Regulation of ventilatory responses in vagotomized cats by caudal brainstem sites.

Respiratory responses to hypercapnia and isocapnic hypoxia were evaluated in vagotomized decerebrate cats prior to and following lesions in the midline at the pontomedullary junction. Subsequent to these lesions, frequency responses to hypercapnia were significantly elevated whereas the concomitantly determined tidal volumes were significantly lowered. In the post-lesion phase, the durations of inspiration, expiration and the total respiratory cycle at maximum hypercapnia-induced tidal volumes were lower than comparable values at maximum tidal volumes in the pre-lesion phase. The same lesions produced no significant alteration of respiratory responses to hypoxia. Mesencephalic or rostral pontile lesions caused no systematic changes in either hypercapnia- or hypoxia-induced responses. It is concluded that the lesions at the pontomedullary junction produce ventilatory alterations by interrupting a pathway interconnecting the caudal pontile apneustic center with the medullary respiratory complex. The possibility that this interruption produces an alteration in the threshold of the inspiratory off-switch mechanism of the brainstem respiratory controller is considered and discussed.     

Functions of the retrofacial nucleus in chemosensitivity and ventilatory neurogenesis

The hypothesis was evaluated that neurons within the retrofacial nucleus of medulla integrate afferent stimuli from the central chemoreceptors. In decerebrate, vagotomized, paralyzed and ventilated cats, activity of the phrenic nerve was monitored. Peak integrated phrenic activity increased in hypercapnia; the frequency of phrenic bursts typically declined slightly. The retrofacial nucleus was ablated by radio-frequency lesions or neurons within this nucleus were destroyed by microinjections of kainic acid. Results were similar following lesions or injections. Following unilateral ablations, peak phrenic activity was greatly reduced at normocapnia and hypercapnia; the frequency of phrenic bursts typically rose. Both frequency and peak phrenic activity fell further after the contralateral destruction with a cessation of all phasic phrenic discharge being observed in most animals. Injections of kainic acid in regions rostral, caudal or medial to the retrofacial nucleus produced no consistent changes in phrenic activity. We conclude that neuronal activities in the region of the retrofacial nucleus are important both in the integration of stimuli from the central chemoreceptors and in defining the discharge patterns of respiratory neurons.     

The role of neural connections crossed at the cervical level in determining rhythm and amplitude of respiration in cats and rabbits

The part played by crossed cervical pathways in determining respiratory rhythm and phrenic nerve amplitude was studied in rabbits and cats. The animals were anesthetized with halothane, paralyzed, vagotomized and mechanically ventilated. All decussating brain stem pathways were surgically interrupted. This resulted in asynchronous firing of the respiratory nerves on the left and right side of the body. However, both frequency and amplitude of the respiratory output depended on the mutual phase relationships due to neural connections crossed at the cervical level. Analysis of the mutual phase and amplitude relationships lead to the following conclusions. a. Phrenic motor neurons receive during inspiration a considerable amount of excitation via diffused pathways crossing the midline at C4-C6 level. b. Phrenic motor neurons are actively inhibited via crossed cervical pathways. c. The level of tonic bias transmitted via descending bulbo-spinal pathways significantly influences the magnitude of PhN output. d. Activity of phrenic motor neurons is transmitted back to the bulbar respiratory centers via an ascending spino-bulbar pathway.     

Properties of postganglionic sympathetic neurons with axons in phrenic nerve.

The aim of the study was to test the reflex and resting properties of postganglionic sympathetic neurons with axons located in the right phrenic nerve. The experiments have been performed on chloralose-anesthetized cats with both vago-aortic nerves cut. The somata or the postganglionic sympathetic neurons were located in the stellate ganglion. Axons of these neurons passed through the upper and lower phrenic nerve roots and through the phrenic nerve itself. The presence of cardiac and respiratory rhythmicities was detected in the activity of the phrenic postganglionic sympathetic neurons. Hyperventilation, which abolished burst discharges of the phrenic nerve, decreased the sympathetic activity by 14%. Systemic hypoxia (ventilating the animals for 2 min with 8% O2 in N2) increased the sympathetic activity threefold. The results of our experiments suggest that axons of the sympathetic neurons located in the right phrenic nerve could possibly be diaphragmatic muscle vasoconstrictors.     

