Calcium and avian intrapulmonary chemoreceptor response to CO2.

Intrapulmonary chemoreceptors (IPC) are highly responsive respiratory chemoreceptors that innervate the lungs of birds and diapsid reptiles. IPC are stimulated by low levels of lung Pco(2), inhibited by high levels of lung Pco(2), and their vagal afferents serve as a sensory limb for reflex adjustments of breathing depth and rate. Most IPC exhibit both phasic and tonic sensitivity to CO(2), and spike frequency adaptation (SFA) contributes to their phasic CO(2) responsiveness. To test whether CO(2) responsiveness and SFA in IPC is modulated by a Ca(2+)-linked mechanism, we quantified the role of transmembrane Ca(2+) fluxes and Ca(2+)-related channels on single-unit IPC function in response to phasic changes in inspired Pco(2). We found that 1) broad-spectrum blockade of Ca(2+) channels using cadmium or cobalt and blockade of L-type Ca(2+) channels using nifedipine increased IPC discharge; 2) activation of L-type Ca(2+) channels using BAY K 8644 reduced IPC discharge; 3) blockade of Ca(2+)-activated potassium channels using charybdotoxin (antagonist of large-conductance Ca(2+)-dependent K(+) channel) increased IPC discharge, but neither charybdotoxin nor apamin affected SFA; and 4) blockade of chloride channels, including Ca(2+)-activated chloride channels, with niflumic acid decreased IPC discharge at low Pco(2) and increased IPC discharge at high Pco(2), resulting in a net attenuation of the IPC CO(2) response. We conclude that Ca(2+) influx through L-type Ca(2+) channels has an inhibitory effect on IPC afferent discharge and CO(2) sensitivity, that spike frequency adaptation is not due to apamin- or charybdotoxin-sensitive Ca(2+)-activated K(+) channels in IPC, and that chloride channels blocked by niflumic acid help modulate IPC CO(2) responses.

Differential expression of connexin26 and connexin32 in the pre-Bötzinger complex of neonatal and adult rat.

The pre-Bötzinger complex (pre-BötC) is hypothesized to be the site for respiratory rhythm generation in mammals. Studies examining the cellular mechanisms mediating rhythm generation have focused on the role of chemically mediated synaptic interactions; however, electrotonic synaptic interactions (i.e., electrotonic coupling), which occur by means of gap junctions, may also play a role. Here, we used immunoblot and immunohistochemical analyses to determine whether the pre-BötC contains the gap junction proteins necessary for electrotonic communication and whether the presence and distribution of these gap junction proteins show a developmental change in expression. We found that both connexin26 (Cx26) and connexin32 (Cx32) were expressed in pre-BötC neurons of neonatal and adult rats; however, the relative amounts and their distribution varied by age. Cx26 labeling was seen in a high proportion of pre-BötC neurons in neonatal rats < or = 7 days postnatal (P7) but declined with increasing age. In contrast, Cx32 labeling was sparse in pre-BötC neurons of neonatal rats < or = P7, but increased with increasing age; the highest proportion was seen in adult rats. These data suggest the potential for gap junctional communication in the pre-BötC of both neonatal and adult rats, and we propose that the gap junction proteins Cx26 and Cx32 form the neuroanatomic substrate for this gap junctional communication, which may be important in the synchronization of neural activity generating respiratory rhythm.

CO(2)/H(+) chemoreception in the cat pre-Bötzinger complex in vivo.

