities were recorded during various stages of regeneration of nerve endings following SLN-crush in rats. The left SLN was crushed surgically and maintained from 4 to 40 days for regeneration of nerve endings. Laryngeal sections were processed for immunohistochemical staining of protein gene product 9.5 to observe regeneration of free nerve endings and taste buds in the epithelium. By day 4 after SLN-crush, both the free nerve endings and taste buds had disappeared. Regeneration of the free nerve endings was recognized from day 8, while that of the taste buds started at day 16. On day 40, the number of taste buds on SLN-crush side was similar to that on the untreated side. Electrophysiological recording of SLN throughout the regeneration period (excluding day 4), showed response to intralaryngeal 9% CO2 (stimulation or inhibition) whether or not taste buds were present. Our results showed intralaryngeal CO2 reception without taste bud involvement, indicating that the free nerve endings in the laryngeal epithelium are receptive to intralaryngeal CO2.     

Response properties of fibers in the hamster superior laryngeal nerve

The purpose of the present investigation was to record electrophysiological responses from single fibers in the hamster superior laryngeal nerve (SLN) that were responsive to chemical stimulation of the larynx. Twenty chemical solutions, commonly used in studies of mammalian gustatory physiology, were applied to taste buds on and around the epiglottis. These stimuli were dissolved in physiological saline. Responses were the number of impulses elicited over a 15-s period following stimulus onset, above or below the background activity elicited by a previous rinse with saline. Unlike fibers in the hamster chorda tympani or glossopharyngeal nerves, SLN units were not easily classifiable into response types. Excitatory stimuli were primarily acids and bitter-tasting stimuli, with the order of their effectiveness being urea tartaric acid > HCl > KCl > citric acid > caffeine > quinine hydrochloride > acetic acid. The sweet-tasting stimuli and most salts other than KCl were primarily inhibitory, with the order of inhibitory effectiveness being CaCl₂ > sucrose > fructose > LiCl > NaNO₃ > Li₂SO₄ > NaCl. A hierarchical cluster analysis of fibers yielded no distinct clusters, yet differing sensitivities across the fibers were suggested. SLN fibers are highly responsive to sour and bitter stimuli, although they are not sensitive to fine differences in taste quality, as are fibers in other gustatory nerves.     

Upper airway motor outputs during vomiting versus swallowing in the decerebrate cat

Swallowing and vomiting are antagonistic motor acts; nevertheless, vomiting can be immediately followed by swallowing. The purpose of this study was to clarify the interrelationship between these two behaviors, particularly in regard to comparing the upper airway motor patterns at the end of the expulsion phase with those during subsequent swallowing. Experiments were conducted using both paralyzed and non-paralyzed decerebrate cats, in which recordings were obtained either from upper airway muscles, the diaphragm and abdominal muscles or from the nerves that innervate those muscles. The activity patterns of most nerves recorded in paralyzed animals were consistent with the behavior recorded in non-paralyzed animals from the muscles innervated by those nerves, with the exception of the cricothyroid and stylopharyngeus muscles. Vomiting can be divided into a series of retches followed by expulsion, which itself can be further subdivided into three phases. The final stage of expulsion, characterized by burst-like exaggerated activity of the laryngeal elevator thyrohyoid and the pharyngeal constrictors, proved to be different from pharyngeal swallowing, as judged from differences in the spatio-temporal patterns of the upper airway motor outputs. However, post-vomiting swallowing activity was still observed even after total deafferentation of the laryngeal and pharyngeal areas in paralyzed animals. It is therefore likely that the central processes for vomiting and swallowing closely relate in generating these two behaviors.     

Presence of neuronal cell bodies in the sympathetic pressor areas of dorsal and ventrolateral medulla inhibiting phrenic nerve discharge in cats

To examine whether neuronal cell bodies (perikarya) in the pressor areas of dorsal medulla or ventrolateral medulla also modulate respiratory function, phrenic nerve activity was monitored and correlated with the pressor response in chloralose-urethane anaesthetized cats. The animals were paralyzed and artificially ventilated maintaining the end-tidal fractional concentration of CO₂ at 0.04–0.05. The same pressor point in the dorsal or ventrolateral medulla was stimulated electrically (rectangular pulse of 20–200µA, 80 Hz and 0.5 ms) and then chemically (0.25–0.5 M sodium glutamate, 80–200 n1). Areas producing pressor effects in either the dorsal or ventrolateral medulla produced a current-dependent decrease of phrenic discharge. The decrease in Pna was significant when the electrical current reached 50µA or above. It occurred immediately before the onset of increase in blood pressure. Injection of glutamate to the same pressor point in either the dorsal or ventrolateral medulla produced a similar decrease in phrenic nerve activity. The results suggest that in the pressor areas of dorsal and ventrolateral medulla there are neuronal perikarya that can increase systemic arterial presssure and inhibit phrenic nerve activity. Whether the same or separate neurones are responsible for these responses is to be determined.     