Effects of hypothalamic lesions on the cardiorespiratory responses to muscular contraction

Recent studies have suggested that descending central command from a hypothalamic site (subthalamic locomotor region) is involved in the control of the respiratory and cardiovascular systems during exercise. The purposes of this study were to determine if this hypothalamic area provides tonic drive to the cardiorespiratory systems and modulates the cardiorespiratory responses to static muscular contraction. Anesthetized cats were studied before and after bilateral electrolytic lesioning of the subthalamic locomotor region. Muscular contraction was evoked by stimulation of the L7-S1 ventral roots. Bilateral hypothalamic lesions produced a fall in resting levels for respiratory frequency, minute ventilation, heart rate and arterial pressure. The lesions did not alter the significant increases in arterial pressure and minute ventilation produced by muscular contraction. However, larger heart rate and respiratory frequency responses to muscular contraction occurred post-lesion. We conclude that this area of the hypothalamus provides tonic drive to the cardiorespiratory systems. Furthermore, the subthalamic locomotor region may participate in the modulation of some cardiorespiratory responses to neural feedback from contracting muscles.     

Respiratory neural activity during long-term facilitation

Intermittent hypoxia results in a long-term facilitation (LTF) of respiratory efferent activity. The studies reviewed here presented data from both anesthetized and decerebrate, paralyzed, vagotomized, artificially ventilated adult cats. Multiple arrays of tungsten microelectrodes were used to record the concurrent responses of brain stem neurons that contribute to respiratory motor pattern generation. Spike trains were analyzed with firing rate histograms, peristimulus time histograms, cycle triggered histograms, spike triggered averages with multiunit phrenic efferent activity, cross correlation histograms, joint peristimulus time histograms and the gravity method. These studies addressed several hypotheses. (1) There is parallel processing of input from carotid chemoreceptors to the brain stem. (2) Respiratory related midline neurons are involved in the induction and maintenance of LTF. (3) There is a change in effective connectivity of brain stem neurons with LTF. (4) Neural networks involved in the induction and maintenance of LTF have patterns of synchrony that recur with a frequency greater than expected by chance.     

Diazepam potentiates postsynaptic inhibition in bulbar respiratory neurons of cats

The purpose of this study was to assess the effect of benzodiazepine on inhibitory postsynaptic potentials (IPSPs) of medullary respiratory neurons in decerebrate, paralyzed cats. Diazepam (0.05 and 0.1 mg/kg i.v.) reversibly increased the IPSP waves occurring during the inactive phase of the respiratory cycle in all inspiratory and postinspiratory neurons examined. Input resistances of these neurons were reduced at that phase. The reversal potential for the IPSP wave was unaltered. Intracellular injection of chloride ions reversed the IPSP to depolarization, and diazepam produced a purely depolarizing effect. The drug effects observed during the active phase of each neuron include a decrease in the firing rate and a shortening of the burst activity. The firing threshold and shape of these spikes, however, remained unaltered. These results suggest that diazepam depresses the bulbar respiratory neuronal activities specifically by potentiating the periodic postsynaptic inhibition.     

CO2-induced c-fos expression in medullary neurons during early development.

In this study, we characterized the responses of brainstem neurons to hypercapnic loading at 5, 15, and 40 postnatal days, using c-fos gene encoded protein (Fos), as a marker of neuronal activity. At any of these studied ages exposure to 10% CO2 for 1 h produced a significant increase in the number of activated neurons within the ventral and the dorsal aspects of the brainstem. In the ventrolateral aspect of the medulla oblongata, Fos positive cells were observed within the ventrolateral medulla, extending from the pontomedullary border to the decussation of the pyramids. In the most rostral regions, within the retrotrapezoid field, the number of Fos positive cells was lower than in caudal ventral medullary regions at the levels of the area postrema and the caudal to it. No age related differences were observed in the number of neurons exhibiting CO2-induced Fos expression. Fos positive cells were additionally observed in the lateral paragigantocellular and gigantocellular reticular nuclei, in the medullary midline complex, in the raphe pallidus and in the raphe obscurus. The number of activated cells in the midline neurons was higher at 5 than at 40 days of age. In the dorsal aspect of the medulla oblongata Fos positive neurons were observed mainly within the caudal nucleus tractus solitarius (nTS). Postnatal age had no effect on the distribution and number of nTS cells activated by hypercapnic loading. These findings indicate that neurons activated by increases in CO2/H+ concentrations appear to be well developed from the first days of postnatal life in maturing rat pups.     