We examined the effects of focal tissue acidosis in the pre-Bötzinger complex (pre-BötC; the proposed locus of respiratory rhythm generation) on phrenic nerve discharge in chloralose-anesthetized, vagotomized, paralyzed, mechanically ventilated cats. Focal tissue acidosis was produced by unilateral microinjection of 10-20 nl of the carbonic anhydrase inhibitors acetazolamide (AZ; 50 microM) or methazolamide (MZ; 50 microM). Microinjection of AZ and MZ into 14 sites in the pre-BötC reversibly increased the peak amplitude of integrated phrenic nerve discharge and, in some sites, produced augmented bursts (i.e., eupneic breath ending with a high-amplitude, short-duration burst). Microinjection of AZ and MZ into this region also reversibly increased the frequency of eupneic phrenic bursts in seven sites and produced premature bursts (i.e., doublets) in five sites. Phrenic nerve discharge increased within 5-15 min of microinjection of either agent; however, the time to the peak increase and the time to recovery were less with AZ than with MZ, consistent with the different pharmacological properties of AZ and MZ. In contrast to other CO(2)/H(+) brain stem respiratory chemosensitive sites demonstrated in vivo, which have only shown increases in amplitude of integrated phrenic nerve activity, focal tissue acidosis in the pre-BötC increases frequency of phrenic bursts and produces premature (i.e., doublet) bursts. These data indicate that the pre-BötC has the potential to play a role in the modulation of respiratory rhythm and pattern elicited by increased CO(2)/H(+) and lend additional support to the concept that the proposed locus for respiratory rhythm generation has intrinsic chemosensitivity.

Stimulation of parabrachial nuclei dilates airways in cats.

Stimulation of the parabrachial nuclei has been shown to increase mean arterial pressure as well as to terminate inspiration. Nevertheless, the effect on airway caliber evoked by stimulation of the parabrachial nuclei is not known. Therefore, in chloralose-anesthetized cats, we microinjected DL-homocysteic acid (25 nl; 100 mM) into 44 sites in or near the lateral and medial parabrachial nuclei while calculating breath-by-breath total lung resistance and dynamic compliance. We found that, in 43 of these sites, microinjection of this excitatory amino acid consistently decreased total lung resistance but had no effect on dynamic compliance. The decrease in lung resistance was caused by a withdrawal of cholinergic tone to the airways. We could find no evidence that the decrease in total lung resistance evoked by stimulation of the parabrachial nuclei was caused by activation of either beta-adrenergic or nonadrenergic noncholinergic pathways. The decrease in total lung resistance evoked by stimulation of the parabrachial nuclei was not secondary to the baroreceptor reflex even though microinjection frequently increased mean arterial pressure. In addition, microinjection did not have consistent effects on phrenic nerve activity, although in individual circumstances the effect on this activity was quite large. We conclude that stimulation of cell bodies and dendrites in the parabrachial nuclei dilates the airways of anesthetized cats and that the effect is not secondary to the baroreceptor reflex.

Blockade of glutamate receptors in CVLM and NTS attenuates airway dilation evoked from parabrachial region.

Previous work from our laboratory has shown that stimulation of cell bodies and dendrites in the medial and lateral parabrachial nuclei dilates the airways. The sites participating in the pathway mediating this airway response are not known. Two likely candidates are the caudal ventrolateral medulla (CVLM) and the nucleus tractus solitarii (NTS). Using chloralose-anesthetized cats, we assessed the airway dilation evoked from the parabrachial region before and during bilateral blockade of the NTS or the CVLM. The airway dilation arising from stimulation of the parabrachial region was evoked by microinjection of DL-homocysteic acid (25 nl, 100 mM). Bilateral blockade of the NTS or CVLM, achieved by microinjection of kynurenic acid (50 nl, 100 mM), reversibly attenuated the airway dilation in every cat tested. On average, kynurenic acid-induced blockade of the NTS caused a more complete attenuation of the dilation evoked from the parabrachial region than did blockade of the CVLM. Bilateral microinjection of cobalt chloride (50 nl, 50 mM) into the CVLM gave inconclusive results, attenuating the airway dilation evoked from the parabrachial region in six cats and potentiating it in three others. We conclude that the CVLM and the NTS participate in the airway dilation arising from the parabrachial region.

NMDA receptors in caudal ventrolateral medulla mediate reflex airway dilation arising from the hindlimb.

