Phrenic Nerve Discharge Research Articles

The temporal relationship between non‐respiratory burst activity of expiratory laryngeal motoneurons and phrenic apnoea during stimulation of the superior laryngeal nerve in rat

A striking effect of stimulating the superior laryngeal nerve (SLN) is its ability to inhibit central inspiratory activity (cause ‘phrenic apnoea'), but the mechanism underlying this inhibition remains unclear. Here we demonstrate, by stimulating the SLN at varying frequencies, that the evoked non-respiratory burst activity recorded from expiratory laryngeal motoneurons (ELMs) has an intimate temporal relationship with phrenic apnoea. During 1–5 Hz SLN stimulation, occasional absences of phrenic nerve discharge (PND) occurred such that every absent PND was preceded by an ELM burst activity. During 10–20 Hz SLN stimulation, more bursts were evoked together with more absent PNDs, leading eventually to phrenic apnoea. Interestingly, subsequent microinjections of isoguvacine (10 mm, 20–40 nl) into ipsilateral Bötzinger complex (BötC) and contralateral nucleus tractus solitarii (NTS) significantly attenuated the apnoeic response but not the ELM burst activity. Our results suggest a bifurcating projection from NTS to both the caudal nucleus ambiguus and BötC, which mediates the closely related ELM burst and apnoeic response, respectively. We believe that such an intimate timing between laryngeal behaviour and breathing is crucial for the effective elaboration of the different airway protective behaviours elicited following SLN stimulation, including the laryngeal adductor reflex, swallowing and cough.

Altered sympathetic reflexes and vascular reactivity in rats after exposure to chronic intermittent hypoxia

Exposure to chronic intermittent hypoxia (CIH) yields persistent elevations in sympathetic nerve activity (SNA) and mean arterial pressure(MAP)with exaggerated sympathetic chemoreflexes. We examined the impact of CIH upon other sympathoexcitatory reflexes and a potential central mechanism underlying the altered regulation of SNA.Male Sprague-Dawley rats were exposed to CIH for 2 weeks (40 s at 6% O2 every 9 min, 8 h day⁻¹). After exposure to CIH, urethane-anaesthetized, vagotomized, ventilated, paralysed rats had significantly elevated MAP, splanchnic SNA, and rate of phrenic nerve discharge (PND; P<0.05). Elimination of SNA by ganglionic blockade produced a larger fall in MAP in rats exposed to CIH (P<0.05). Like acute hypoxia, stimulation of the sciatic nerve or the nasal mucosa evoked greater increases in SNA after exposure to CIH (P<0.05). In addition, acute hypoxia promoted exaggerated increases in PND amplitude after CIH (P<0.05). In contrast, the nasopharyngeal reflex evoked exaggerated increases in SNA during apnoea. These sympathoexcitatory reflexes are mediated by glutamatergic activation of the rostral ventrolateral medulla (RVLM), and accordingly, microinjections of glutamate into RVLM evoked larger increases in SNA after CIH (P<0.05). Paradoxically, none of these exaggerated acute rises in SNA was accompanied by enhanced pressor responses. Reduced adrenergic vascular reactivity may contribute to the blunted sympathetically mediated pressor responses, because bolus doses of phenylephrine evoked attenuated pressor responses after CIH (P<0.01).These data suggest exposure to CIH facilitates activation of SNA, potentially by changes within the RVLM. However, the exaggerated rises in SNA are not dependent upon stimulation of inspiratory drive. Although elevated SNA may contribute to CIH-induced hypertension, reduced adrenergic vascular reactivity buffers the cardiovascular impact of exaggerated acute rises in SNA.

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.