Comparison of activities of medullary respiratory neurons in eupnea and apneusis

We evaluated patterns of antidromic latencies of medullary respiratory neurons in eupnea and apneusis to define how afferent influences from the pneumotaxic center regulate their activities. Apneusis was reversibly produced by cold block in decerebrate, vagotomized, paralyzed and ventilated cats. Most neurons, which discharged during all of eupneic inspiration or expiration, maintained the same pattern in apneusis. However, those active during only portions of these phases or spanning both changed markedly with alterations in periods of discharge, including tonic patterns or cessations of activity. Such marked changes were observed for activities of all laryngeal expiratory neurons. Upon termination of eupneic discharge, most bulbospinal and laryngeal neurons had transient peaks of latencies, indicating hyperpolarizations; declines from these peak values were greatly reduced in apneusis. Moreover, reflecting depolarizations, latencies of some inspiratory and expiratory neurons declined during eupneic expiration and inspiration, respectively; these declines were much reduced in apneusis. We conclude that the pneumotaxic center influences medullary respiratory neuronal activities not only at end-inspiration, but throughout the entire respiratory cycle.     

Ventrolateral pons mediates short-term depression of respiratory frequency after brief hypoxia

The respiratory response to hypoxia is dynamic in the adult anesthetized Sprague-Dawley rat. Hypoxia elicits acute increases in both tidal volume (VT) and respiratory frequency (fR) followed by short-term increases in VT and short-term decreases in fR. After brief hypoxia (<1 min), recovery of the breathing pattern is again dynamic, where both VT and fR decrease immediately, but where VT remains above, and fR drops below, baseline. These acute changes are followed by a short-term progressive decrease in VT and increase in fR to baseline. We have identified a potential neural mechanism that depends on the integrity of the ventrolateral (vl) pons. Our studies show that: (a) blockade of activity in the vl pons prevents the short-term decrease in fR after hypoxia (b) stimulation of the vl pons decreases fR, and (c) vl pontine expiratory neurons are activated after hypoxia. These neurons may not be acting through alpha(2) -adrenergic receptors, but their effect does depend on NMDA-type receptor function. We conclude that the vl pons is a critical element in the pontomedullary network that generates and modulates the fR response to acute hypoxia.     

Hyperoxic ventilatory responses of high altitude acclimatized cats

We have examined the effect of steady-state hyperoxia on the ventilation of sea level (SL) cats and cats acclimatized to simulated high altitude (HA) at 5500 m for three weeks. Three groups of cats were studied. In group I, the ventilatory responses to 10%, 21% and 100% O2 were studied at SL, and after acclimatization to HA, the ventilatory responses to 10% and 100% O2 were measured. In group II the ventilatory responses and femoral artery and superior sagittal sinus blood gases were measured in two sets of cats, one at SL and one at HA, during exposure to the gases outlined in group I. In group III, we examined the effect of chronic vagotomy on the ventilatory responses to the gas mixtures outlined in group I. Breathing 100% O2 at SL had no significant effect on ventilation, tidal volume, respiratory frequency, or cerebral blood flow (inferred from the cerebral veno-arterial CO2 difference). Ventilation was constant in the HA acclimatized cats while breathing 10% and 100% O2, but the ventilatory pattern changed dramatically during hyperoxia: respiratory frequency increased and tidal volume fell. Breathing 100% O2 was associated with changes in CBF, and venous PCO2 that might be expected to stimulate ventilation, but the change in ventilatory pattern suggests to us that hyperoxic disinhibition of central respiratory processes (which were modified by HA acclimatization) is the mechanism whereby ventilation is sustained during hyperoxia at HA. After vagotomy at HA, ventilation remained constant while breathing 100% O2, but the changes in respiratory pattern were no longer apparent. Therefore, vagal afferents seems to have a role in determining the pattern, but not necessarily the absolute level, of ventilation during hyperoxia. Cats vagotomized at SL prior to HA exposure did not show any evidence of HA ventilatory acclimatization; thus, the vagi may also play a heretofore unrecognized role in the process of acclimatization.     

