Purinergic receptor blockade in the retrotrapezoid nucleus attenuates the respiratory chemoreflexes in awake rats.

AIM: Recent evidence suggests that adenosine triphosfate (ATP)-mediated purinergic signalling at the level of the rostral ventrolateral medulla contributes to both central and peripheral chemoreceptor control of breathing and blood pressure: neurones in the retrotrapezoid nucleus (RTN) function as central chemoreceptors in part by responding to CO2 -evoked ATP release by activation of yet unknown P2 receptors, and nearby catecholaminergic C1 neurones regulate blood pressure responses to peripheral chemoreceptor activation by a P2Y1 receptor-dependent mechanism. However, potential contributions of purinergic signalling in the RTN to cardiorespiratory function in conscious animals have not been tested. METHODS: Cardiorespiratory activity of unrestrained awake rats was measured in response to RTN injections of ATP, and during exposure to hypercapnia (7% CO2 ) or hypoxia (8% O2 ) under control conditions and after bilateral RTN injections of P2 receptor blockers (PPADS or MRS2179). RESULTS: Unilateral injection of ATP into the RTN increased cardiorespiratory output by a P2-receptor-dependent mechanism. We also show that bilateral RTN injections of a non-specific P2 receptor blocker (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) reduced the ventilatory response to hypercapnia (7% CO2 ) and hypoxia (8% O2 ) in unanesthetized rats. Conversely, bilateral injections of a specific P2Y1 receptor blocker (MRS2179) into the RTN had no measurable effect on ventilatory responses elicited by hypercapnia or hypoxia. CONCLUSION: These data exclude P2Y1 receptor involvement in the chemosensory control of breathing at the level of the RTN and show that ATP-mediated purinergic signalling contributes to central and peripheral chemoreflex control of breathing and blood pressure in awake rats.

Respiratory deficits in a rat model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of the dopaminergic nigrostriatal pathway. In addition to deficits in voluntary movement, PD involves a disturbance of breathing regulation. However, the cause and nature of this disturbance are not well understood. Here, we investigated breathing at rest and in response to hypercapnia (7% CO2) or hypoxia (8% O2), as well as neuroanatomical changes in brainstem regions essential for breathing, in a 6-hydroxydopamine (6-OHDA) rat model of PD. Bilateral injections of 6-OHDA (24µg/µl) into the striatum decreased tyrosine hydroxylase (TH(+))-neurons in the substantia nigra pars compacta (SNpc), transcription factor phox2b-expressing neurons in the retrotrapezoid nucleus and neurokinin-1 receptors in the ventral respiratory column. In 6-OHDA-lesioned rats, respiratory rate was reduced at rest, leading to a reduction in minute ventilation. These animals also showed a reduction in the tachypneic response to hypercapnia, but not to hypoxia challenge. These results suggest that the degeneration of TH(+) neurons in the SNpc leads to impairment of breathing at rest and in hypercapnic conditions. Our data indicate that respiratory deficits in a 6-OHDA rat model of PD are related to downregulation of neural systems involved in respiratory rhythm generation. The present study suggests a new avenue to better understand the respiratory deficits observed in chronic stages of PD.

Brainstem areas activated by intermittent apnea in awake unrestrained rats.

We investigated the role of the autonomic nervous system to cardiovascular responses to obstructive apnea in awake, unrestrained rats, and measured expression of Fos induced by apnea in the brainstem. We implanted a tracheal balloon contained in a rigid tube to allow the induction of apnea without inducing pain in the trachea. During bouts of 15s of apnea, heart rate fell from 371±8 to 161±11bpm (mean±SEM, n=15, p<0.01) and arterial pressure increased from 115±2 to 131±4mmHg (p<0.01). Bradycardia was due to parasympathetic activity because it was blocked by the muscarinic antagonist, methylatropine. The pressor response was due to vasoconstriction caused by sympathetic activation because it was blocked by the α1 antagonist, prazosin. Apnea induced Fos expression in several brainstem areas involved in cardiorespiratory control such as the nucleus of the solitary tract (NTS), ventrolateral medulla (VLM), and pons. Ligation of the carotid body artery reduced apnea-induced bradycardia, blocked heart rate responses to i.v. injection of cyanide, reduced Fos expression in the caudal NTS, and increased Fos expression in the rostral VLM. In conclusion, apnea activates neurons in regions that process signals from baroreceptors, chemoreceptors, pulmonary receptors, and regions responsible for autonomic and respiratory activity both in the presence and absence of carotid chemoreceptors.

