Medullary Respiratory Nuclei Research Articles

Opioid inhibition of projection‐specific, respiratory‐related pontomedullary neurons

Opioids depress breathing by inhibition of inter‐connected respiratory nuclei in the pons and medulla. We have previously shown that mu opioid receptor (MOR) agonists hyperpolarize a population of neurons in the dorsolateral pons due to activation of G protein‐mediated inwardly rectifying potassium conductance. However, we do not know the projection target of MOR‐expressing, dorsolateral pontine neurons, if they project at all. Here, we show that MOR‐expressing, dorsolateral pontine neurons project to core respiratory nuclei in the ventrolateral medulla, identifying projection‐related heterogeneity of dorsolateral pontine neurons vulnerable to opioid inhibition. First, retrograde DIO‐eGFP injections into the ventrolateral medulla of oprm1‐cre mice showed that dorsolateral pontine neurons project to the Bötzinger complex (BötC), pre‐Bötzinger complex (preBötC), and rostral ventral respiratory group (rVRG), and are distinct from calcitonin gene‐related peptide (CGRP)‐expressing parabrachial neurons. Second, we performed whole‐cell, voltage‐clamp recordings from dorsolateral pontine Kölliker‐Fuse neurons that were retrogradely labeled by fluorescent bead injections into the ventrolateral medulla of wild‐type mice. MOR‐mediated outward currents were identified in dorsolateral pontine neurons that project to the BötC, preBötC, and rVRG, confirming that these projection neurons are functionally inhibited by MORs and vulnerable to opioid inhibition. Together, these approaches demonstrate that MOR‐expressing, dorsolateral pontine neurons project to medullary respiratory nuclei and further unravel mechanisms of opioid‐induced respiratory depression.

Inspiratory and Phase‐Spanning Dorsolateral Pontine Neurons Are Silent During Fentanyl‐Induced Apnea

Overdoses caused by the opioid agonist fentanyl have increased exponentially in recent years. These overdose deaths are the result of inadequate ventilation. While much research has focused on opioid modulation of medullary respiratory nuclei, pontine mediated effects of opioids have received less attention. Given the interconnectivity of the pontine Kölliker-Fuse/Parabrachial complex (KF/PB) with rhythmogenic nuclei in the medulla, their involvement in controlling respiratory rate and pattern, and their sensitivity to opioids, we investigate the role they play in opioid mediated respiratory depression and hypothesize that dorsolateral pontine inspiratory, phase-spanning, and post-inspiratory neurons are silenced by opioids. To address this, we have done single unit recordings in the working heart brainstem in situ rat preparation. Dorsolateral pontine neural units (n=48) were recorded extracellularly and systemically exposed to fentanyl and then the opioid antagonist naloxone. A phase-fraction 2-D histogram (heatmap) was generated to compare all unit's action potential spiking over respiratory phases and during periods following fentanyl administration. Each neural unit was categorized at baseline into 4 main groups of discharge identity: Inspiratory, Expiratory, Phase-spanning, and Tonic. Systemic application of fentanyl (300nM) resulted in a consistent decline in fictive respiratory output, transitioned to fast shallowing breathing, and eventually resulted in cessation of respiratory output (apnea). Out of 48 extracellular single unit recordings in the dorsolateral pons, 25 (52%) were completely silent (firing frequency of 0 Hz) suggesting post-synaptic opioid receptor activation during fentanyl-induced apnea; 17/20 inspiratory, 6/12 phase spanning (4/9 IE & 2/3 EI), 1/13 expiratory (1/5 Post I Dec) and 1/3 tonic neurons. To note, many neurons that were not completely silenced by opioids did fire at a much lower firing frequency, suggesting pre-synaptic opioid receptors also contribute to opioid-induced loss of pontine neural activity. Our hypothesis was partially supported, as the majority of inspiratory neurons (17/20) and a subpopulation of phase-spanning neurons (6/12) were completely silenced by fentanyl. Surprisingly post-inspiratory neurons were mainly insensitive to opioids (4/5). These results suggest that loss of post-inspiratory activity with DAMGO injection into the KF may depend critically on post-synaptic inhibition of inspiratory and a subset of IE phase-spanning neurons, which in turn, could decrease local pre-synaptic excitation of post-inspiratory neurons. The majority of pontine expiratory neurons were not silenced by fentanyl (12/13). Due to the KF/PB role in expiratory duration, sustained activity of pontine expiratory neurons may facilitate sustained apnea during opioid overdose. These results warrant further investigation into the contribution of the dorsolateral pons in respiratory pattern formation and opioid-induced respiratory depression.

Oxidative stress in the medullary respiratory neurons contributes to respiratory dysfunction in the 6-OHDA model of Parkinson's disease.