The caudal ventrolateral medulla (CVLM) has been shown to participate in the reflex airway dilation evoked by stimulation of thin fiber afferents innervating the hindlimb of anesthetized dogs. Nevertheless, the pharmacological mechanism in the CVLM by which hindlimb afferents evoke this reflex airway dilation is not known. Therefore, we examined the role played by excitatory amino acid receptors in the CVLM in the reflex airway dilation arising from the hindlimb. Using chloralose-anesthetized dogs, we found that bilateral microinjections into the CVLM of either (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (25 mM, 50 nl) or (+/-)-2-amino-5-phosphonovaleric acid (50 mM, 50 nl), both of which block N-methyl-D-aspartate (NMDA) receptors, reversibly attenuated the decrease in total lung resistance that was evoked by either electrical stimulation of C-fibers in the sciatic nerve or by static contraction of both gastrocnemius muscles. In contrast, bilateral microinjection into the CVLM of 6-cyano-7-nitroquinoxaline-2,3-dione (39 microM, 50 nl), which blocks non-NMDA receptors, augmented the reflex decrease in total lung resistance that was evoked by either sciatic nerve stimulation or contraction of the gastrocnemius muscles. Bilateral microinjections of xanthurenic acid (100 mM, 50 nl) into the CVLM had no effect on the decrease in total lung resistance that was evoked by sciatic nerve stimulation. We conclude that NMDA, but not non-NMDA, receptors in the CVLM play an important role in the reflex arc that dilates the airways when hindlimb afferents are stimulated by either muscular contraction or electrical stimulation.

Bronchomotor vagal preganglionic cell bodies in the dog: an anatomic and functional study.

A previous study in our laboratory demonstrated that the stimulation with microinjection of DL-homocysteic acid of cell bodies in the rostral portion of the external formation of the nucleus ambiguus (Aext) increased total lung resistance in dogs. In the present study anatomic experiments were conducted in dogs to determine if the rostral Aext contains vagal preganglionic cell bodies that give rise to axons in the pulmonary branches of the vagus nerve. The application of horseradish peroxidase (HRP) to either the pulmonary branches or the vagus at a point between the pulmonary branches and the cardiac branches resulted in retrograde labeling of cell bodies in both rostral Aext and the dorsal motor nucleus of the vagus (DMN). On the other hand, application of HRP to the vagus at a point below the pulmonary branches did not result in any retrogradely labeled cell bodies in rostral Aext but did result in labeled cell bodies in DMN. In another series of experiments DL-homocysteic acid (2.5 nmol in 25 nl) was microinjected at sites in rostral Aext and DMN. As we previously reported the injection of DL-homocysteic acid in rostral Aext increased total lung resistance. In contrast, in the same animals, the injection of DL-homocysteic acid in DMN did not change total lung resistance. We conclude that bronchomotor vagal preganglionic cell bodies are located in rostral Aext but not in DMN. The functional significance of vagal preganglionic cell bodies in DMN whose axons contribute to the pulmonary branches of the vagus nerve remains to be determined.

Role of the parabrachial nuclei in the airway dilation evoked by the Hering-Breuer reflex.

We tested the hypothesis that blockade of glutamatergic receptors in the parabrachial nucleus (PBN) of chloralose-anesthetized cats attenuated the reflex airway dilation evoked by activation of pulmonary stretch receptors. Unilateral microinjection of kynurenic acid (50 nl, 100 mM) into the PBN reversibly attenuated the reflex relaxation of the trachealis muscle in 7 cats. These findings suggest that the PBN is part of the central pathway mediating the airway dilation component of the Hering-Breuer reflex.

Reconfiguration of the pontomedullary respiratory network: a computational modeling study with coordinated in vivo experiments.

A large body of data suggests that the pontine respiratory group (PRG) is involved in respiratory phase-switching and the reconfiguration of the brain stem respiratory network. However, connectivity between the PRG and ventral respiratory column (VRC) in computational models has been largely ad hoc. We developed a network model with PRG-VRC connectivity inferred from coordinated in vivo experiments. Neurons were modeled in the "integrate-and-fire" style; some neurons had pacemaker properties derived from the model of Breen et al. We recapitulated earlier modeling results, including reproduction of activity profiles of different respiratory neurons and motor outputs, and their changes under different conditions (vagotomy, pontine lesions, etc.). The model also reproduced characteristic changes in neuronal and motor patterns observed in vivo during fictive cough and during hypoxia in non-rapid eye movement sleep. Our simulations suggested possible mechanisms for respiratory pattern reorganization during these behaviors. The model predicted that network- and pacemaker-generated rhythms could be co-expressed during the transition from gasping to eupnea, producing a combined "burst-ramp" pattern of phrenic discharges. To test this prediction, phrenic activity and multiple single neuron spike trains were monitored in vagotomized, decerebrate, immobilized, thoracotomized, and artificially ventilated cats during hypoxia and recovery. In most experiments, phrenic discharge patterns during recovery from hypoxia were similar to those predicted by the model. We conclude that under certain conditions, e.g., during recovery from severe brain hypoxia, components of a distributed network activity present during eupnea can be co-expressed with gasp patterns generated by a distinct, functionally "simplified" mechanism.

