Neonatal maturation of the hypercapnic ventilatory response and central neural CO2 chemosensitivity.

The ventilatory response to CO2 changes as a function of neonatal development. In rats, a ventilatory response to CO2 is present in the first 5 days of life, but this ventilatory response to CO2 wanes and reaches its lowest point around postnatal day 8. Subsequently, the ventilatory response to CO2 rises towards adult levels. Similar patterns in the ventilatory response to CO2 are seen in some other species, although some animals do not exhibit all of these phases. Different developmental patterns of the ventilatory response to CO2 may be related to the state of development of the animal at birth. The triphasic pattern of responsiveness (early decline, a nadir, and subsequent achievement of adult levels of responsiveness) may arise from the development of several processes, including central neural mechanisms, gas exchange, the neuromuscular junction, respiratory muscles and respiratory mechanics. We only discuss central neural mechanisms here, including altered CO2 sensitivity of neurons among the various sites of central CO2 chemosensitivity, changes in astrocytic function during development, the maturation of electrical and chemical synaptic mechanisms (both inhibitory and excitatory mechanisms) or changes in the integration of chemosensory information originating from peripheral and multiple central CO2 chemosensory sites. Among these central processes, the maturation of synaptic mechanisms seems most important and the relative maturation of synaptic processes may also determine how plastic the response to CO2 is at any particular age.

Sites of respiratory rhythmogenesis during development in the tadpole.

During ontogeny, amphibian larvae experience a dramatic alteration in the motor act of breathing as the premetamorphic gill breather develops into the postmetamorphic lung ventilator. We tested the hypothesis that the site of lung rhythmogenesis relocates during metamorphosis by recording fictive lung ventilation before and after transecting the in vitro brain stem of pre- and postmetamorphic Rana catesbeiana into four segments. In premetamorphic tadpoles, the two caudalmost brain stem segments combined proved to be the minimum brain stem configuration necessary and sufficient for lung burst generation. In the postmetamorphic counterpart, this function was supplied by the combination of the two rostralmost brain stem segments. In the postmetamorphic brain stem, a 500-microm segment lying just rostral to cranial nerve IX conveys rhythmogenic capability to neighboring rostral or caudal segments. We conclude that lung rhythmogenic capability translocates rostrally during development as the tadpole shifts from gill to lung ventilation.

Location of central respiratory chemoreceptors in the developing tadpole.

The location of central respiratory chemoreceptors in amphibian larvae may change as the central chemoreceptive function shifts from driving gill to driving lung ventilation during metamorphosis. We examined this possibility in the in vitro brain stem of the pre- and postmetamorphic Rana catesbeiana tadpole by microinjecting hypercapnic artificial cerebrospinal fluid (aCSF) while recording fictive lung ventilation. The rostral and caudal brain stem were separately explored systematically using injections of 11 nl of aCSF equilibrated with 100% CO2 that transiently acidified a 500-microm region, producing a maximum reduction in pH of 0.23 +/- 0.06 at the site of injection. In postmetamorphic tadpoles, chemoreceptive sites were concentrated in the rostral compared with the caudal brain stem. No such segregation was observed in the premetamorphic tadpole. We conclude that, as in lung rhythmogenic function, respiratory chemosensitivity emerges rostrally in the amphibian brain stem during development.

The fictively breathing tadpole brainstem preparation as a model for the development of respiratory pattern generation and central chemoreception.

Spontaneous high-frequency, low-amplitude and low-frequency, high-amplitude efferent bursting patterns of cranial and spinal motor nerve activity in the in vitro brainstem preparation of the bullfrog tadpole Rana catesbeiana have been characterized as fictive gill and lung ventilation, respectively (Gdovin MJ, Torgerson CS, Remmers JE). Characterization of gill and lung ventilatory activity in cranial nerves in the spontaneously breathing tadpole Rana catesbeiana, FASEB J 1996;10(3):A642; Gdovin MJ, Torgerson CS, Remmers JE. Neurorespiratory pattern of gill and lung ventilation in the decerebrate spontaneously breathing tadpole, Respir Physiol 1998;113:135 146; Pack AI, Galante RJ, Walker RE, Kubin LK, Fishman AP. Comparative approach to neural control of respiration, In: Speck DF, Dekin MS, Revelette WR, Frazier DT, editors. Respiratory Control Central and Peripheral Mechanisms. Lexington: University of Kentucky Press, 1993:52-57). In addition, the ontogenetic dependence of central respiratory chemoreceptor stimulation on fictive gill and lung ventilation has been previously described (Torgerson CS, Gdovin MJ, Remmers JE. Fictive gill and lung ventilation in the pre- and post-metamorphic tadpole brainstem, J Neurophysiol 1998, in press). To investigate the neural substrates responsible for central respiratory rhythm generation of gill and lung ventilation in the developing tadpole, we recorded efferent activities of cranial nerve (CN) V, VII, and X and spinal nerve (SN) II during changes in superfusate PCO2 before and after multiple transection of the in vitro brainstem. The brainstem was transected between CN VIII and IX and the response to changes in PCO2 was recorded. A second transection was then made between the caudal margin of CN X and rostral to SN II. Preliminary data reveal that robust gill ventilation was recorded consistently only if the segment of brainstem included CN X, whereas the loci capable of eliciting fictive lung bursting patterns appeared to differ depending on developmental stage. These data demonstrate that the neural substrate required for fictive gill and lung ventilation exists in anatomically separate regions such that the gill central pattern generator (CPG) is located in the caudal medulla at the level of CN X throughout development, whereas the location of the lung CPG is located more rostrally at the level of CN VII in the post-metamorphic larva. Both in vivo and in vitro studies revealed two distinct neural bursting patterns associated with gill and lung ventilation. Sequential activation of CN V, VII, X were observed during gill ventilation of in vivo and fictive gill ventilation in vitro, whereas these nerve activities, along with SN II displayed more synchronous bursting patterns of activation during lung ventilation and fictive lung breaths.

