Amplitude Of Phrenic Nerve Activity Research Articles

A Novel Rat Model of Sleep Apnea Reveals Increased Sensitivity to Opioid‐Induced Respiratory Depression and an Unexpected Role for Endogenous Opioids

Opioid‐induced respiratory depression (OIRD) underlies opioid lethality. Sleep disorders, including sleep apnea (SA), increase OIRD susceptibility, but mechanisms remain unknown. Here, we mimicked blood gas fluctuations of SA by exposing conscious rats to chronic intermittent hypoxia with hypercapnia (CIHHC), which consisted of 10 cycles/h of hypoxia (FiO2 reduced from 21% to 8%) with hypercapnia (FiCO2 increased from ~0% to 8%) for 8 h/day for 1 week. OIRD was first assessed in anesthetized (urethane/chloralose), vagotomized, paralyzed, and artificially ventilated rats and was quantified as reduced burst frequency and/or amplitude of phrenic nerve activity (PNA) to graded systemic doses of fentanyl (0, 2, 20, 50 mg/kg, iv; n=2–6/group), the most lethal clinically‐relevant opioid. Fentanyl dose‐dependently reduced PNA burst frequency, amplitude, and width, and increased the duration of PNA quiescence in control and CIHHC exposed rats. CIHHC caused a significant left‐shift of the fentanyl dose‐PNA burst frequency inhibition curve, suggesting increased OIRD potency of fentanyl in our model of SA. To compare endogenous opioid suppression of respiratory drive, the opioid receptor antagonist naloxone (1 mg/kg, iv) was administered. Naloxone increased PNA burst amplitude (40.4 ± 10.6%, n=3) in CIHHC rats compared to controls (3.8 ± 2.0%, n=3) (P<0.01). Additionally, naloxone increased PNA burst amplitude to a greater extent in CIHHC rats (54.1 ± 11.2%, n=2) than controls (9.1 ± 6.7%, n=3; P<0.01) following a 20 mg/kg dose of iv fentanyl – a dose of fentanyl that alone reduced PNA burst frequency (CIHHC: −97.4 ± 2.9%, n=5; Control: −79.2 ± 22.4%, n=3; P<0.01) for at least 5 min in both groups. These data reveal an underlying endogenous opioid inhibitory tonus in our rat model of SA. To exclude the possibility that increased sensitivity to OIRD in our SA model was related to differential anesthesia‐induced respiratory depression relative to controls, unrestrained conscious rats were instrumented with indwelling venous catheters and radio‐frequency telemetry transmitters to measure subpleural pressure as an index of ventilatory movements. In normoxic controls, vehicle did not change respiratory frequency (−1.7 ± 0.9%, n=2), and a 20 μg/kg dose of fentanyl induced a modest bradypnea (−19.3 ± 12.7%, n=3) not accompanied by apnea/hypopnea. In CIHHC rats, vehicle similarly did not change respiratory frequency (−0.9 ± 0.9%, n=2), but fentanyl caused significant bradypnea (−56.2 ± 13.3%, n=3; P<0.05) as well as apnea/hypopnea. These results are consistent with those in anesthetized rats. Collectively, the present findings indicate that CIHHC increases endogenous opioid inhibitory tonus and respiratory depressant effects of a clinically‐relevant opioid, fentanyl. Our CIHHC model mimics clinical findings of increased sensitivity to OIRD amongst SA patients, and provides a model system to investigate mechanisms of opioid respiratory neuromodulation to improve analgesic safety in SA patients.Support or Funding InformationSupport: NIH T32‐HL007446 (ADB), HL088052 (GMT)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Testing the hypothesis of neurodegeneracy in respiratory network function with a priori transected arterially perfused brain stem preparation of rat.