Acute exercise-induced activation of Phox2b-expressing neurons of the retrotrapezoid nucleus in rats may involve the hypothalamus.

The rat retrotrapezoid nucleus (RTN) contains neurons that have a well-defined phenotype characterized by the presence of vesicular glutamate transporter 2 (VGLUT2) mRNA and a paired-like homeobox 2b (Phox2b)-immunoreactive (ir) nucleus and the absence of tyrosine hydroxylase (TH). These neurons are important to chemoreception. In the present study, we tested the hypothesis that the chemically-coded RTN neurons (ccRTN) (Phox2b(+)/TH(-)) are activated during an acute episode of running exercise. Since most RTN neurons are excited by the activation of perifornical and lateral hypothalamus (PeF/LH), a region that regulates breathing during exercise, we also tested the hypothesis that PeF/LH projections to RTN neurons contribute to their activation during acute exercise. In adult male Wistar rats that underwent an acute episode of treadmill exercise, there was a significant increase in c-Fos immunoreactive (c-Fos-ir) in PeF/LH neurons and RTN neurons that were Phox2b(+)TH(-) (p<0.05) compared to rats that did not exercise. Also the retrograde tracer Fluoro-Gold that was injected into RTN was detected in c-Fos-ir PeF/LH (p<0.05). In summary, the ccRTN neurons (Phox2b(+)TH(-)) are excited by running exercise. Thus, ccRTN neurons may contribute to both the chemical drive to breath and the feed-forward control of breathing associated with exercise.

Commissural nucleus of the solitary tract regulates the antihypertensive effects elicited by moxonidine.

The rostral ventrolateral medulla (RVLM) contains the presympathetic neurons involved in cardiovascular regulation that has been implicated as one of the most important central sites for the antihypertensive action of moxonidine (an α2-adrenergic and imidazoline agonist). Here, we sought to evaluate the cardiovascular effects produced by moxonidine injected into another important brainstem site, the commissural nucleus of the solitary tract (commNTS). Mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and activity of putative sympathoexcitatory vasomotor neurons of the RVLM were recorded in conscious or urethane-anesthetized, and artificial ventilated male Wistar rats. In conscious or anesthetized rats, moxonidine (2.5 and 5 nmol/50 nl) injected into the commNTS reduced MAP, HR and sSNA. The injection of moxonidine into the commNTS also elicited a reduction of 28% in the activity of sympathoexcitatory vasomotor neurons of the RVLM. To further assess the notion that moxonidine could act in another brainstem area to elicit the antihypertensive effects, a group with electrolytic lesions of the commNTS or sham and with stainless steel guide-cannulas implanted into the 4th V were used. In the sham group, moxonidine (20 nmol/1 µl) injected into 4th V decreased MAP and HR. The hypotension but not the bradycardia produced by moxonidine into the 4th V was reduced in acute (1 day) commNTS-lesioned rats. These data suggest that moxonidine can certainly act in other brainstem regions, such as commNTS to produce its beneficial therapeutic effects, such as hypotension and reduction in sympathetic nerve activity.

Arterial chemoreceptor activation reduces the activity of parapyramidal serotonergic neurons in rats.