Time–frequency energy distribution of phrenic nerve discharges during aspiration reflex, cough and quiet inspiration

Aspiration reflex (AspR) represents a specific inspiratory motor behavior expressed by short, powerful inspiratory activity without subsequent active expiration and characterized by the ability to interrupt strong tonic inspiratory activity, as well as hypoxic apnea and several other functional disorders. Multiresolution analysis-based determination of spectral features arising during AspR has not yet been satisfactorily investigated. The time–frequency energy distribution of phrenic nerve electrical activity was compared during the AspR, inspiratory phase of tracheobronchial cough and quiet inspiration. Data obtained from 16 adult cats anesthetized with chloralose or pentobarbital were analyzed using a wavelet transformation, a sensitive method suitable for processing of the non-stationary respiratory output signal. Phrenic nerve energy was accumulated within lower frequency bands in AspR bursts. In AspR, higher frequencies contributed less to the total power, when compared to cough inspiration. Moreover, AspR indicated a stable time–frequency energy distribution, regardless of which of the two types of anesthesia were used. Chloralose anesthesia induced a decrease of parameters in cough and quiet inspiration related to the quantity of energy. The results indicate a specific method of information processing during generation of AspR, underlying its powerful ability to influence various severe functional disorders with potential implications for model experiments and clinical practice.

Tonic and reflex control of the cardio-respiratory system by neurons in the ventral medulla

To investigate the channels and neurotransmitters in the ventrolateral medulla (VLM) oblongata that are responsible for the maintenance of sympathetic tone and cardio-respiratory reflex regulation. Microinjections of excitant amino acid (glutamate, 100 nl, 100 mM), calcium channel blockers, agonists and antagonists were made throughout the VLM in anaesthetized rats. Arterial blood pressure, sympathetic nerve activity and phrenic nerve discharge was recorded. Responses to activation of the baroreceptor, somatosympathetic, and chemoreceptor reflexes were recorded. Injection sites were marked with albumin-colloidal gold or methylene blue. At the end of recording, brains were removed, fixed, sectioned and stained for gold spots, and counterstained to localize injection sites. Bilateral microinjection of calcium channel antagonists into the rostral VLM (RVLM) had little effect on arterial blood pressure with the exception of Ni++ (an agent that blocks low voltage T-type calcium channels). Ni++ caused a profound fall in arterial blood pressure and sympathetic nerve activity, but did not affect reflex function. By contrast the high voltage channel (P, Q, L, N) blockers increased blood pressure and sympathetic activity following injection into the caudal VLM (CVLM). The agents applied were 8-OHDPAT; 5HT1a agonist, DAMGO; mu opioid agonist, DPDPE; delta opioid agonist and sar9-substance P; substance P agonist. In addition, the effect of changing the inhaled concentration of carbon dioxide was tested. In all cases except injection DAMGO, there was an attenuation of the somato-sympathetic reflex. DAMGO attenuated the baroreceptor reflex. None of these agents affected the chemoreceptor reflex. The data suggest that tonic maintenance of sympathetic activity is mediated, at least in part, by a Ni++ sensitive conductance in RVLM and that the neurochemical heterogeneity found within the inputs to sympathoexcitatory and inhibitory neurons in the RVLM has functional significance, with different neurotransmitters affecting cardiorespiratory reflexes differentially. Further functional studies are required to determine the precise connectivity between different neuronal populations that are subserving different functions in the RVLM.

Neurochemistry and anatomy of the ventral medulla

The relationship between the anatomy and neurochemistry of neurons in the ventral medulla oblongata in regions that is responsible for cardiovascular, airways, and respiratory regulation was investigated. Microinjections of excitant amino acid (glutamate, 100 nl, 100 mM) were made throughout the ventral medulla in anesthetized rats. Arterial blood pressure, sympathetic nerve activity and phrenic nerve discharge was recorded. Injection sites were marked with albumin-colloidal gold or methylene blue. At the end of recording, brains were removed, fixed, sectioned and stained for gold spots, dopamine-beta-hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT) and serotonin. In the ventrolateral medulla (VLM), several interesting features emerged; first, the entire region corresponding to the pressor and depressor regions of the rostral and caudal VLM (RVLM and CVLM) lay in the area containing PNMT-immunoreactive neurons (C1 cell group). Caudal to the CVLM pressor sites were identified as far caudally as the spino-medullary junction. Activation of the B?tzinger region consistently inhibited phrenic amplitude and frequency whereas activation of the pre B?tzinger increased the basal level of phrenic nerve discharge as well as its frequency and amplitude. The PreBotzinger is found at the same level as the most rostral part of the CVLM. Medial sites in CVLM tended to be excitatory while lateral sites evoked inhibition. In the midline, few sites were found where excitation elicited large (±10%) changes in blood pressure or sympathetic nerve activity. However at one site (11.3 mm caudal to bregma) injection of glutamate elicited phrenic apnea in both paralyzed and non-paralyzed animals. The ventral medulla contains many areas from which responses that affect the control of airways, breathing and circulation exist; these responsive sites are coextensive with populations of neurons that are immunoreactive for many neurochemicals including amines and peptides. Functional studies are required to determine the individual roles played by these different neurotransmitters.