Degeneracy of respiratory network function would imply that anatomically discrete aspects of the brain stem are capable of producing respiratory rhythm. To test this theory we a priori transected brain stem preparations before reperfusion and reoxygenation at 4 rostrocaudal levels: 1.5 mm caudal to obex (n = 5), at obex (n = 5), and 1.5 (n = 7) and 3 mm (n = 6) rostral to obex. The respiratory activity of these preparations was assessed via recordings of phrenic and vagal nerves and lumbar spinal expiratory motor output. Preparations with a priori transection at level of the caudal brain stem did not produce stable rhythmic respiratory bursting, even when the arterial chemoreceptors were stimulated with sodium cyanide (NaCN). Reperfusion of brain stems that preserved the pre-Bötzinger complex (pre-BötC) showed spontaneous and sustained rhythmic respiratory bursting at low phrenic nerve activity (PNA) amplitude that occurred simultaneously in all respiratory motor outputs. We refer to this rhythm as the pre-BötC burstlet-type rhythm. Conserving circuitry up to the pontomedullary junction consistently produced robust high-amplitude PNA at lower burst rates, whereas sequential motor patterning across the respiratory motor outputs remained absent. Some of the rostrally transected preparations expressed both burstlet-type and regular PNA amplitude rhythms. Further analysis showed that the burstlet-type rhythm and high-amplitude PNA had 1:2 quantal relation, with burstlets appearing to trigger high-amplitude bursts. We conclude that no degenerate rhythmogenic circuits are located in the caudal medulla oblongata and confirm the pre-BötC as the primary rhythmogenic kernel. The absence of sequential motor patterning in a priori transected preparations suggests that pontine circuits govern respiratory pattern formation.

Acute intermittent optogenetic stimulation of nucleus tractus solitarius neurons induces sympathetic long-term facilitation.

Acute intermittent hypoxia (AIH) induces sympathetic and phrenic long-term facilitation (LTF), defined as a sustained increase in nerve discharge. We investigated the effects of AIH and acute intermittent optogenetic (AIO) stimulation of neurons labeled with AAV-CaMKIIa, hChR2(H134R), and mCherry in the nucleus of the solitary tract (NTS) of anesthetized, vagotomized, and mechanically ventilated rats. We measured renal sympathetic nerve activity (RSNA), phrenic nerve activity (PNA), power spectral density, and coherence, and we made cross-correlation measurements to determine how AIO stimulation and AIH affected synchronization between PNA and RSNA. Sixty minutes after AIH produced by ventilation with 10% oxygen in balanced nitrogen, RSNA and PNA amplitude increased by 80% and by 130%, respectively (P < 0.01). Sixty minutes after AIO stimulation, RSNA and PNA amplitude increased by 60% and 100%, respectively, (P < 0.01). These results suggest that acute intermittent stimulation of NTS neurons can induce renal sympathetic and phrenic LTF in the absence of hypoxia or chemoreceptor afferent activation. We also found that while acute intermittent optogenetic and hypoxic stimulations increased respiration-related RSNA modulation (P < 0.01), they did not increase synchronization between central respiratory drive and RSNA. We conclude that mechanisms that induce LTF originate within the caudal NTS and extend to other interconnecting neuronal elements of the central nervous cardiorespiratory network.

Respiratory and sympathetic chemoreflex regulation by Kölliker-Fuse neurons in rats

Chemoreceptor activation increases phrenic nerve activity (PNA) and sympathetic nerve activity (SNA). The dorsolateral pontine neurons, including the parabrachial nucleus and the Kölliker-Fuse (KF) region project to several brainstem areas involved in autonomic and respiratory regulation. Here the objective was to further test the hypothesis that the KF region could contribute to central and peripheral sympathetic chemoreflex activation. In urethane-anesthetized sino-aortic denervated or intact and vagotomized male Wistar rats (N = 7-8/group), hypercapnia (end-expiratory CO2 from 5 to 10 %) or KCN increased mean arterial pressure (MAP), splanchnic SNA, and PNA frequency and amplitude. Bilateral injection of muscimol (GABA-A agonist; 2 mM-50 nl) into the KF region increased resting PNA amplitude and reduced resting PNA frequency, without significant changes in resting MAP and SNA. Bilateral blockade of the KF region reduced the rise in MAP, sSNA, and PNA frequency and amplitude produced by hypercapnia or hypoxia. Our data suggest that the KF neurons could integrate and modulate breathing and sympathetic outflow during chemoreceptor activation.

