Multiple pontomedullary mechanisms of respiratory rhythmogenesis.

Mammalian central pattern generators producing rhythmic movements exhibit robust but flexible behavior. However, brainstem network architectures that enable these features are not well understood. Using precise sequential transections through the pons to medulla, it was observed that there was compartmentalization of distinct rhythmogenic mechanisms in the ponto-medullary respiratory network, which has rostro-caudal organization. The eupneic 3-phase respiratory pattern was transformed to a 2-phase and then to a 1-phase pattern as the network was physically reduced. The pons, the retrotrapezoid nucleus and glycine mediated inhibition are all essential for expression of the 3-phase rhythm. The 2-phase rhythm depends on inhibitory interactions (reciprocal) between Bötzinger and pre-Bötzinger complexes, whereas the 1-phase-pattern is generated within the pre-Bötzinger complex and is reliant on the persistent sodium current. In conditions of forced expiration, the RTN region was found to be essential for the expression of abdominal late expiratory activity. However, it is unknown whether the RTN generates or simply relays this activity. Entrained with the central respiratory network is the sympathetic nervous system, which exhibits patterns of discharge coupled with the respiratory cycle (in terms of both gain and phase of coupling) and dysfunctions in this coupling appear to underpin pathological conditions. In conclusion, the respiratory network has rhythmogenic capabilities at multiple levels of network organization, allowing expression of motor patterns specific for various physiological and pathophysiological respiratory behaviors.

Maintenance of eupnea and gasping following alterations in potassium ion concentration of perfusates of in situ rat preparation.

Levels of extracellular potassium ion, above those in vivo, are required for the generation of rhythmic activities of in vitro preparations. Our purpose was to define whether hyperkalemia in the perfusate of an in situ preparation was likewise necessary for the expression of eupnea and gasping. Studies were performed using the in situ preparation of the juvenile rat, in which activity of the phrenic nerve was recorded as an index of the respiratory rhythm. Eupnea and gasping were impervious to modifications of potassium ion of the perfusate. Eupnea was maintained uninterrupted for more than 60 min whether the total concentration of potassium was hypokalemic (2.75 mM), normokalemic (4.0 mM), or hyperkalemic (6.25 and 7.75 mM). Gasping, with identical characteristics, was elicited at any concentration of potassium ion. We conclude that both eupnea and gasping are unaffected by modest changes in the concentration of potassium ion in the perfusate with which the in situ rat preparation is maintained. These results add further support to the conclusion that the in situ preparation represents a model that accurately reproduces mechanisms of rhythm generation of both eupnea and gasping in vivo.

Modeling the ponto-medullary respiratory network.

The generation and shaping of the respiratory motor pattern are performed in the lower brainstem and involve neuronal interactions within the medulla and between the medulla and pons. A computational model of the ponto-medullary respiratory network has been developed by incorporating existing experimental data on the medullary neural circuits and possible interactions between the medulla and pons. The model reproduces a number of experimental findings concerning alterations of the respiratory pattern following various perturbations/stimulations applied to the pons and pulmonary afferents. The results of modeling support the concept that eupneic respiratory rhythm generation requires contribution of the pons whereas a gasping-like rhythm (and the rhythm observed in vitro) may be generated within the medulla and involve pacemaker-driven mechanisms localized within the medullary pre-Botzinger Complex. The model and experimental data described support the concept that during eupnea the respiration-related pontine structures control the medullary network mechanisms for respiratory phase transitions, suppress the intrinsic pacemaker-driven oscillations in the pre-BotC and provide inspiration-inhibitory and expiration-facilitatory reflexes which are independent of the pulmonary Hering-Breuer reflex but operate through the same medullary phase switching circuits.

Alpha2 receptor binding in the medulla oblongata in the sudden infant death syndrome.