Activation of NMDA and non-NMDA receptors in the caudal ventrolateral medulla dilates the airways.

The caudal ventrolateral medulla (CVLM) participates in the central control of airway caliber. For example, both electrical and chemical stimulation of the CVLM decrease total lung resistance by withdrawing cholinergic input to airway smooth muscle. Although cell bodies in the CVLM have been shown to play an important role in mediating the central control of airway caliber, the pharmacological mechanism in this brainstem region responsible for causing this airway dilation is unknown. We, therefore, examined the role played by ionotropic excitatory amino acid receptors in the CVLM in the control of airway caliber in chloralose-anesthetized dogs. We found that microinjection of 3.9 pmol of NMDA or AMPA or quisqualate into 12 sites in the CVLM decreased total lung resistance by 1.5 +/- 0.2 cm H2O l(-1) s(-1) (p < 0.05), and that microinjection of 3.9 pmol of kainic acid into 9 in the CVLM decreased total lung resistance by 0.5 +/- 0.1 cm H2O l(-1) s(-1) (p < 0.05). The decrease in total lung resistance evoked by either NMDA or AMPA or quisqualate was not different (p > 0.05) while that evoked by kainic acid was significantly smaller. Additionally, microinjection of NMDA or AMPA or quisqualate caused a small but significant decrease in mean arterial pressure and heart rate (p < 0.05). These experiments demonstrate that the airway dilation evoked by stimulation of excitatory amino acid receptors in the CVLM is mediated by both NMDA and non-NMDA receptors.

Excitation of phrenic and sympathetic output during acute hypoxia: contribution of medullary oxygen detectors.

Severe brain hypoxia results in respiratory excitation and an increase in sympathetic nerve activity. Respiratory excitation takes the form of gasping which is characterized by an abrupt onset, high amplitude, short duration burst of inspiratory activity. Recent evidence suggests that centrally-mediated hypoxic respiratory and sympathetic excitation may result from direct hypoxic stimulation of discrete hypoxia chemosensitive sites in the medulla. Thus, medullary regions involved in the generation and modulation of respiratory and sympathetic vasomotor output may contain neurons which function as central oxygen detectors, acting as medullary analogs to the peripheral (arterial) chemoreceptors. This review focuses on the medullary sites and mechanisms proposed to mediate hypoxic respiratory and sympathetic excitation in anesthetized, chemodeafferented animals, and provides the evidence suggesting a role for central oxygen detectors in the control of breathing and sympathetic vasomotor output.

Effects of low intrapulmonary PCO2 on ventilatory sensitivity to PaCO2 in chickens.

We found conflicting reports regarding the relationship between intrapulmonary PCO2 and sensitivity to PaCO2 in the chicken. To resolve this, we anesthetized eight cockerels with sodium pentobarbital (25-35 mg/kg), cannulated the cutaneous ulnar vein and carotid artery, opened the thorax and ventilated right and left lungs independently. We established PaCO2 by over-ventilating the denervated, perfused right lung with the following: 94.7, 39.9, 31.6 and 19.0 Torr PCO2 balanced with oxygen. At each right lung CO2 tension (PRCO2), we measured blood pressure and ventilatory responses to five CO2 tensions (PLCO2) ventilating the non-perfused left lung. PLCO2 ranged from 70.3 to 15.7 Torr. We found a linear relationship between the amplitude of sternal deflections and PaCO2-1, and that the slope depended upon PLCO2. Respiratory period was also linearly related to PaCO2-1, however the intercept, not the slope, was a altered by PLCO2. Stepwise regression analyses revealed an important interaction term (that is, (PaCO2.PLCO2)-1) in the determination of respiratory amplitude, but not in the determination of period. We conclude that low intrapulmonary PCO2 enhances the sensitivity of respiratory amplitude to changes in PaCO2. Further, we propose that intrapulmonary chemoreceptor discharge mediates this change in sensitivity to PaCO2.