Acute intermittent hypoxia induces sympathetic long‐term facilitation but does not alter synchronization between phrenic and sympathetic nerves (1130.15)

Acute intermittent hypoxia (AIH) can induce phrenic and sympathetic long‐term facilitation (LTF). Respiration modulates the sympathetic nervous system, leading to an idea that respiratory‐sympathetic coupling plays a role in the generation of sympathetic LTF. To determine if augmentation of synchronization between phrenic and sympathetic nerve activities is necessary to generate sympathetic LTF, we investigated the effect of AIH on phrenic nerve activity (PNA) and renal sympathetic nerve activity (RSNA) using spectral analysis in anesthetized, vagotomized and artificially ventilated Sprague‐Dawley rats (n=6). AIH was performed by ventilation with 10% oxygen for 1min every 6min for a total of 10 hypoxic exposures within an hour. PNA amplitude and RSNA increased by 110% and 100% respectively measured 60 min after AIH (P&lt;0.05). We estimated spectral power of PNA and RSNA and coherence between PNA and RSNA at central respiratory drive frequency, defined as frequency of the main peak in PNA. Spectral power of PNA and RSNA increased by 640% and 270% respectively measured 60 min after AIH (P&lt;0.05). However, coherence between PNA and RSNA did not change from baseline (0.62 +/‐ 0.34) to 60 min after AIH (0.71 +/‐ 0.25). These results suggest that AIH can induce sympathetic LTF in the absence of augmented synchronization between central respiratory drive and RSNA.Grant Funding Source: HL088052

Sevoflurane and isoflurane monoanesthesia abolished the phrenic long-term facilitation in rats

Phrenic long-term facilitation (pLTF) is a form of respiratory plasticity, manifested by prolonged increase in peak amplitude of phrenic nerve activity (PNA) after episodes of acute intermittent hypoxia (AIH). The aim was to investigate the effects of sevoflurane and isoflurane monoanesthesia at equipotent anesthetic doses on the expression of amplitude pLTF. Experiments were performed on Sprague-Dawley anesthetized, vagotomized, and mechanically ventilated rats. Two experimental groups, sevoflurane and isoflurane, and a control group (urethane-anesthetized) were formed. Peak amplitude of phrenic nerve activity, respiratory frequency and breathing rhythm parameters (Ti, inspiratory duration; Te, expiratory duration; and Ttot, total respiratory time duration) were analyzed during hypoxic episodes, and at 15, 30, and 60min after the last hypoxic episode. In the control group average PNA increased by 173.03±70.16% (p<0.001), at 60min after the last hypoxic episode compared to baseline values. Therefore amplitude pLTF was induced. In the sevoflurane and isoflurane groups PNA failed to increase, and in fact decreased by 15.79±15.18% and 31.00±11.00%, respectively (p>0.05). Amplitude pLTF was abolished during sevoflurane and isoflurane monoanesthesia.

Purinergic signaling in the retrotrapezoid nucleus (RTN) contributes to central and peripheral chemoreflexes by divergent mechansims

Evidence suggests that purinergic signaling in the retrotrapezoid nucleus (RTN) can influence both respiratory and sympathetic activity. Here we evaluate the extent to which purinergic signaling contributes to central and peripheral chemoreception integration at the level of the RTN in vivo. Mean arterial pressure (MAP) and phrenic nerve activity (PNA) were recorded in urethane‐anaesthetized, vagotomized and artificial ventilated male Wistar rats (n = 6–7/group). Bilateral RTN injections of a non‐specific P2‐receptor blocker (PPADS) decreased pressor (8 ± 2 mmHg, vs. saline: 22 ± 4 mmHg) and respiratory (PNA amplitude = 62 ± 3%, vs. saline: 101 ± 5% and PNA frequency = 77 ± 3%, vs. saline: 101 ± 5%) responses to 10% CO2. Conversely, bilateral RTN injections of MRS2179 (P2Y1 receptor antagonist) attenuated cardiorespiratory responses to peripheral chemoreceptor activation, but did not affect CO2‐responsivness. Further, 63% of BDA‐labeled NTS terminals in the RTN are immunoreactive for vesicular glutamate transporter‐2 (VGLUT2) and vesicular nucleotide transporter (VNUT) but only 5% contained glutamic acid decarboxylase‐67 (GAD67). These data indicate that purinergic signaling in the RTN contributes to central and peripheral chemoreception by different, but potentially interactive, mechanisms