Activity of abdominal muscle motoneurons during hypercapnia.

Our purpose was to examine the influence of hypercapnia on the activity of motoneurons innervating the transversus abdominis and internal oblique abdominal muscles, and of integrated phrenic and abdominal motor nerve activities. Studies were done in nine adult cats that were decerebrated, vagotomized, thoracotomized, paralyzed and ventilated mechanically. Of 42 motoneurons examined, 24 showed strong respiratory modulation (RM neurons), with the discharge confined primarily to the central expiratory period. The remaining 18 motoneurons discharged tonically, and failed to show respiratory modulation even at increased levels of central respiratory drive. Hyperoxic hypercapnia augmented the activities of the phrenic and abdominal nerves and increased the early expiratory discharge frequency of the RM neurons. The hypercapnia-induced increase in firing frequency during early expiration was accompanied by a corresponding decline in late expiration, and a virtual abolition of the inspiratory activity in the few neurons that discharged in this phase under normocapnic conditions. Finally, hypercapnia induced an increase in the number of spikes generated during each expiratory period in about half of the RM neurons, whereas the remaining cells showed a decrease. Thus, the increased peak activity of the integrated whole abdominal nerve burst with hypercapnia was brought about by a shift in the temporal pattern of motoneuron firing, or by an increase in the number of spikes generated during the expiratory period. The steep rate of rise and the pronounced early expiratory peak observed in the integrated abdominal nerve burst during hypercapnia in this preparation are consistent with the increase in motoneuron firing frequency during the early stages of the expiratory phase.     

Differing responses to hypercapnia and hypoxia following pneumotaxic center ablation.

The respiratory responses of 21 cats were examined upon exposure to hypercapnia and isocapnic hypoxia. Animals having bilateral electrolytic lesions localized in the pontile pneumotaxic center exhibited hypercapnia-induced minute volumes which were significantly less than those of unlesioned control cats. The hypoxia-induced minute volumes of pneumotaxic lesioned animals, examined at isocapnic alveolar gas partial pressures, were likewise significantly less than control animals at end-expired oxygen partial pressures (PAO2) in excess of 65.0 mm Hg. At PAO2 levels below 65.0 mmHg. the minute volume of experimental animals rose sharply and became statistically indistinguishable from that of the unlesioned cats. The placement of control brain stem lesions typically produced no significant alterations in the respiratory responses to hypoxia or hypercapnia. It was concluded that the pneumotaxic center constitutes an integral component of the central chemoreceptor CO1-H+ CONTROLLING SUBSYSTEM. The concept of differing anatomical sites within the brain stem serving integrative functions for central chemoreceptor and peripheral chemoreceptor afferent stimuli is also supported.     

Respiratory-associated thalamic activity is related to level of respiratory drive.

We recorded phrenic nerve activity and thalamic single unit firing in unanesthetized, suprathalamically decerebrated, paralyzed and ventilated cats, in which vagi and carotid sinus nerves (CSN) had been ablated. Seventy-six (14%) of 545 neurons in regions of the thalamus related to the ascending reticular system, which had been tonically firing at low respiratory drives, developed rhythmic increases of firing associated with each respiration when drive had been increased by CSN stimulation or hypercapnia. The increases of neuronal firing occurred in late inspiration/post-inspiration but sometimes lasted into expiration; the magnitude of change was graded according to the magnitude of respiratory activity. Thalamic neurons also fired with a rhythm related to ventilator-induced chest expansion, some units showing both the respiratory-associated and the ventilator-related rhythms. Simultaneously recorded mesencephalic and thalamic neurons developed similar rhythms when drive was increased. We suggest that these neuronal activities reflect the conveyance of information about respiration to the cortex, where it may lead to the sensation of dyspnea and perhaps to arousal.     