The sudden infant death syndrome (SIDS) is the leading cause of postnatal infant mortality in the United States. Its etiology remains unknown. We propose that SIDS, or a subset of SIDS, is due to a failure of autoresuscitation, a protective brainstem response to asphyxia or hypoxia, in a vulnerable infant during a critical developmental period. Gasping is an important component of autoresuscitation that is thought to be mediated by the "gasping center" in the lateral tegmentum of the medulla, a region homologous in its cytoarchitecture and chemical anatomy to the intermediate reticular zone (IRZ) in the human. Since we found that [3H]para-aminoclonidine ([3H]PAC) binding to alpha2-adrenergic receptors localizes to this region in human infants and, thereby provides a neurochemical marker for it, we tested the hypothesis that [3H]PAC binding to alpha2-adrenergic receptors is decreased in the IRZ in SIDS victims. Using quantitative tissue autoradiography with [3H]PAC as the radioligand and phentolamine as the displacer, we analyzed alpha2-receptor binding density in the IRZ, as well as in 7 additional sites for comparison, in 10 SIDS and 10 control medullae. There were no significant differences in alpha2 receptor binding in the IRZ, vagal nuclei, or other medullary sites examined between SIDS and control cases. These results suggest that the putative gasping defect in the IRZ in SIDS victims is not related to [3H]PAC binding to alpha2-adrenergic receptors.

Characterizations of eupnea, apneusis and gasping in a perfused rat preparation.

In vivo mammalian preparations can exhibit eupnea, apneusis and gasping. In vitro mammalian preparations exhibit only a single invariant pattern, which appears identical to gasping. We characterized the patterns of ventilatory activity of a perfused heart-brainstem preparation of the juvenile rat. In this preparation, phrenic activity has a 'ramp-like' rise similar to eupnea in vivo. Peak phrenic activity declines and ultimately disappears in hypocapnia. In hypercapnia, both frequency and peak of phrenic bursts increase. In hypoxia, such increases are transient. The phrenic burst is terminated by electrical stimulation of the pontile 'pneumotaxic center' and, as in apneusis, is prolonged by lesions in this region. With severe hypoxia or ischemia, the 'ramp-like' phrenic activity is replaced by the 'decrementing' pattern of gasping. Variables of phrenic activity in gasping produced in hypoxia and ischemia are identical. We conclude that the perfused juvenile rat preparation exhibits patterns of eupnea, apneusis and gasping which are similar to in vivo mammalian preparations.

Sensitivities of eupnea and gasping to alterations in temperature of in vivo and perfused rat preparations.

Severe hypoxia or ischemia causes an alteration from eupnea to gasping. At body temperatures approximating 37 degrees C in vivo, the frequency of gasping is much less than that of eupnea. However in a perfused juvenile rat preparation, which is maintained at 30-31 degrees C, the frequency of eupnea and gasping is the same. We hypothesized that brainstem mechanisms responsible for the neurogenesis of eupnea and gasping might have different sensitivities to alterations in temperature. In both decerebrate adult rats in vivo and in a perfused juvenile rat preparation, eupnea and gasping had different frequencies at rectal or perfusate temperatures in excess of 34 degrees C, whereas, at lower temperatures, eupnea and gasping had identical frequencies in both preparations. These findings support the conclusion that different brainstem mechanisms underlie the neurogenesis of eupnea and gasping. In addition, these findings have implications for interpretation of results from in vitro mammalian preparations, which are examined at temperatures at which the frequency of eupnea and gasping are indistinguishable.

Transient, reversible apnoea following ablation of the pre-Bötzinger complex in rats.