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 β-hydroxylase-saporin (anti-DβH-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-DβH-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.

Attenuated phrenic long-term facilitation in orexin neuron-ablated mice

We examined phrenic long-term facilitation (LTF) in urethane-anesthetized, vagotomized, paralyzed, and artificially ventilated orexin neuron-ablated mice and their wild-type littermates. Effect of isocapnic single hypoxic episode (SHE, for 45 s) and intermittent hypoxia (IH, 5 times of SHE separated by 5 min) on phrenic nerve activity (PNA) was measured for 1-2h. In wild-type mice, amplitude of PNA gradually increased after cessation of IH and reached 55+/-15% above the baseline (n=7, p<0.05) whereas the burst rate of PNA did not change. Qualitatively similar but significantly attenuated response (16+/-8%) was observed in orexin neuron-ablated mice. SHE did not affect amplitude nor frequency in both animals. We conclude that orexin contributes to eliciting phrenic LTF at least in part in mice. This study also showed, for the first time, phrenic LTF following IH in WT mice. Characteristics of phrenic and ventilatory LTF in mice were similar to those in rats.

Defining ventral medullary respiratory compartments with a glutamate receptor agonist in the rat.

The regional organization of the ventral respiratory group (VRG) was examined with respect to generation of respiratory rhythm (breathing frequency) versus control of the respiratory motor pattern on individual nerves. In urethane-anaesthetized, neuromuscularly blocked and vagotomized Sprague-Dawley rats, arterial blood pressure (ABP) and respiratory motor outputs (phrenic, pharyngeal branch of the vagus, or superior laryngeal nerves) were recorded. The VRG organization was mapped systematically using injections of the excitatory amino acid DL-homocysteic acid (DLH; 5-20 mM, 2-6 nl) from single- or double-barrel pipettes at 100-200 microm intervals between the facial nucleus and the calamus scriptorius. Recording of respiratory neurons through the injection pipette ensured that the pipette was located within the VRG. At the end of each experiment, the injection pipette was used to make an electrical lesion, thereby marking the electrode position for subsequent histological reconstruction of injection sites. Four rostrocaudal regions were identified: (1) a rostral bradypnoea area, at the level of the Bötzinger complex, in which respiratory rhythm slowed and ABP increased, (2) a tachypnoea/dysrhythmia area, at the level of the preBötzinger complex, in which breathing rate either increased or became irregular, with little or no change in ABP, (3) a caudal bradypnoea area at the level of the anterior part of the rostral VRG in which ABP decreased and (4) a caudal 'no effect' region in the posterior part of the rostral VRG. The peak amplitude of phrenic nerve activity decreased with injections into all three rostral regions. Changes in respiratory rhythm were associated with opposite changes in inspiratory (TI) and expiratory (TE) durations after injections into either the Bötzinger complex or anterior rostral VRG, while both TI and TE decreased after injections into the preBötzinger complex. Effects on selected cranial nerves were similar to those on the phrenic nerve except that tonic activity was elicited on the superior larygneal nerve ipsilateral to injections in the Bötzinger complex and on the pharyngeal branch of the vagus ipsilateral to injections in the preBötzinger complex. These data reinforce the subdivision of the VRG into functionally distinct compartments and suggest that a further subdivision of the rostral VRG is warranted. They also suggest that region-specific influences, especially on the pattern of cranial motor discharge, can be used to assist the identification of recording sites within the VRG. However, the postulated clear functional separation of rhythm- versus pattern-generating regions was not supported.