The parapyramidal (ppy) region targets primarily the intermediolateral cell column and is probably involved in breathing and thermoregulation. In the present study, we tested whether ppy serotonergic neurons respond to activation of central and peripheral chemoreceptors. Bulbospinal ppy neurons (n=30) were recorded extracellularly along with the phrenic nerve activity in urethane/α-chloralose-anesthetized, paralyzed, intact (n=7) or carotid body denervated (n=6) male Wistar rats. In intact animals, most of the ppy neurons were inhibited by hypoxia (n=14 of 19) (8% O2, 30s) (1.5 ± 0.03 vs. control: 2.4 ± 0.2 Hz) or hypercapnia (n=15 of 19) (10% CO2) (1.7 ± 0.1 vs. control: 2.2 ± 0.2 Hz), although some neurons were insensitive to hypoxia (n=3 of 19) or hypercapnia (n=4 of 19). Very few neurons (n=2 of 19) were activated after hypoxia, but not after hypercapnia. In carotid body denervated rats, all the 5HT-ppy neurons (n=11) were insensitive to hypercapnia (2.1 ± 0.1 vs. control: 2.3 ± 0.09 Hz). Biotinamide-labeled cells that were recovered after histochemistry were located in the ppy region. Most labeled cells (90%) showed strong tryptophan hydroxylase immunocytochemical reactivity, indicating that they were serotonergic. The present data reveal that peripheral chemoreceptors reduce the activity of the serotonergic premotor neurons located in the ppy region. It is plausible that the serotonergic neurons of the ppy region could conceivably regulate breathing automaticity and be involved in autonomic regulation.

Pontomedullary and hypothalamic distribution of Fos-like immunoreactive neurons after acute exercise in rats.

During exercise, intense brain activity orchestrates an increase in muscle tension. Additionally, there is an increase in cardiac output and ventilation to compensate the increased metabolic demand of muscle activity and to facilitate the removal of CO(2) from and the delivery of O(2) to tissues. Here we tested the hypothesis that a subset of pontomedullary and hypothalamic neurons could be activated during dynamic acute exercise. Male Wistar rats (250-350 g) were divided into an exercise group (n=12) that ran on a treadmill and a no-exercise group (n=7). Immunohistochemistry of pontomedullary and hypothalamic sections to identify activation (c-Fos expression) of cardiorespiratory areas showed that the no-exercise rats exhibited minimal Fos expression. In contrast, there was intense activation of the nucleus of the solitary tract, the ventrolateral medulla (including the presumed central chemoreceptor neurons in the retrotrapezoid/parafacial region), the lateral parabrachial nucleus, the Kölliker-Fuse region, the perifornical region, which includes the perifornical area and the lateral hypothalamus, the dorsal medial hypothalamus, and the paraventricular nucleus of the hypothalamus after running exercise. Additionally, we observed Fos immunoreactivity in catecholaminergic neurons within the ventrolateral medulla (C1 region) without Fos expression in the A2, A5 and A7 neurons. In summary, we show for the first time that after acute exercise there is an intense activation of brain areas crucial for cardiorespiratory control. Possible involvement of the central command mechanism should be considered. Our results suggest whole brain-specific mobilization to correct and compensate the homeostatic changes produced by acute exercise.

Control of the central chemoreflex by A5 noradrenergic neurons in rats.

Central chemoreflex stimulation produces an increase in phrenic nerve activity (PNA) and sympathetic nerve activity (SNA). The A5 noradrenergic region projects to several brainstem areas involved in autonomic regulation and contributes to the increase in SNA elicited by peripheral chemoreflex activation. The aim of the present study was to further test the hypothesis that the A5 noradrenergic region could contribute to central chemoreflex activation. In urethane-anesthetized, sino-aortic denervated, and vagotomized male Wistar rats (n=6-8/group), hypercapnia (end-expiratory CO2 from 5% to 10%) increased mean arterial pressure (MAP; Δ=+33±4 mmHg, P<0.05), splanchnic SNA (sSNA; Δ=+97±13%, P<0.05), and PNA frequency and amplitude. Bilateral injection of muscimol (GABA-A agonist; 2 mM) into the A5 noradrenergic region reduced the rise in MAP (Δ=+19±3 mmHg, P<0.05), sSNA (Δ=+63±5%, P<0.05), and PNA frequency and amplitude produced by hypercapnia. Injections of the immunotoxin anti-dopamine ß-hydroxylase-saporin (anti-DßH-SAP) into the A5 region destroyed TH⁺ neurons but spared facial motoneurons and the chemosensitive neurons in the retrotrapezoid nucleus that express the transcription factor Phox2b and that are non-catecholaminergic (TH⁻Phox2b⁺). Two weeks after selective destruction of the A5 region with the anti-DßH-SAP toxin, the increase in MAP (Δ=+22±5 mmHg, P<0.05), sSNA (Δ=+68±9%, P<0.05), and PNA amplitude was reduced after central chemoreflex activation. These results suggest that A5 noradrenergic neurons contribute to the increase in MAP, sSNA, and PNA activation during central chemoreflex stimulation.