Static muscular contraction elicits a pressor reflex in the chicken.

Static muscular contraction has been shown to increase arterial blood pressure and heart rate in humans and other mammals. It is not clear, however, whether birds exhibit a similar response to this maneuver. Therefore, we designed these experiments to determine if the chicken exhibits a cardiovascular response to static muscular contraction and if the observed responses are evoked through a reflex involving muscle afferents. Static contraction of the gastrocnemius muscle was evoked by electrically stimulating the sciatic nerve at 1.5-3.0 times motor threshold (30-40 Hz; 0.025 ms) in 13 chloralose-anesthetized cockerels. We measured arterial blood pressure and muscle tension before and during static contraction and calculated mean arterial pressure and heart rate from the arterial pressure trace. We found that static contraction of the gastrocnemius muscle increased mean arterial pressure from 71 +/- 4 to 95 +/- 4 mmHg (P < 0.05) and increased heart rate from 304 +/- 8 to 345 +/- 10 beats/min (P < 0.05). Furthermore, we found that stimulation of the sciatic nerve after paralysis of the birds with vecuronium bromide or stimulation of the cut peripheral end of the sciatic nerve (using the same stimulation parameters described above) evoked no change in mean arterial pressure or heart rate. We conclude that static muscular contraction of the gastrocnemius muscle in the chicken elicits a pressor response and that this response is due to a reflex arising from the contracting muscles.

Pre-Bötzinger complex functions as a central hypoxia chemosensor for respiration in vivo.

Recently, we identified a region located in the pre-Bötzinger complex (pre-BötC; the proposed locus of respiratory rhythm generation) in which activation of ionotropic excitatory amino acid receptors using DL-homocysteic acid (DLH) elicits a variety of excitatory responses in the phrenic neurogram, ranging from tonic firing to a rapid series of high-amplitude, rapid rate of rise, short-duration inspiratory bursts that are indistinguishable from gasps produced by severe systemic hypoxia. Therefore we hypothesized that this unique region is chemosensitive to hypoxia. To test this hypothesis, we examined the response to unilateral microinjection of sodium cyanide (NaCN) into the pre-BötC in chloralose- or chloralose/urethan-anesthetized vagotomized, paralyzed, mechanically ventilated cats. In all experiments, sites in the pre-BötC were functionally identified using DLH (10 mM, 21 nl) as we have previously described. All sites were histologically confirmed to be in the pre-BötC after completion of the experiment. Unilateral microinjection of NaCN (1 mM, 21 nl) into the pre-BötC produced excitation of phrenic nerve discharge in 49 of the 81 sites examined. This augmentation of inspiratory output exhibited one of the following changes in cycle timing and/or pattern: 1) a series of high-amplitude, short-duration bursts in the phrenic neurogram (a discharge similar to a gasp), 2) a tonic excitation of phrenic neurogram output, 3) augmented bursts in the phrenic neurogram (i.e., eupneic breath ending with a gasplike burst), or 4) an increase in frequency of phrenic bursts accompanied by small increases or decreases in the amplitude of integrated phrenic nerve discharge. Our findings identify a locus in the brain stem in which focal hypoxia augments respiratory output. We propose that the respiratory rhythm generator in the pre-BötC has intrinsic hypoxic chemosensitivity that may play a role in hypoxia-induced gasping.

Chicken intrapulmonary and cardiac nerve afferents interact in ventilatory control.