Respiratory-related bronchial rhythmic constrictions in the dog with extracorporeal circulation.

Respiratory-related bronchial rhythmic contraction was quantitatively analyzed in eight paralyzed dogs. The caliber of the fifth-generation bronchus was continuously measured as the pressure (Pbr) of a balloon-tipped catheter under the condition of complete immobilization due to extracorporeal oxygenation. Pbr changed rhythmically in synchrony with phrenic nerve activity (PNA) bursts. Rhythmic bronchial constriction started at 1.4 +/- 0.49 (SD) s after onset of PNA, reached a maximum level at 2.8 +/- 1.6 s after termination of PNA, and then decreased exponentially with a time constant of 6.9 +/- 2.5 s. When the respiratory rate of dogs increased at hypercapnia, the various bronchial contractions fused to behave like a tonic contraction. The rhythmic component of this contraction was separated and quantitatively analyzed. Each rhythmic Pbr amplitude linearly increased with increases in PNA amplitude, whereas the end-expiratory Pbr level was not significantly changed. Bilateral efferent nerve transection did not decrease the end-expiratory Pbr level. In response to electric stimulation of efferent nerve fibers, the bronchus did not maintain tonic contraction. We concluded that vagally mediated commands contract bronchial smooth muscle only intermittently and that most of bronchial resting tension may thus be attributed to the summation of rhythmic contractions.

Selective lesioning of the cat pre-Bötzinger complex in vivo eliminates breathing but not gasping.

1. To examine the functional importance of the pre-Bötzinger complex for breathing we micro-injected, under in vivo conditions, the calcium channel blocker omega-conotoxin GVIA and the sodium channel blocker tetrodotoxin (TTX) into the ventrolateral medulla of adult cats, while monitoring respiratory rhythmic motor output in the phrenic nerve. 2. omega-Conotoxin GVIA caused a highly localized synaptic ablation by blocking presynaptic N-type calcium channels. When injecting 5-60 fmol omega-conotoxin GVIA unilaterally, the amplitude of phrenic nerve activity decreased bilaterally and sometimes disappeared, indicating central apnoea. These effects were reversible and could only be induced in a very localized area of the pre-Botzinger complex. By injecting omega-conotoxin GVIA several times during an experiment and analysing the areas where injections affected respiratory activity, it was possible to map exactly the anatomical extent of the area critical for respiratory rhythm generation. 3. Following the precise localization of the pre-Bötzinger complex with omega-conotoxin GVIA, we injected TTX to induce an irreversible inactivation of this region. TTX injected unilaterally into the pre-Bötzinger complex irreversibly reduced the amplitude of phrenic nerve activity. Bilateral TTX injections eliminated respiratory rhythmic activity, causing a persistent central apnoea. 4. After bilateral lesioning of the pre-Bötzinger complex, it was still possible to induce gasping during hypoxia or asphyxia, indicating that respiration and gasping are generated by two different neuronal networks. 5. We propose that omega-conotoxin GVIA as employed in this study to investigate the functional role of the pre-Bötzinger complex can also be used as a general pharmacological approach to map other neuronal networks. We call this the 'omega-conotoxin GVIA tracing' method.

GABAA receptor mediated fast synaptic inhibition in the rabbit brain-stem respiratory system.

The involvement of GABA mediated neurotransmission in the central control of respiration was investigated by administration of the specific GABAA receptor agonist muscimol and the specific GABAA receptor antagonist biculline into the fourth cerebral ventricle of the rabbit. Cycle-triggered averaging of the phrenic nerve activity (PNA) was used to quantify drug-induced changes of the central respiratory pattern. Muscimol reduced the peak amplitude of PNA and increased the duration of the respiratory phases. High amounts of muscimol led to a long-lasting but reversible central apnea. Bicuculline very effectively blocked the effects of externally applied muscimol. Blockade of intrinsically active GABAergic neurotransmission by bicuculline resulted in a multitude of effects. Peak amplitude of PNA increased whereas the duration of both inspiration and expiration decreased. In this respect, effects of bicuculline and muscimol were complementary. Bicuculline reduced the slope of the inspiratory ramp, increased postinspiratory activity and induced an augmenting type of discharge activity in the last part of expiration resulting in a smooth transition between expiration and inspiration. In some cases the respiratory modulation was completely lost and PNA became perfectly tonic. This 'apneustic' type of respiratory pattern could be transformed into rhythmic breathing by increasing the respiratory drive. We conclude that neurotransmission via GABAA receptors is important for the maintenance of respiratory rhythm as well as the generation of normal respiratory pattern.