Ventrolateral medulla mechanisms involved in cardiorespiratory responses to central chemoreceptor activation in rats

A rise in arterial Pco(2) stimulates breathing and sympathetic activity to the heart and blood vessels. In the present study, we investigated the involvement of the retrotrapezoid nucleus (RTN) and glutamatergic mechanisms in the Bötzinger/C1 region (Bötz/C1) in these responses. Splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane-anesthetized, sino-aortic-denervated, vagotomized, and artificially ventilated rats subjected to hypercapnia (end-expiratory CO(2) from 5% to 10%). Phrenic activity was absent at end-expiratory CO(2) of 4%, and strongly increased when end-expiratory CO(2) reached 10%. Hypercapnia also increased sSND by 103 ± 7%. Bilateral injections of the GABA-A agonist muscimol (2 mM) into the RTN eliminated the PND and blunted the sSND activation (Δ = +56 ± 8%) elicited by hypercapnia. Injections of NMDA receptor antagonist AP-5 (100 mM), non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 100 mM) or metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100 mM) bilaterally into the Bötz/C1 reduced PND (Δ = +43 ± 7%, +52 ± 6% or +56 ± 11%, respectively). MCPG also reduced sSND (Δ = +41 ± 7%), whereas AP-5 and DNQX had no effect. In conclusion, the increase in sSND caused by hypercapnia depends on increased activity of the RTN and on metabotropic receptors in the Bötz/C1, whereas PND depends on increased RTN activity and both ionotropic and metabotropic receptors in the Bötz/C1.

Essential Role of Phox2b-Expressing Ventrolateral Brainstem Neurons in the Chemosensory Control of Inspiration and Expiration

Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN), located in the ventrolateral brainstem, are sensitive to changes in PCO(2)/pH, have excitatory projections to the central respiratory rhythm/pattern generator, and their activation enhances central respiratory drive. Using in vivo (conscious and anesthetized rats) and in situ (arterially perfused rat brainstem-spinal cord preparations) models, we evaluated the functional significance of this neuronal population for both resting respiratory activity and the CO(2)-evoked respiratory responses by reversibly inhibiting these neurons using the insect peptide allatostatin following transduction with a lentiviral construct to express the G-protein-coupled Drosophila allatostatin receptor. Selective inhibition of the Phox2b-expressing neurons in the ventrolateral brainstem, including the RTN, using allatostatin was without effect on resting respiratory activity in conscious rats, but decreased the amplitude of the phrenic nerve discharge in anesthetized rats and the in situ rat preparations. Postinspiratory activity was also reduced in situ. In the absence or presence of the peripheral chemoreceptor input, inhibiting the Phox2b-expressing neurons during hypercapnia abolished the CO(2)-evoked abdominal expiratory activity in anesthetized rats and in situ preparations. Inspiratory responses evoked by rising levels of CO(2) in the breathing air were also reduced in anesthetized rats with denervated carotid bodies and conscious rats with peripheral chemoreceptors intact (by 28% and 60%, respectively). These data indicate a crucial dependence of central expiratory drive upon Phox2b-expressing neurons of the ventrolateral brainstem and support the hypothesis that these neurons contribute in a significant manner to CO(2)-evoked increases of inspiratory activity.