Intrapulmonary chemoreceptors (IPC) and extrapulmonary afferents (EPA) have been shown to interact in ventilatory control in the chicken. It is not clear, however, if all or only some of the EPA are involved in this interaction with IPC. We therefore designed this study to determine if afferents carried in the middle cardiac nerve (MCN) interact centrally with IPC. We anesthetized six cockerels with sodium pentobarbital (ca. 30 mg/kg), cannulated the cutaneous ulnar vein and carotid artery, opened the thorax, and unidirectionally ventilated each lung separately. The right, denervated lung was used to fix PaCO2 at the following levels: 89.5 +/- 1.1, 41.9 +/- 0.6, 35.9 +/- 0.8, and 23.6 +/- 0.5 Torr. At each PaCO2, we measured ventilatory and blood pressure responses while the left, non-perfused lung was ventilated with gases ranging from 70.2 to 15.7 Torr PCO2. We then cut the MCN and repeated the protocol. We found that MCN section increased the amplitude of sternal deflections (SD/SDmax) at all levels of intrapulmonary PCO2 and reduced the respiratory period (T/Tmax) at all but the highest level of intrapulmonary PCO2. Mean arterial pressure increased following MCN section, and this increase was independent of intrapulmonary PCO2. Multiple regression analyses of SD/SDmax revealed that MCN section reduced both arterial CO2 sensitivity and the interaction between EPA and IPC. These findings suggest that afferents carried in the MCN interact with IPC to reduce ventilatory amplitude during hypocapnia.

Patterns of phrenic motor output evoked by chemical stimulation of neurons located in the pre-Bötzinger complex in vivo.

The pre-Bötzinger complex (pre-BötC) has been proposed to be essential for respiratory rhythm generation from work in vitro. Much less, however, is known about its role in the generation and modulation of respiratory rhythm in vivo. Therefore we examined whether chemical stimulation of the in vivo pre-BötC manifests respiratory modulation consistent with a respiratory rhythm generator. In chloralose- or chloralose/urethan-anesthetized, vagotomized cats, we recorded phrenic nerve discharge and arterial blood pressure in response to chemical stimulation of neurons located in the pre-BötC with DL-homocysteic acid (DLH; 10 mM; 21 nl). In 115 of the 122 sites examined in the pre-BötC, unilateral microinjection of DLH produced an increase in phrenic nerve discharge that was characterized by one of the following changes in cycle timing and pattern: 1) a rapid series of high-amplitude, rapid rate of rise, short-duration bursts, 2) tonic excitation (with or without respiratory oscillations), 3) an integration of the first two types of responses (i.e., tonic excitation with high-amplitude, short-duration bursts superimposed), or 4) augmented bursts in the phrenic neurogram (i.e., eupneic breath ending with a high-amplitude, short-duration burst). In 107 of these sites, the phrenic neurogram response was accompanied by an increase or decrease (>/=10 mmHg) in arterial blood pressure. Thus increases in respiratory burst frequency and production of tonic discharge of inspiratory output, both of which have been seen in vitro, as well as modulation of burst pattern can be produced by local perturbations of excitatory amino acid neurotransmission in the pre-BötC in vivo. These findings are consistent with the proposed role of this region as the locus for respiratory rhythm generation.

Localization of connexin26 and connexin32 in putative CO(2)-chemosensitive brainstem regions in rat.

Recent studies have suggested that cell-to-cell coupling, which occurs via gap junctions, may play a role in CO(2) chemoreception. Here, we used immunoblot and immunohistochemical analyses to investigate the presence, distribution, and cellular localization of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) in putative CO(2)-chemosensitive brainstem regions in both neonatal and adult rats. Immunoblot analyses revealed that both Cx subtypes were expressed in putative CO(2)-chemosensitive brainstem regions; however, regional differences in expression were observed. Immunohistochemical experiments confirmed Cx expression in each of the putative CO(2)-chemosensitive brainstem regions, and further demonstrated that Cx26 and Cx32 were found in neurons and Cx26 was also found in astrocytes in these regions. Thus, our findings suggest the potential for gap junctional communication in these regions in both neonatal and adult rats. We propose that the gap junction proteins Cx26 and Cx32, at least in part, form the neuroanatomical substrate for this gap junctional communication, which is hypothesized to play a role in central CO(2) chemoreception.