Glycine receptor-mediated fast synaptic inhibition in the brainstem respiratory system

The involvement of glycinergic neurotransmission in the central regulation of respiration was investigated by administration of specific receptor agonists and antagonists into the 4th cerebral ventricle of the rabbit. Central respiratory activity was assessed by cycle-triggered averaging of phrenic nerve activity (PNA). Glycine reduced the peak amplitude of PNA and increased the duration of the respiratory phases. Conversely, strychnine decreased the duration of both phases, being more effective in expiration. Strychnine converted inspiratory ramp activity to a decrescendo type of pattern, with the highest discharge activity present at the onset of the inspiratory phase. Strychnine induced tonic PNA lasting throughout expiration with a decrescendo type of activity. In many cases strychnine induce apneusis with tonic PNA being at a level of about 2/3 of its maximum value during control. We conclude that glycinergic neurotransmission is important for the maintenance of the respiratory rhythm as well as the generation of normal respiratory pattern. Glycine could be involved in phasic as well as tonic inhibitory neurotransmission within the central respiratory system.

Altered breathing pattern elicited by stimulation of abdominal visceral afferents.

The effect of stimulation of afferent mesenteric nerves on tidal volume (VT), phrenic nerve, and external intercostal muscle activities was studied in anesthetized spontaneously breathing cats. Both mechanical distension of the small intestine and electrical stimulation of the mesenteric nerves resulted in an initial inspiratory inhibition of VT followed by a gradual recovery above the prestimulus controls. Changes in VT were accompanied by a depression of phrenic nerve activity and an excitation of external intercostal muscle activity. During the recovery phase of VT, the amplitude of phrenic nerve activity returned only partially, whereas the activity of the external intercostal muscle was greater than the prestimulus controls. In a second group of experiments, brief tetanic stimulation at the beginning of inspiration led to a complete and maintained inhibition of phrenic nerve activity but with a simultaneous excitation of external intercostal muscle activity and without any change in VT; whereas expiratory stimulation caused a decrease in expiratory abdominal muscle activity, without changing the peak amplitude of phrenic nerve activity. The respiratory changes observed with distension of the small intestine were abolished after denervation of the mesenteric plexus. It is concluded that activation of the visceral afferents of the mesenteric region reflexly changes diaphragmatic breathing to intercostal breathing. It is assumed that such a type of breathing pattern may occur in pregnancy and in pathophysiological situations involving splanchnic viscera.

Differentiation of two respiratory areas in the cat medulla using kainic acid

Kainic acid (KA) was used to destroy neuronal perikarya in different areas of the brainstem. Single KA microinjections were performed in 30 anaesthetized, vagotomized, artificially ventilated cats. Consequences were studied on the phrenic nerve activity (PNA) and blood pressure. We observed changes of the PNA unrelated to blood pressure alteration. Destruction of the dorsal respiratory area (DRA) including the nucleus tractus solitarius at the obex level produced a 40% decrease of the PNA frequency. Destruction restricted to the lateral part of the ventral respiratory area (VRA1) including the ambiguus nucleus induced a 60% decrease of the integrated PNA amplitude followed by a 40% increase of PNA frequency. These latter effects were also observed after destruction inside the infra solitary reticular formation (ISRF). No effect was observed after destruction in other brain structures. We concluded that ISRF and VRA1 form a single ventral bulbar respiratory area. This area controls respiration in a way different from that of the dorsal respiratory area (DRA).