Parkinson's disease (PD) is associated with respiratory dysfunction. In the 6-OHDA rat model of PD this is seen as a reduction in respiratory frequency and minute ventilation during normoxia and hypercapnia stimulus. Respiratory dysfunction is caused by neuronal death of medullary respiratory nuclei in the 6-OHDA model of PD. Oxidative stress can be considered a strong candidate for neurodegeneration via miR-34c downregulation and pro-apoptotic signalling in respiratory neurons, preceding the functional impairment observed in the 6-OHDA model of PD. Parkinson's disease (PD) is a neurodegenerative disease caused by dopaminergic neuron death in the substantia nigra (SN). New evidence has revealed that this neurodegeneration is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction and disruption of the blood-brain barrier (BBB) in the SN. In addition to classic symptoms, PD patients also exhibit respiratory failure. Here, we investigated whether oxidative stress was associated with neurodegeneration in a respiratory group (RG) of 6-OHDA-treated rats, which act as a model of PD. We analysed how oxidative stress affected apoptotic signalling in the RG 30days after 6-OHDA treatment, shortly before commencement of breathing impairment (40days). After 30days, a dihydroethidium assay showed increased oxidative stress in the RG, anti-apoptotic signalling, as shown by an increase in p-Akt and BcL-2 and a decrease in Bax in the caudal aspect of the nucleus of the solitary tract (cNTS), and a decrease in p-p38 and Bax levels in the retrotrapezoid nucleus (RTN); pro-apoptotic signalling was indicated by a decrease in p-Akt and BcL-2 and an increase in Bax in the rostral ventral respiratory group (rVRG) and pre-Botzinger complex (preBotC). miR-34c, a known oxidative stress protector, was downregulated in 6-OHDA animals in the RC. After 40days of 6-OHDA, the NTS, rVRG, preBotC and RTN exhibited reduced NeuN immunoreactivity, no BBB disruption and an increase in thiobarbituric acid reactivity. We conclude that in the 6-OHDA model of PD, oxidative stress contributes to neurodegeneration in medullary respiratory neurons.

Development of a Novel FIJI-Based Method to Investigate Neuronal Circuitry in Neonatal Mice.

The emergence of systems neuroscience tools requires parallel generation of objective analytical workflows for experimental neuropathology. We developed an objective analytical workflow that we used to determine how specific autonomic neural lineages change during postnatal development. While a wealth of knowledge exists regarding postnatal alterations in respiratory neural function, how these neural circuits change and develop in the weeks following birth remains less clear. In this study, we developed our workflow by combining genetic mouse modeling and quantitative immunofluorescent confocal microscopy and used this to examine the postnatal development of neural circuits derived from the transcription factors NKX2.2 and OLIG3 into three medullary respiratory nuclei. Our automated FIJI-based image analysis workflow rapidly and objectively quantified synaptic puncta in user-defined anatomic regions. Using our objective workflow, we found that the density and estimated total number of Nkx2.2-derived afferents into the pre-Bötzinger Complex significantly decreased with postnatal age during the first three weeks of postnatal life. These data indicate that Nkx2.2-derived structures differentially influence pre-Bötzinger Complex respiratory oscillations at different stages of postnatal development.

Expression of the transcription factor FOXP2 in brainstem respiratory circuits of adult rat is restricted to upper-airway pre-motor areas

Expression of the transcription factor FOXP2 is linked to brain circuits that control motor function and speech. Investigation of FOXP2 protein expression in respiratory areas of the ponto-medullary brainstem of adult rat revealed distinct rostro-caudal expression gradients. A high density of FOXP2 immunoreactive nuclei was observed within the rostral pontine Kölliker-Fuse nucleus, compared to low densities in caudal pontine and rostral medullary respiratory nuclei, including the: (i) noradrenergic A5 and parafacial respiratory groups; (ii) Bötzinger and pre-Bötzinger complex and; (iii) rostral ventral respiratory group. Moderate densities of FOXP2 immunoreactive nuclei were observed in the caudal ventral respiratory group and the nucleus retroambiguus, with significant density levels found in the caudal half of the dorsal respiratory group and the hypoglossal pre-motor area lateral around calamus scriptorius. FOXP2 immunoreactivity was absent in all cranial nerve motor nuclei. We conclude that FOXP2 expression in respiratory brainstem areas selectively delineates laryngeal and hypoglossal pre-motor neuron populations essential for the generation of sound and voice.

Desogestrel enhances ventilation in ondine patients: Animal data involving serotoninergic systems

Congenital central hypoventilation syndrome (CCHS) is a neurorespiratory disease characterized by life-threatening sleep-related hypoventilation involving an alteration of CO2/H+ chemosensitivity. Incidental findings have suggested that desogestrel may allow recovery of the ventilatory response to CO2. The effects of desogestrel on resting ventilation have not been reported. This study was designed to test the hypothesis that desogestrel strengthens baseline ventilation by analyzing the ventilation of CCHS patients. Rodent models were used in order to determine the mechanisms involved. Ventilation in CCHS patients was measured with a pneumotachometer. In mice, ventilatory neural activity was recorded from ex vivo medullary-spinal cord preparations, ventilation was measured by plethysmography and c-fos expression was studied in medullary respiratory nuclei. Desogestrel increased baseline respiratory frequency of CCHS patients leading to a decrease in their PETCO2. In medullary spinal-cord preparations or in vivo mice, the metabolite of desogestrel, etonogestrel, induced an increase in respiratory frequency that necessitated the functioning of serotoninergic systems, and modulated GABAA and NMDA ventilatory regulations. c-FOS analysis showed the involvement of medullary respiratory groups of cell including serotoninergic neurons of the raphe pallidus and raphe obscurus nuclei that seem to play a key role. Thus, desogestrel may improve resting ventilation in CCHS patients by a stimulant effect on baseline respiratory frequency. Our data open up clinical perspectives based on the combination of this progestin with serotoninergic drugs to enhance ventilation in CCHS patients.