Orexin‐A and respiration in a rat model of smoke‐induced chronic obstructive pulmonary disease

1. Orexins are neuropeptides synthesized in the hypothalamus that regulate many physiological functions, including energy homeostasis, stress responses, sleep/wake states etc. It is now emerging that orexins may also regulate breathing, but little is known as to how they do this, particularly in chronic obstructive pulmonary disease (COPD). In the present study, we used a rat model of cigarette smoke-induced COPD to investigate orexin-A expression in the hypothalamus and medulla and its effect on respiration. 2. Sprague-Dawley rats were exposed to cigarette smoke (1 h twice daily) for 12 weeks. Lung function and pathological changes associated with inflammation and emphysema were determined to confirm the validity of the COPD model. 3. Hypothalamic and medullary orexin-A levels, as determined by radioimmunoassay, were higher in smoke-exposed than control rats. Furthermore, the expression of prepro-orexin (PPO) mRNA in the hypothalamus and orexin OX(1) receptor mRNA in the medulla, as determined by real-time quantitative polymerase chain reaction, was higher in smoke-exposed than control rats. 4. The number of orexin-A-positive neurons in the hypothalamus and OX(1) and OX(2) receptor-positive neurons in the ventrolateral medulla was higher in smoke-exposed than control rats. 5. Microinjection of orexin-A (1 μmol/L, 0.1 μL) into the pre-Bötzinger complex enhanced phrenic nerve discharge to a greater extent in smoke-exposed compared with control rats (61% vs 36%, respectively). 6. The findings of the present study demonstrate that the increased respiratory activity in smoke-exposed rats is due to an increase in orexin-A as well as upregulation of orexin receptors in the ventrolateral medulla.

Effects of anesthetic state on properties of fast inspiratory rhythms in bilateral phrenic discharges of currarized adult rats

In both cat and rat, fast rhythms during inspiration are seen in bilateral phrenic nerve discharges. These oscillations have been classified into two types: (a) high‐frequency (HFO), present in both autospectra and in cross‐spectra (coherences) of a signal pair; (b) medium frequency (MFO), present only in autospectra of a signal pair. To analyze these effects in rats, we recorded from bilateral phrenic discharges in two types of curarized rat preparation (male Sprague‐Dawley, 280–350 gm): 1) Urethane‐anesthetized (1.8 gm/kg). In 18 rats, the opposite phrenic nerves had similar autospectral dominant peaks (distribution: 55 mean + 13 SD Hz); but there was no significant coherence between opposite phrenic discharges at this dominant frequency. 2) Supracollicularly decerebrated. In 3 rats, phrenic discharges exhibited fast rhythms (HFO, high‐frequency oscillations, range 132 –170 Hz) that were seen in the autospectra of activities on opposite sides with very similar peak frequency and which also had a significant coherence peak at the dominant frequency. The lack of significant bilateral coherence peaks under anesthesia indicates that there is a tendency to reduce the interactions between inspiratory neurons that are involved in HFO generation and suggests that such interactions are weaker between neurons on opposite sides than between neurons on the same side.(Supported by N.I.H. Grant NS‐043940.)

Activity of the respiratory muscles during natural defecation: a study on experimental animals

Activity of the respiratory muscles during natural defecation was studied in two anesthetized and two decerebrate dogs. In anesthetized dogs, excitation of the abdominal muscles and an increase in gastric pressure were observed during defecation. However, pleural pressure was little influenced by such increase in abdominal pressure, maintaining the same rhythmic changes as observed during spontaneous respiration. The rhythmic changes in pleural pressure were associated with rhythmic activity of the diaphragm. When gastric pressure increased during defecation, the diaphragmatic activity also increased during both the inspiratory and expiratory phases. In a decerebrate dog, airflow and airway pressure changed similarly to during defecation. The diaphragm was continuously active, with superimposed rhythmic augmentation. In a paralyzed and artificially ventilated dog with open-chest, the phrenic nerve similarly developed discharges. We conclude that the non-respiratory activity and rhythmic augmentation of phrenic nerve discharge during defecation is pre-programmed in the command for defecation. The activity of phrenic motoneurons may be further modulated by changes in thoracic and abdominal pressure. These mechanisms may act together to coordinate respiration and defecation.