1. In some anaesthetized preparations, eupnoea is eliminated following a blockade or destruction of neurons in a rostral medullary pre-Botzinger complex. 2. Neurons in this region might underlie the neurogenesis of eupnoea, or be the source of an input which is necessary for eupnoea to be expressed. If the latter, any apnoea following ablation of the pre-Botzinger complex might be reversed by an augmentation in 'tonic input.' Contrariwise, this apnoea should be permanent if the neuronal activities of the pre- Botzinger complex are an exclusive generator of the eupnoeic rhythm. 3. Decerebrate, vagotomized, paralysed and ventilated adult rats were studied. Efferent activity of the phrenic nerve was recorded as an index of ventilatory activity. 4. Blockade or destruction of neuronal activities of the pre-Botzinger complex by unilateral and/or bilateral injections of muscimol or kainic acid eliminated eupnoea only transiently. Eupnoea returned following activation of the peripheral chemoreceptors and spontaneously over time. 5. Results do not support the concept that neuronal activities of the pre-Botzinger complex play an exclusive role in the neurogenesis of eupnoea in vivo. Rather, these neuronal activities appear to provide a tonic input to the ponto-medullary circuit which generates eupnoea and/or appear to be one component of this circuit.

Rostral medullary respiratory neuronal activities of decerebrate cats in eupnea, apneusis and gasping.

Eupnea is generated by mechanisms within the pons and medulla. Following removal of pons or exposure to anoxia, gasping is elicited. Eupnea and gasping are markedly different ventilatory patterns. The genesis of gasping is dependent upon rostral medullary neuronal activities. To generate the gasp, these activities should commence before the phrenic burst. In decerebrate, vagotomized, paralyzed and ventilated cats, eupnea was altered to gasping in anoxia. Rostral medullary neuronal activities had inspiratory, expiratory and phase-spanning patterns in eupnea. During gasping, some inspiratory neuronal activities commenced before the phrenic gasp; these same neurons had commenced activities after the onset of the eupneic phrenic burst. Expiratory and phase-spanning neurons did not discharge. Neuronal activities which are consonant with a role in the neurogenesis of gasping had very different discharge patterns in eupnea. Results support the concept that medullary mechanisms for gasping are incorporated in the ponto-medullary circuit responsible for the neurogenesis and expression of eupnea.

Maternal nicotine depresses eupneic ventilation of neonatal rats.

Maternal smoking is a risk factor for the sudden infant death syndrome (SIDS). We hypothesized that pre-natal exposure to nicotine would result in abnormalities of ventilatory activity in newborns. Neonatal rats which had been exposed to nicotine had significantly lower minute ventilation breathing air and hypoxic gas mixtures than did control animals. In both groups, anoxia elicited gasping which was equally effective in restoring eupnea. Maternal exposure to nicotine may result in a reduced metabolic rate and/or chronic hypoventilation in the newborn.

Modulation of hypoxic depressions of ventilatory activity in the newborn piglet by mesencephalic mechanisms.

In neonates, ventilatory responses to hypoxia are 'biphasic,' with an augmentation followed by a decline. The hypoxia-induced augmentations in ventilation are attenuated and the depressions are accentuated following denervation of the peripheral chemoreceptors. Piglets that were decerebrated at a rostral mesencephalic level exhibited these hypoxia-induced depressions. These depressions were lessened following transection through the caudal mesencephalon. Mesencephalic mechanisms play a fundamental role in the brainstem regulation of ventilatory responses to hypoxia.

Alterations in respiratory neuronal activities in the medullary 'pre-Bötzinger' region in hypocapnia.

'Pre-inspiratory' neuronal activities in a rostral ventrolateral medullary 'pre-Bötzinger' complex have been hypothesized to generate eupnea. Respiratory-modulated neuronal activities were recorded in this region in decerebrate, vagotomized, paralyzed, and ventilated cats, having bilateral carotid sinus nerve sections. As end-tidal partial pressures of carbon dioxide were reduced to hypocapnic levels, all neuronal activities which were tonic or expiratory inspiratory ('pre-inspiratory') either ceased or lost respiratory-modulation. Similarly, most expiratory and inspiratory expiratory activities did not maintain a phasic discharge. Half of the inspiratory neuronal activities did continue a phasic discharge, which commenced after phrenic activity or became independent of the phrenic rhythm. Results do not support a fundamental role of the 'pre-Bötzinger' complex in the neurogenesis of eupnea. Some neuronal activities can establish a phasic discharge in hypocapnia which is independent of the central respiratory rhythm. At normocapnia, this independent discharge is superseded and incorporated into the ponto-medullary respiratory neuronal circuit which generates eupnea.