Neurorespiratory pattern of gill and lung ventilation in the decerebrate spontaneously breathing tadpole.

A decerebrate, spontaneously breathing tadpole preparation (Taylor-Kollros stages 16-19) was used to test the general hypothesis that the efferent bursting activities of cranial nerves (CN) V, VII and spinal nerve (SN) II are respiratory in nature, and, in particular, to identify separate and specific neural correlates of gill and lung ventilation. Oropharyngeal pressure (POP), intrapulmonary pressure (PIP), electromyogram (EMG) of the buccal levator muscle (interhyoideus), and efferent neural activities of CN V, CN VII and SN II were recorded while the animal was exposed to hyperoxia (100% inspired O2), normoxia (21% inspired O2), and hypoxia (10, 5 and 0% inspired O2). Gill ventilation, indicated by fluctuations in POP at constant PIP, was characterized by high-frequency, low-amplitude bursts of action potentials in CN V and VII and interhyoideus EMG without phasic activity in SN II. Lung breaths, indicated by oscillations in POP and PIP were characterized by large bursts in EMG, CN V and VII together with a large burst in SN II. The amplitude of the moving average of nerve activities associated with lung ventilation was significantly larger than those associated with gill ventilation. During gill ventilation, the burst in CN V led that in CN VII, and both preceded the rise in POP. By contrast, a more synchronous neural burst onset pattern was observed during lung ventilation. The results document the neural, muscular, and mechanical characteristics of gill and lung ventilation in the tadpole, and establish bursting activity in SN II as a specific marker for lung ventilation in the metamorphic tadpole.

Fictive gill and lung ventilation in the pre- and postmetamorphic tadpole brain stem.

The pattern of efferent neural activity recorded from the isolated brain stem preparation of the tadpole Rana catesbeiana was examined to characterize fictive gill and lung ventilations during ontogeny. In vitro recordings from cranial nerve (CN) roots V, VII, and X and spinal nerve (SN) root II of premetamorphic tadpoles showed a coordinated sequence of rhythmic bursts occurring in one of two patterns, pattern1, high-frequency, low-amplitude bursts lacking corresponding activity in SN II and pattern 2, low-frequency, high-amplitude bursts with coincident bursts in SN II. These two patterns corresponded to gill and lung ventilatory burst patterns, respectively, recorded from nerve roots of decerebrate, spontaneously breathing tadpoles. Similar patterns were observed in brain stem preparations from postmetamorphic tadpoles except that they showed a greater frequency of lung bursts and they expressed fictive gill ventilation in SN II. The laryngeal branch of the vagus (Xl) displayed efferent bursts in phase with gill and lung activity, suggesting fictive glottal constriction during gill ventilation and glottal dilation during lung ventilation. The fictive gill ventilatory cycle of pre- and postmetamorphic tadpoles was characterized by a rostral to caudal sequence of CN bursts. The fictive lung ventilatory pattern in the premetamorphic animal was initiated by augmenting CN VII discharge followed by synchronous bursts in CN V, X, SN II, and Xl. By contrast, postmetamorphic patterns of fictive lung ventilation were characterized by lung burst activity in SN II that preceded burst onset in CN V and followed the lead burst in CN VII. We conclude that recruitment and timing of pattern 1 and pattern 2 rhythmic bursts recorded in vitro closely resemble that recorded during spontaneous respiratory behavior, indicating that the two patterns are the neural equivalent of gill and lung ventilation, respectively. Further, fictive gill and lung ventilatory patterns in postmetamorphic tadpoles differ in burst onset latency from premetamorphic tadpole patterns and resemble fictive oropharyngeal and pulmonary burst cycles in adult frogs.

Depth profiles of pH and PO2 in the in vitro brainstem preparation of the tadpole Rana catesbeiana.