Somatostatin selectively ablates post-inspiratory activity after injection into the Bötzinger complex

Somatostatin (SST) neurons in the ventral respiratory column (VRC) are essential for the generation of normal breathing. Little is known about the neuromodulatory role of SST on ventral respiratory neurons other than that local administration induces apnoea. Here, we describe the cardiorespiratory effects of microinjecting SST into the preBötzinger and Bötzinger complexes which together elaborate a normal inspiratory augmenting and expiratory respiratory pattern, and on spinally projecting respiratory subnuclei (rostral ventral respiratory group; rVRG). Microinjections (20–50 nl) of SST (0.15, 0.45, 1.5 mM) were made into respiratory subnuclei of urethane-anaesthetized, paralysed, vagotomized and artificially ventilated Sprague–Dawley rats (n=46). Unilateral microinjection of SST into the Bötzinger complex converted the augmenting activity of phrenic nerve discharge into a square-wave apneustic pattern associated with a lengthening of inspiratory period and shortening of expiratory time. Following bilateral microinjection the apneusis became pronounced and was associated with a dramatic variability in inspiratory duration. Microinjection of SST into the Bötzinger complex also abolished the post-inspiratory (post-I) motor activity normally observed in vagal and sympathetic nerves. In the preBötzinger complex SST caused bradypnoea and with increasing dose, apnoea. In the rVRG SST reduced phrenic nerve amplitude, eventually causing apnoea. In conclusion, SST powerfully inhibits respiratory neurons throughout the VRC. Of particular interest is the finding that chemical inhibition of the Bötzinger complex with SST ablates the post-I activity that is normally seen in respiratory activity and leads to apneusis. This loss of post-I activity is a unique feature of inhibition with SST and is not seen following inhibition with other agents such as galanin, GABA and endomorphin. The effect seen on post-I activity is similar to the effect of inhibiting the Kölliker–Fuse nucleus in the pons. The mechanism by which SST exerts this effect on Bötzinger neurons remains to be determined.

Donepezil reverses buprenorphine-induced central respiratory depression in anesthetized rabbits.

Buprenorphine is a mixed opioid receptor agonist-antagonist used in acute and chronic pain management. Although this agent's analgesic effect increases in a dose-dependent manner, buprenorphine-induced respiratory depression shows a marked ceiling effect at higher doses, which is considered to be an indicator of safety. Nevertheless, cases of overdose mortality or severe respiratory depression associated with buprenorphine use have been reported. Naloxone can reverse buprenorphine-induced respiratory depression, but is slow-acting and unstable, meaning that new drug candidates able to specifically antagonize buprenorphine-induced respiratory depression are needed in order to enable maximal analgesic effect without respiratory depression. Acetylcholine is an excitatory neurotransmitter in central respiratory control. We previously showed that a long-acting acetylcholinesterase inhibitor, donepezil, antagonizes morphine-induced respiratory depression. We have now investigated how donepezil affects buprenorphine-induced respiratory depression in anesthetized, paralyzed, and artificially ventilated rabbits. We measured phrenic nerve discharge as an Index of respiratory rate and amplitude, and compared discharges following the injection of buprenorphine with discharges following the injection of donepezil. Buprenorphine-induced suppression of the respiratory rate and respiratory amplitude was antagonized by donepezil (78.4 +/- 4.8 %, 92.3% +/- 22.8 % of control, respectively). These findings indicate that systemically administered donepezil restores buprenorphine-induced respiratory depression in anesthetized rabbits.

Defective Respiratory Rhythmogenesis and Loss of Central Chemosensitivity in Phox2b Mutants Targeting Retrotrapezoid Nucleus Neurons

The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as Phox2b-, Vglut2-, neurokinin1 receptor-, and Atoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional Phox2b mutations, which target different subsets of Phox2b-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously described Phox2b(27Ala) mutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the Phox2b-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.

Activation of the retrotrapezoid nucleus by posterior hypothalamic stimulation.