Central chemoreceptors and neural mechanisms of cardiorespiratory control

The arterial partial pressure (P CO2) of carbon dioxide is virtually constant because of the close match between the metabolic production of this gas and its excretion via breathing. Blood gas homeostasis does not rely solely on changes in lung ventilation, but also to a considerable extent on circulatory adjustments that regulate the transport of CO2 from its sites of production to the lungs. The neural mechanisms that coordinate circulatory and ventilatory changes to achieve blood gas homeostasis are the subject of this review. Emphasis will be placed on the control of sympathetic outflow by central chemoreceptors. High levels of CO2 exert an excitatory effect on sympathetic outflow that is mediated by specialized chemoreceptors such as the neurons located in the retrotrapezoid region. In addition, high CO2 causes an aversive awareness in conscious animals, activating wake-promoting pathways such as the noradrenergic neurons. These neuronal groups, which may also be directly activated by brain acidification, have projections that contribute to the CO2-induced rise in breathing and sympathetic outflow. However, since the level of activity of the retrotrapezoid nucleus is regulated by converging inputs from wake-promoting systems, behavior-specific inputs from higher centers and by chemical drive, the main focus of the present manuscript is to review the contribution of central chemoreceptors to the control of autonomic and respiratory mechanisms.

Central chemoreceptors and neural mechanisms of cardiorespiratory control.

The arterial partial pressure (P(CO)(2)) of carbon dioxide is virtually constant because of the close match between the metabolic production of this gas and its excretion via breathing. Blood gas homeostasis does not rely solely on changes in lung ventilation, but also to a considerable extent on circulatory adjustments that regulate the transport of CO(2) from its sites of production to the lungs. The neural mechanisms that coordinate circulatory and ventilatory changes to achieve blood gas homeostasis are the subject of this review. Emphasis will be placed on the control of sympathetic outflow by central chemoreceptors. High levels of CO(2) exert an excitatory effect on sympathetic outflow that is mediated by specialized chemoreceptors such as the neurons located in the retrotrapezoid region. In addition, high CO(2) causes an aversive awareness in conscious animals, activating wake-promoting pathways such as the noradrenergic neurons. These neuronal groups, which may also be directly activated by brain acidification, have projections that contribute to the CO(2)-induced rise in breathing and sympathetic outflow. However, since the level of activity of the retrotrapezoid nucleus is regulated by converging inputs from wake-promoting systems, behavior-specific inputs from higher centers and by chemical drive, the main focus of the present manuscript is to review the contribution of central chemoreceptors to the control of autonomic and respiratory mechanisms.

Inhibition of the caudal pressor area reduces cardiorespiratory chemoreflex responses.