Extracellular pH and PO2 was recorded in the isolated in vitro brainstem of the metamorphic tadpole, Rana catesbeiana while the brainstem preparation was superfused with oxygenated mock cerebrospinal fluid of pH = 7.8, PCO2 = 17 Torr, PO2 = 600 Torr at 23 degrees C. Using pH and PO2 microelectrodes, the ventral medullary surface was penetrated at midline and lateral sites between cranial nerves V and X. Mean pH and PO2 gradients of 0.07 pH units/100 microns and 60 Torr/100 microns were detected in the superfusate, 100-200 microns above the ventral surface of the brainstem. These gradients remained virtually constant for the first 100-200 microns below the medullary surface. Beyond this level, pH and PO2 gradients decreased in a curvilinear fashion. For midline tracts, minimum values of pH and PO2 (7.58 +/- 0.05 and 323 +/- 31 Torr) were reached at a depth of 500-750 microns, whereas for lateral tracts, mean minimum values of pH and PO2 (7.34 +/- 0.12 and 240 +/- 68 Torr), were recorded at 850-900 microns. With further electrode advancement, pH and PO2 gradients in both midline and lateral tracts reversed as levels began to increase. Between CN V and X, lateral width was 4.34 +/- 0.57 mm, while dorsal-ventral thickness in midline and lateral regions was 0.92 +/- 0.21 and 1.31 +/- 0.22 mm, respectively. Overall, the in vitro tadpole brainstem provides a robust neural preparation which, although moderately acidic, is well oxygenated throughout all tissue layers.

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.

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.

Respiratory motor nerve activities during experimental seizures in cats.

We evaluated respiratory motor nerve activities during experimental seizures induced with subcortical penicillin. The activities of the phrenic (PH), nasolabial (NL), and hypoglossal (HG) nerves and the recurrent laryngeal motor branches to the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles were analyzed in 13 anesthetized, vagotomized, paralyzed, and ventilated cats. During ictal and interictal phases of seizures, nerve activities became irregular and peak integrated nerve activities increased, particularly in the case of the PH nerve. The ictal phase of seizures was associated with increased tonic activity and decreased phasic respiratory discharges, particularly in the cases of the HG, NL, and PCA nerves. During some prolonged ictal discharges, entrainment of nerve activities by cortical spiking was associated with irregular uncoordinated activation, particularly in the TA nerve. These studies help explain respiratory impairment during seizures by providing evidence of impaired coordination between activation of muscles that regulate upper airway patency and activation of the diaphragm.

Respiratory motor nerve activities during spontaneous bladder contractions.

We monitored spontaneous bladder contractions (SBCs) in decerebrate vagotomized paralyzed ventilated cats while recording respiratory motor nerve activities and intravesical pressure under isovolumetric conditions. Phrenic nerve discharge diminished during SBCs, as did the activities of the hypoglossal nerve, the nasolabial branch of the facial nerve, and inspiratory (posterior cricoarytenoid) and expiratory (thyroarytenoid) branches of the recurrent laryngeal nerve. Hypoglossal activity was most strikingly reduced during SBCs, disappearing completely in some animals. The triangularis sterni nerve exhibited an initial decrease, followed by an increase in activity during SBCs, whereas the cranial iliohypogastric nerve showed increased activity. The changes in nerve activities during SBCs could also be elicited by passive distension of the bladder and were abolished by bilateral section of the pelvic nerves. These findings extend the understanding of reflexes originating from the urinary bladder to include a coordinated respiratory response and suggest that these reflexes may compromise upper airway patency under some conditions.

Nerve growth factor increases nicotinic ACh receptor gene expression and current density in wild-type and protein kinase A-deficient PC12 cells.

Although neuronal nicotinic ACh receptors (nAChR) play a key role in synaptic transmission and information transfer in the nervous system, little is known about the molecular mechanisms that govern the expression of the multiple subunits that form the receptors and determine their functional properties. Using electrophysiological and molecular biological approaches, we have investigated the NGF-mediated regulation of nAChR expression in rat pheochromocytoma (PC12) cells and protein kinase A (PKA)-deficient PC12 cells. We report that NGF treatment increases steady state levels of mRNA encoding the alpha 3, alpha 5, alpha 7, beta 2, and beta 4 subunits, increases the occurrence of ACh-induced single-channel activity in excised patches, and increases ACh-induced macroscopic current density, all by mechanisms independent of PKA activity.

Influence of laryngeal CO2 on respiratory activities of motor nerves to accessory muscles.

Intralaryngeal CO2 in decerebrate, vagotomized cats decreases phrenic nerve activity and increases the respiratory activity of the hypoglossal (HG) nerve. These responses are mediated by afferents in the superior laryngeal nerves. To explore the responses of other respiratory motor nerves to this stimulus, we have recorded the activities of the nasolabial (NL) branch of the facial nerve, the posterior cricoarytenoid (PCA) and thyroarytenoid (TA) branches of the recurrent laryngeal nerve and the nerve to triangularis sterni (TS) muscle. In response to 5 and 10% CO2 in the surgically isolated upper airway, we found dose-related decreases in phrenic activity, increases in HG and NL activity and characteristic, but intermittent, exaggeration of early expiratory bursts of TA activity. The activities of the PCA and TS nerves showed no consistent responses. These results broaden the definition of the reflex response to intralaryngeal CO2, revealing components that reflect ventilatory inhibition, upper airway dilation and laryngeal protection.