The retrotrapezoid nucleus (RTN) contains chemically defined neurons (ccRTN neurons) that provide a pH-regulated excitatory drive to the central respiratory pattern generator. Here we test whether ccRTN neurons respond to stimulation of the perifornical hypothalamus (PeF), a region that regulates breathing during sleep, stress and exercise. PeF stimulation with gabazine increased blood pressure, phrenic nerve discharge (PND) and the firing rate of ccRTN neurons in isoflurane-anaesthetized rats. Gabazine produced an approximately parallel upward shift of the steady-state relationship between ccRTN neuron firing rate and end-tidal CO(2), and a similar shift of the relationship between PND and end-tidal CO(2). The central respiratory modulation of ccRTN neurons persisted after gabazine without a change in pattern. Morphine administration typically abolished PND and reduced the discharge rate of most ccRTN neurons (by 25% on average). After morphine administration, PeF stimulation activated the ccRTN neurons normally but PND activation and the central respiratory modulation of the ccRTN neurons were severely attenuated. In the same rat preparation, most (58%) ccRTN neurons expressed c-Fos after exposure to hypercapnic hyperoxia (6-7% end-tidal CO(2); 3.5 h; no hypothalamic stimulation) and 62% expressed c-Fos under hypocapnia (approximately 3% end-tidal CO(2)) after PeF stimulation. Under baseline conditions (approximately 3% end-tidal CO(2), hyperoxia, no PeF stimulation) few (11%) ccRTN neurons expressed c-Fos. In summary, most ccRTN neurons are excited by posterior hypothalamic stimulation while retaining their normal response to CNS acidification. ccRTN neurons probably contribute both to the chemical drive of breathing and to the feed-forward control of breathing associated with emotions and or locomotion.

P7.7 Exposure to chronic intermittent hypoxia promotes exaggerated sympathetic reflexes but blunted adrenergic vascular reactivity in Sprague–Dawley rats

Chronic intermittent hypoxia (CIH) yields persistent elevations in sympathetic nerve activity (SNA) and arterial pressure (AP). In addition, chemoreflex activation by acute hypoxia or cyanide evokes a larger rise in SNA in rats after CIH due to enhanced sensory and central mechanisms. We examined the hypothesis that other sympatho-excitatory reflexes are also augmented after CIH. Male Sprague–Dawley rats (4 wks old) were exposed to CIH (6% O2 for 40 s, every 9 min, 8 h/day, 2 wks). Isoflurane-anesthetized, ventilated, paralyzed rats were prepared for recording AP, splanchnic SNA, phrenic nerve discharge and then switched urethane anesthesia (1.5 g/kg, iv).

Optical stimulation of Channel Rhodopsin2(ChR2)‐transfected retrotrapezoid nucleus (RTN) neurons activates breathing

The role of the RTN neurons in breathing is not definitely established. RTN neurons are Phox2b‐expressing, glutamatergic, non‐TH, acid‐sensitive and innervate regions involved in respiratory rhythm generation. Using a lentivirus with the ChR2‐mCherry fusion protein under the PRSX8 promoter (with 8 copies of the Phox2 transcription factor response site), we drove expression of ChR2‐mCherry to Phox2b neurons with &gt;92% selectivity. Appropriately targeted microinjections of the virus restricted transgene expression to the RTN and nearby C1 adrenergic neurons (2.7/1 non TH/TH in control rats and 5/1 in rats with selective lesions of C1 cells). Activation of the ChR2‐transfected neurons with 470 nm; 20 Hz, 10 ms pulses, 30s trains of pulsed laser light (LL) produced large increases in phrenic nerve discharge (PND), a 5mmHg increase in BP and a 10% rise in sympathetic nerve discharge (SND). PND activation decayed slowly after turning off LL (t1/2=11.8s) whereas SND activation returned to control within seconds. LL increased PND equally in control and C1‐lesion rats with no BP change in the latter. LL had no effect on PND or BP in animals with RTN transfection using control lentivirus of comparable titer. In summary, activation of RTN stimulates respiratory activity with a slow time course consistent with a chemoreceptor role, whereas C1 neuron stimulation primarily affects BP.