The caudal pressor area (CPA) is a brainstem area located close to the spinal cord. The activation of the CPA increases sympathetic activity and mean arterial pressure (MAP) by mechanisms dependent on the commissural nucleus of the solitary tract (commNTS) and rostroventrolateral medulla, however, the signals that activate the CPA to produce these responses are still unknown. Therefore, in the present study, we investigated the activity of glutamatergic and GABAergic mechanisms from the CPA and commNTS in rats exposed to hypoxia and the effects of the inhibition of CPA neurons on cardiorespiratory responses to peripheral chemoreceptor activation with i.v. sodium cyanide (NaCN). Male Sprague-Dawley rats (250-280 g, n=5-8/group) were used. In conscious rats, most of the commNTS neurons (66±11%) and part of the CPA neurons (36±7%) activated by hypoxia (8% O2) were glutamatergic (contained VGLUT2mRNA). Small part of the neurons activated during hypoxia was GABAergic (contained GAD-67mRNA) in the commNTS (9±4%) or the CPA (6±2%). In urethane anesthetized rats, the inhibition of CPA neurons with bilateral injections of muscimol (GABA-A agonist, 2 mM) reduced baseline MAP, splanchnic sympathetic nerve discharge (SND) and phrenic nerve discharge (PND). Muscimol into the CPA also reduced by around 50% the pressor and sympathoexcitatory responses and the increase in PND to peripheral chemoreceptor activation with NaCN (50 µg/kg i.v.), without changing sympathetic baroreflex responses. These data suggest that CPA mechanisms facilitate cardiorespiratory responses to peripheral chemoreflex activation. Immunohistochemistry results also suggest that at least part of the CPA mechanisms activated by hypoxia is glutamatergic.

Central nervous system distribution of the transcription factor Phox2b in the adult rat.

Phox2b is required for development of the peripheral autonomic nervous system and a subset of cranial nerves and lower brainstem nuclei. Phox2b mutations in man cause diffuse autonomic dysfunction and deficits in the automatic control of breathing. Here we study the distribution of Phox2b in the adult rat hindbrain to determine whether this protein is selectively expressed by neurons involved in respiratory and autonomic control. In the medulla oblongata, Phox2b-immunoreactive nuclei were present in the dorsal vagal complex, intermediate reticular nucleus, dorsomedial spinal trigeminal nucleus, nucleus ambiguus, catecholaminergic neurons, and retrotrapezoid nucleus (RTN). Phox2b was expressed by both central excitatory relays of the sympathetic baroreflex (nucleus of the solitary tract and C1 neurons) but not by the inhibitory relay of this reflex. Phox2b was absent from the ventral respiratory column (VRC) caudal to RTN and rare within the parabrachial nuclei. In the pons, Phox2b was confined to cholinergic efferent neurons (salivary, vestibulocochlear) and noncholinergic peritrigeminal neurons. Rostral to the pons, Phox2b was detected only in the oculomotor complex. In adult rats, Phox2b is neither a comprehensive nor a selective marker of hindbrain autonomic pathways. This marker identifies a subset of hindbrain neurons that control orofacial movements (dorsomedial spinal trigeminal nucleus, pontine peritrigeminal neurons), balance and auditory function (vestibulocochlear efferents), the eyes, and both divisions of the autonomic efferent system. Phox2b is virtually absent from the respiratory rhythm and pattern generator (VRC and dorsolateral pons) but is highly expressed by neurons involved in the chemical drive and reflex regulation of this oscillator.

Central muscarinic receptors signal pilocarpine-induced salivation.

Although cholinergic agonists such as pilocarpine injected peripherally can act directly on salivary glands to induce salivation, it is possible that their action in the brain may contribute to salivation. To investigate if the action in the brain is important to salivation, we injected pilocarpine intraperitoneally after blockade of central cholinergic receptors with atropine methyl bromide (atropine-mb). In male Holtzman rats with stainless steel cannulas implanted into the lateral ventricle and anesthetized with ketamine, atropine-mb (8 and 16 nmol) intracerebroventricularly reduced the salivation induced by pilocarpine (4 micro mol/kg) intraperitoneally (133 + 42 and 108 + 22 mg/7 min, respectively, vs. saline, 463 + 26 mg/7 min), but did not modify peripheral cardiovascular responses to intravenous acetylcholine. Similar doses of atropine-mb intraperitoneally also reduced pilocarpine-induced salivation. Therefore, systemically injected pilocarpine also enters the brain and acts on central muscarinic receptors, activating autonomic efferent fibers to induce salivation.