Preoptic LH-RH and somatostatin in the rat median eminence. An experimental light and electron microscopic immunocytochemical study

Glass microknife lesions and immunocytochemistry were used to evaluate luteinizing hormone-releasing hormone (LH-RH)- and somatostatin (SS)-immunoreactive pathways from the preoptic region to the rat median eminence. Cuts were so placed that axons of more caudally located neurons in the periventricular hypothalamic areas were spared. Light and EM observations of LH-RH-immunostained preparations indicated that following the midline periventricular cuts the density of LH-RH labelled axons and axon terminals in the ME appeared similar to that of nonlesioned animals. Following bilateral lateral hypothalamic cuts placed between the preoptic area and the ME, LH-RH immunostaining in the ME was markedly reduced. This provides evidence that the preponderance of LH-RH axons originating from the preoptic area reach the ME by a lateral hypothalamic route. In contrast to the LH-RH findings, midline lesions made using the same coordinates caused a noticeable reduction in SS immunostaining in the accurate nucleus and ME. There was either no change or only minimal change after the lateral cut. Somatostatin axons arising from the preoptic periventricular nucleus take a periventricular route and contribute to median eminence innervation, but much less extensively than the more caudally located somatostatin neurons in the hypothalamic periventricular nucleus [19].     

Influence of lung volume on respiratory responses to spontaneous bladder contractions

Spontaneous bladder contractions (SBCs) in decerebrate, vagotomized, paralyzed, ventilated cats have been shown to decrease phrenic and hypoglossal inspiratory nerve activities, as well as the activities of other respiratory motor nerves. To determine whether vagal afferents from the lung influence the respiratory inhibition associated with SBCs, we recorded phrenic and hypoglossal nerve activities in decerebrate, paralyzed, vagally intact cats. The animals were ventilated by a servo-respirator, which inflated the lungs in accordance with integrated phrenic nerve activity. Maintained increases in end-expiratory lung volume were produced by the application of 2–10 cm H₂O positive end-expiratory pressure (PEEP). SBCs were accompanied by decreases in both phrenic and hypoglossal peak integrated nerve activities, as well as by marked decreases in respiratory frequency. The reduction of respiratory frequency was greater with higher levels of PEEP, a few animals becoming apneic during SBCs. After bilateral vagotomy, SBCs continued to decrease phrenic and hypoglossal peak integrated nerve activities as previously reported, but the reduction of respiratory frequency was much less striking than when the vagi were intact. These results indicate that activity of vagal afferents from the lung augments the respiratory influence of SBCs. Furthermore, SBCs in vagally intact animals can induce periodic breathing.     

Role of the carotid bodies in ventilatory acclimation to chronic hypoxia by the awake cat

Steady-state breathing patterns during air and hypoxia (PIO2 = 84 Torr) were measured in awake cats in the following conditions: (1) during 7 months of exposure to air following carotid body resection (CBR; N = 6); (2) during 7 months of hypobaric hypoxia (PIO2 = 84 Torr; N = 5) following CBR; (3) during 5 months of exposure to hypobaric hypoxia (N = 4) while intact and then following CBR. Also, in groups (1) and (2) the aortic nerves were sectioned (ANX) at the end of the acclimation periods. The results show that the awake cat hypoventilates if the carotid bodies have been removed, and hypoxic sensitivity is reduced during long-term exposures to either hypoxia or normoxia. ANX caused a slight increase in respiratory frequency, indicating a minor role for the aortic bodies. CBR after acclimation to hypoxia resulted in decreased tidal volume but no change in respiratory frequency. The slight ventilatory acclimation to hypoxia in CBR cats was solely due to increased respiratory frequency. The phenomenon of 'hypoxic tachypnea' was modulated by acclimation, indicating that the effect of hypoxic acclimation upon respiratory frequency is due to central mechanisms.     