Fast rhythms in the discharges of medullary inspiratory neurons

The discharges of 44 medullary inspiratory (I) neurons in decerebrate paralyzed cats were studied using interval and spectral analysis. Most neurons had a rhythm in their discharge. In 31 the rhythm was at the frequency of, and coherent to, the high-frequency oscillations (HFOs) of I nerves, and in 7 the rhythm was in the range of medium-frequency oscillations (MFOs), with no coherence to nerve MFOs. Thus, correlated HFOs are characteristic of the I system at all levels, whereas MFOs are uncommon in medullary neurons and seem to be unrelated to general mechanisms.     

Projections of the internal branch of the superior laryngeal nerve of the cat

The internal branch of the superior laryngeal nerve (iSLN) conveys sensory afferent information from receptors located in the laryngeal mucosa. The objectives of this study were: to determine the specific anatomical location of iSLN cell bodies within the nodose ganglion; to ascertain whether the jugular ganglion might also contain iSLN afferent bodies; to determine whether the iSLN contains sympathetic efferents originating in the cervical sympathetic ganglion; to determine whether the cell bodies of these efferents, if present, are localized within a specific region of this ganglion and to trace the transganglionic projection of iSLN afferents into the brain stem. Horseradish peroxidase was applied to the iSLN in ten adult cats. Following a survival period of 72 hours, the animals were sacrificed and the tissue was processed according to the tetramethylbenzidine method. Reaction product was localized in the rostral end of the nodose ganglion extending into the exiting vagus nerve, in the caudal end of the jugular ganglion and in the posterior portion of the cervical sympathetic ganglion. Transganglionic projections to the nucleus tractus solitarius were localized primarily in the dorsolateral subnucleus with substantial amounts of reaction product also in the intermediate and interstitial subnuclei. Except for a small bilateral projection observed in the commissural subnucleus, no other projections were seen to any other brain stem structures.     

Anatomical localization of the cells of origin of efferent fibers in the superior laryngeal and recurrent laryngeal nerves of the dogs

Using horseradish peroxidase, we identified the cells of origin of motor fibers in the superior laryngeal nerves of dogs. Cells giving rise to fibers in the superior laryngeal nerve were found in the dorsal motor nucleus and the nucleus ambiggus, whereas cells giving rise to fibers in the recurrent laryngeal nerve were found in the nucleus ambiguus and nucleus retroambigualis, but usually not in the dorsal motor nucleus.     

Respiratory rhythmicity after extensive lesions of the dorsal and ventral respiratory groups in the decerebrate cat

It was previously demonstrated that extensive destruction of the regions of the dorsal (DRG) and rostral portions of the ventral respiratory groups (VRG) in the medulla does not disrupt respiratory rhythmicity in the anesthetized cat. The present experiments examined if either higher CNS structures or the caudal expiratory VRG might have been responsible for preserving rhythm in those studies. Results indicate that the DRG and VRG are not required for respiratory rhythmicity in the midcollicularly decerebrated cat.     

Role of nucleus retroambigualis in respiratory reflexes evoked by superior laryngeal and vestibular nerve afferents and in emesis

An ascending projection from the medullary nucleus retroambigualis (NRA) has recently been described as important for the control of the upper airway during vocalization. We evaluated the importance of this projection in other behaviors by making localized injections of the neurotoxin kainic acid in the NRA in decerebrate cats, most of which were paralyzed and artificially ventilated. In contrast to its importance for vocalization, the NRA is not essential for activation of upper airway musculature during respiration, swallowing, vomiting, or reflexes elicited by superior laryngeal or vestibular nerve afferents. However, kainic acid injections in the NRA and adjacent reticular formation prolonged the inhibitory phrenic motoneuronal response to superior laryngeal nerve stimulation and abolished or reduced abdominal motoneuronal responses during respiration, vomiting, and superior laryngeal nerve stimulation. Thus, of the behaviors we investigated, the importance of the ascending projection from the NRA appears to be limited to vocalization, while descending projections from the NRA region are important in a number of behaviors.