Blockade of neuronal gap junctions modifies slow and fast oscillatory rhythms in phrenic nerve discharge in arterially‐perfused adult rat

We have previously demonstrated that pharmacological blockade of gap junctions alters respiratory rhythm, phrenic burst pattern, and inspiratory‐phase synchronization. The pharmacological agents used to block functional gap junction coupling, however, were not specific to gap junctions composed of a specific connexin (Cx) isoform, and therefore may have exerted their effects on both neuronal and astrocytic gap junctions. To further delineate the role of neuronal gap junctions in central respiratory control, we examined the effects of mefloquine (MEQ; 0.5‐1.0 μM), which blocks neuron‐specific Cx36‐containing gap junctions, on phrenic nerve discharge in 5 arterially‐perfused adult rat preparations. We found that perfusion with MEQ increased phrenic burst frequency by 35±4% (P&lt;0.001) due to decreases in both TI (P=0.001) and TE (P&lt;0.001). No significant changes in burst amplitude were observed; however, time‐to‐peak activity (Tpeak/TI) was shifted from 85±2 to 72±5% (P&lt;0.01). Perfusion with MEQ also produced a small increase in HFO spectral power (P=0.07), shifted HFO onset to an earlier time point in the phrenic burst (from 62±3 to 36±7% TI; P&lt;0.01), and prolonged the duration of significant HFO activity. No changes in MFO activity were seen. These data suggest that neuronal gap junctions participate in both the slow and fast oscillatory rhythms underlying phrenic nerve discharge. Supported by NS045321

Role of GABAergic receptors within the nucleus of the solitary tract in spontaneously hypertensive rats

Inhibitory neurotransmission plays a crucial role in the processing of sensory afferent signals in the nucleus of the solitary tract (NTS). Gamma‐aminobutyric acid (GABA) neurons are scattered throughout NTS. Previous studies demonstrated that alterations in GABA mediated neural transmission in the NTS contribute to the attenuation of the baroreceptor reflex observed in SHR. Here, we test the hypothesis that enhanced stimulation of GABA‐A and B receptors in the NTS in SHR are responsible for the attenuation of the sympathetic baroreflex, but not the chemoreflex. Under urethane/chloralose anesthesia, baseline arterial pressure (AP) in SHR was higher than the Wistar Kyoto (WKY) rats (SHR: 146 ± 5, vs. WKY: 112 ± 6 mmHg). The effects of baroreflex and chemoreflex on AP, splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were test before and after bilateral injection of the GABA‐A antagonist (bicuculline) or the GABA‐B antagonist (CGP 35348) within the NTS. The baroreflex range and gain were increased after bicuculline and CGP 35348 injection into the NTS, but had no effect on sympathoexcitatory and increase PND produced by chemoreflex activation. Bilateral injection of bicuculline and GCP 35348 within the NTS in SHR produced a pronounced decrease in AP compared with WKY (Δ = ‐15 ± 4 and ‐33 ± 6 mmHg, respectively vs. WKY: Δ = ‐5 ± 4 mmHg), but did not change PND. These results suggest that alterations in GABA‐mediated neural transmission in the NTS contribute to the attenuation of the baroreceptor reflex observed in SHR.

Mechanisms of the ventrolateral medulla on the control of sympathetic activity by central chemoreflex in rats

In the absence of inputs from carotid bodies and cardiopulmonary receptors hypercapnia markedly increases sympathetic nerve discharge (SND) to the heart and blood vessels. In the present study, splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane anaesthetized sino‐aortic denervated and vagotomized rats. Hypercapnia (end‐expiratory CO2 from 5% to 10%) increased sSND (δ= 103 ± 7%). Bilateral injection of muscimol (GABA‐A agonist ‐ 2 mM in 30 nl) into the retrotrapezoid nucleus (RTN) eliminated PND and reduced the effect of CO2 on sSND (δ= 56 ± 8%). Injection of AP‐5 (NMDA glutamate receptor antagonist ‐ 100 mM in 50 nl) or DNQX (non NMDA glutamate receptor antagonist ‐ 100 mM in 50 nl) bilaterally into rostroventrolateral medulla (RVLM) reduced PND, but did not change the effect of CO2 on sSND. Bilateral injection of MCPG (metabotropic glutamate receptor antagonist ‐ 100 mM in 50 nL) into the RVLM reduced PND and the effect of CO2 on sSND (δ= 41 ± 7%). In conclusion, RVLM metabotropic receptors and RTN are involved in the increase in sSND produced by CO2.