Respiratory effects of pontine, medullary and spinal cord midline sections in the rabbit

Experiments were performed on halothane anaesthetized, paralyzed rabbits with vagi intact or cut. In vagotomized rabbits separation of both halves of the medulla by a midline section resulted in a 'desynchronization' of both phrenic and both efferent vagal inspiratory volleys. Activities of all these nerves fall and respiratory frequency decreased. In animals with intact vagi the inspiratory volleys generated by each half of the brainstem were locked to the respirator (and consequently to each other). Elimination of the phasic feedback by stopping the respirator led to 'desynchronization'. Extension of the incision through the pons and midbrain increased phrenic amplitude almost to control values. During 'desynchronized' firing the coincident volleys exhibited markedly bigger amplitude than those which appeared in the opposite phase, i.e. during the silent period of the other phrenic. Hemisection at C1 level or a cervical spinal cord midline myelotomy eliminated the amplitude differences between coinciding and non-coinciding volleys. Amplitude of efferent vagal activity was always constant and synchronous with the ipsilateral phrenic. Our findings indicate the existence of bulbo-spinal excitatory and inhibitory pathways which affect phrenic motoneurones from the opposite side. We conclude that integration at pontine and cervical spinal cord level may significantly influence the respiratory output.     

Monoclonal antibody R2D5 reveals midsagittal radial glial system in postnatally developing and adult brainstem.

Radial glial cells and their processes play critical roles in organizing the spatial arrangement of the nervous system in the embryonic brain. It has been thought that following completion of their roles in the embryo, most of the radial glial processes disappear before or shortly after birth. Here we use R2D5, a monoclonal antibody to a soluble cytosolic protein, to demonstrate that a specific system of midsagittal radial glial cells persists in postnatal and adult brain. In the brainstem of postnatally developing and adult rabbits and cats, the R2D5-positive processes of radial glial cells were observed to be arranged in a precisely parallel array at the midsagittal seam. These radial glial processes formed a continuous palisade separating the right and left brainstem. In early postnatal animals, R2D5-positive radial processes were found to reach the pial surface and to cover the entire midsagittal seam of the brainstem. These processes embraced dendrites and somata of neurons in almost all of the midsagittal nuclei, including the raphe nuclei, suggesting that the radial glial cells may interact with the midsagittal groups of neurons. In addition, the palisade of R2D5-positive radial processes formed loose openings for crossing axonal bundles at the midline decussations of fiber tracts. In more mature brains, somata of R2D5-positive radial glial cells that had migrated ventrally were observed within the palisades, and in adult cats, most of the R2D5-positive radial processes were found to have retracted from the ventral parts of the midsagittal seam. The spatial arrangement of R2D5-positive processes suggests that they may have persistent functional roles as an interface between ventricular humoral signals and midsagittal groups of neurons in the postnatally developing brainstem and in the adult brainstem. The structure of the midline glial system suggests also that it plays a role in organizing the spatial arrangement of decussating axons during development.     

Roles of the pontine pneumotaxic and micturition centers in respiratory inhibition during bladder contractions

In decerebrate or anesthetized cats with moderately distended urinary bladders, spontaneous bladder contractions (SBCs) have been shown to decrease phrenic and hypoglossal nerve activities. To determine the involvement of both the pontine micturition center (PMC) and the pneumotaxic center in the respiratory response to SBCs, we recorded phrenic and hypoglossal nerve activities in decerebrate, paralyzed, vagotomized, artificially ventilated cats. Electrical stimulation of the PMC in cats with subthreshold bladder volumes below the threshold for SBCs elicited both increases in intravesical pressure (IVP) and attenuation of respiratory motor nerve activities. Respiration was not altered after PMC lesions, which abolished SBCs, contractions in response to PMC stimulation, and respiratory inhibition due to passive bladder distension. Electrical stimulation of the pneumotaxic center altered respiratory motor nerve activities and increased IVP in cats with subthreshold bladder volumes. Pneumotaxic center lesions caused apneusis, but did not abolish the SBCs, which continued to attenuate the apneustic respiratory motor nerve activity. These results indicate that the PMC is an important component of the reflex pathway from urinary bladder distension to respiratory inhibition, whereas the pneumotaxic center does not appear to be an essential part of this pathway.