Maternal cocaine alters eupneic ventilation but not gasping of neonatal rats.

In severe hypoxia, normal eupneic respiration is replaced by gasping. Gasping provides for 'autoresuscitation' such that, if air is available, normal breathing returns. As maternal use of cocaine may increase the incidence of the Sudden Infant Death Syndrome (SIDS), prenatal exposure to cocaine was hypothesized to result in a failure of gasping in the newborn. Cocaine was administered daily to pregnant rats. In the newborn, no impairment of gasping as a mechanism of autoresuscitation was detected. However, on the first and second days after birth, ventilation in hypoxia was less in newborns having exposure to cocaine than in the control rats. Maternal use of cocaine may retard the development of the ventilatory control system. However, gasping mechanisms are not included in this retardation.

Neurogenesis of patterns of automatic ventilatory activity.

Normal respiration, termed eupnea, is characterized by periodic filling and emptying of the lungs. Eupnea can occur 'automatically' without conscious effort. Such automatic ventilation is controlled by the brainstem respiratory centers of pons and medulla. Following removal of the pons, eupnea is replaced by gasping, marked by brief but maximal inspiratory efforts. The mechanisms by which the respiratory rhythms are generated have been examined intensively. Evidence is discussed that ventilatory activity can be generated in multiple regions of pons and medulla. Eupnea and gasping represent fundamentally different ventilatory patterns. Only for gasping has a critical region for neurogenesis been identified, in the rostral medulla. Gasping may be generated by the discharge of 'pacemaker' neurons. In eupnea, this pacemaker activity is suppressed and incorporated into the pontile and medullary neuronal circuit responsible for the neurogenesis of eupnea. Evidence for ventilatory neurogenesis which has been obtained from a number of in vitro preparations is discussed. A much-used preparation is that of a 'superfused' brainstem of the neonatal rat. However, activities of this preparation differ greatly from those of eupnea, as recorded in vitro or in arterially perfused in vitro preparations. Activities of this 'superfused' preparation are identical with gasping and, hence, results must be reinterpreted accordingly. The possibility is present that mechanisms responsible for generating respiratory rhythms may differ from those responsible for shaping respiratory-modulated discharge patterns of cranial and spinal nerves. The importance of pontile mechanisms in the neurogenesis and control of eupnea is reemphasized.

Pontine cholinergic respiratory depression in neonatal and young rats.

We postulated that activation of pontine cholinergic mechanisms would cause respiratory depression in neonatal and young rats. Phrenic activity was recorded in decerebrate, paralyzed, ventilated and vagotomized rats of 4 to 22 days after birth. Small volumes (10-60 nl) of carbachol (44-88 mM) were injected into the medial portion of the rostral pons. The injection of carbachol, but not saline, decreased phrenic peak activity (83 +/- 6% of control) and respiratory frequency (64 +/- 9.5% of control) within 2 min following the injection in neonates and the depression lasted for less than 10 min. The site of injection in the pontine reticular formation was confirmed by histology. Results suggest that cholinergic mechanisms in the medial pons depress respiratory activity in the neonate.

The morphology and connections of neurons in the gasping centre of adult rats.

Neuronal activities in the intermediate reticular nucleus and adjacent lateral tegmental field are critical for the neurogenesis of the ventilatory pattern of gasping. We report herein the anatomical features of these neurons, their axonal projections and the location of neurons providing afferent inputs. These neuroanatomical evaluations were performed by iontophoretic injection of the tracer Neurobiotin into the region of the intermediate reticular nucleus of the rat. At the site of injection, neurons having soma of 30-50 microns were filled. Labelled axons and terminals were observed in ipsilateral regions which contain neurons having established functions in the control of ventilatory activity. These regions include the nucleus ambiguous and motor nuclei of the hypoglossal and facial nerves. In addition, axonal projections extended to the contralateral region of the intermediate reticular nucleus. From this contralateral region, retrograde tracing revealed projections to the site of injection. Similarly, many ipsilateral regions which received axonal terminals from the region of the intermediate reticular nucleus had reciprocal projections to this region. These anatomical results support the physiological observation that the neurogenesis of gasping involves a synchronized activation of diverse components of the brainstem ventilatory control system.

Lesions of regions for in vitro ventilatory genesis eliminate gasping but not eupnea.

Medullary regions, termed 'pre-inspiratory' and 'pre-Bötzinger', are considered critical for the neurogenesis of rhythmic ventilatory activity of in vitro preparations of the neonatal rat. We examined the influence of destruction of neurons in these regions, by microinjections of kainic acid, upon eupnea and gasping in vivo. Decerebrate, vagotomized, paralyzed and ventilated rats of age 8-15 days were used; the phrenic nerve activity was recorded. Eupnea was not consistently altered following destruction of neurons in any region. However, in the majority of animals, anoxia-induced gasping was not observed following injections of kainic acid into the 'pre-inspiratory' region, 'pre-Bötzinger' complex or lateral tegmental field; the latter region is important for the neurogenesis of gasping in adults. Injections into other regions did not prevent the elicitation of gasping. These results do not support the possibility that neuronal activities which are responsible for respiratory rhythm generation in vitro underlie the neurogenesis of eupnea in vivo.

Medullary regions for neurogenesis of gasping: noeud vital or noeuds vitals?

Gasping is a critical mechanism for survival in that it serves as a mechanism for autoresuscitation when eupnea fails. Eupnea and gasping are separable patterns of automatic ventilatory activity in all mammalian species from the day of birth. The neurogenesis of the gasp is dependent on the discharge of neurons in the rostroventral medulla. This gasping center overlaps a region termed "the pre-Bötzinger complex." Neuronal activities of this complex, characterized in an in vitro brain stem spinal cord preparation of the neonatal rat, have been hypothesized to underlie respiratory rhythm generation. Yet, the rhythmic activity of this in vitro preparation is markedly different from eupnea but identical with gasping in vivo. In eupnea, medullary neuronal activities generating the gasp and the identical rhythm of the in vitro preparation are incorporated into a portion of the pontomedullary circuit defining eupneic ventilatory activity. However, these medullary neuronal activities do not appear critical for the neurogenesis of eupnea, per se.

Phrenic response to hypercapnia in the unanesthetized, decerbrate, newborn rat.

We developed a decerebrate, vagotomized, newborn rat preparation to investigate brainstem respiratory control mechanisms without the influence of anesthesia, supra-pontine structures, or vagally mediated feedback mechanisms. We measured the changes in phrenic nerve electrical activity in response to breathing 3% and 5% CO2 in unanesthetized, vagotomized, decerebrate newborn rats from 0 to 10 days of age and compared them with the changes in anesthetized, vagotomized, newborn rats and adult, vagotomized, decerebrate or anesthetized, animals. Phrenic nerve activity was irregular in the young newborn rats and became more regular between 7 and 10 days of age. T1 and T1/Ttot increased with age but increasing age had no influence on the response to CO2. The response to CO2 was dominated by increases in phrenic amplitude, minute activity, and inspiratory slope with no change in timing variables. These responses are similar to those that have been reported previously in vagally intact animals, suggesting that vagal feedback contributes little to the response to hypercapnia in the newborn rat. In summary, decerebrate newborn rats consistently respond to hypercapnia by increasing inspiratory drive similar to conscious animals.