Lethal H5N1 Virus Invades Medullary Respiratory‐related Neurons Responsible for Respiratory Chemoreflexes and Rhythm/Pattern Generation

Highly pathogenic H5N1 avian influenza virus is neurotropic and its infection is lethal mainly due to the respiratory failure. Studies have shown that lethal H5N1 HK483 (A/HK/483/97) virus invades the central nervous system, blunts ventilatory responses to hypoxia and hypercapnia, and induces apneas, leading to death at 6–8 days postinfection (dpi) in mice (Gao et al, Journal of virology 1999, Zhuang et al., ATS 2013, EB 2015). However, the relevant mechanisms remain unclear. We asked if HK483 H5N1 virus: 1) invaded neurons and/or astrocytes in the medullary respiratory‐related nuclei; 2) infected those respiratory neurons expressing neurokinin 1 receptor (NK1R), tyrosine hydroxylase (TH, a marker of catecholaminergic neuron), or tryptophan hydroxylase (TPH, a marker of serotonergic neuron); and 3) altered the these neural expressions of NK1R, TH, or TPH. Female Balb/c mice were intranasally inoculated with vehicle (Ctrl) or HK483 virus (100 PFU) and euthanized at 6–7 dpi. After fixation, the medullary sections were immunohistochemically processed to detect the nucleoprotein of H5N1 influenza A (NP) in neurons and/or astrocytes marked by glial fibrillary acidic protein (GFAP) and its co‐labeling with immunoreactivity (IR) of NK1R, TH, or TPH. We found that: 1) HK483 virus mainly invaded the respiratory‐related nuclei including the nucleus of solitary tract (NTS), ambiguus nucleus (Amb), pre‐Botzinger complex (PBC), Botzinger complex (BC), retrotrapzoid nucleus (RTN) and their neighboring areas with the raphe less infected and the invasion in astrocytes was limited; 2) approximately 20–50% of NK1R neurons (the NTS, PBC, and RTN), 10–30% of TH‐IR neurons (C1/2, A1 areas, the NTS, and locus ceruleus), and < 10% TPH (the raphe) were co‐labeled with NP; and 3) significantly reduced the density of NK1R‐IR in the corresponding nuclei with less effect on reduction of the raphe neural population expressing TPH and no effect on TH‐IR. We conclude that HK483 is capable of invading the medullary respiratory nuclei, particularly those neurons expressing NK1R or TPH to depress NK1R or TPH expression, which likely contributes to depression of the ventilatory responses to hypoxia and hypercapnia and introduction of apneas/respiratory failure, leading to the death.Support or Funding InformationSupported by HL119683 and HL107462

Changes in glutamate receptor subunits within the medulla in goats after section of the carotid sinus nerves.

The mechanisms which contribute to the time-dependent recovery of resting ventilation and the ventilatory CO2 chemoreflex after carotid body denervation (CBD) are poorly understood. Herein we tested the hypothesis that there are time-dependent changes in the expression of specific AMPA, NMDA, and/or neurokinin-1 (NK1R) receptors within respiratory-related brain stem nuclei acutely or chronically after CBD in adult goats. Brain stem tissues were collected acutely (5 days) or chronically (30 days) after sham or bilateral CBD, immunostained with antibodies targeting AMPA (GluA1 or GluA2), NMDA (GluN1), or NK-1 receptors, and optical density (OD) compared. Physiological measurement confirmed categorization of each group and showed ventilatory effects consistent with bilateral CBD (Miller et al. J Appl Physiol 115: 1088-1098, 2013). Acutely after CBD, GluA1 OD was unchanged or slightly increased, but GluA2 and GluN1 OD were reduced 15-30% within the nucleus tractus solitarius (NTS) and in other medullary respiratory nuclei. Chronically after CBD, GluA1 was reduced (P < 0.05) within the caudal NTS and in other nuclei, but there was significant recovery of GluA2 and GluN1 OD. NK1 OD was not significantly different from control after CBD. We conclude that the initial decrease in GluA2 and GluN1 after CBD likely contributes to hypoventilation and the reduced CO2 chemoreflex. The partial recovery of ventilation and the CO2 chemoreflex after CBD parallel a time-dependent return of these receptors to near control levels but likely depend upon additional initiating and maintenance factors for neuroplasticity.

Increased Ca2+ permeable ampa receptor expression following carotid body denervation in goats (713.2)

The mechanisms which contribute to the time‐dependent recovery of resting ventilation and the ventilatory CO2 chemoreflex after carotid body denervation (CBD) are poorly understood. The initiation of neuronal plasticity often involves changes in Ca2+ permeability. Therefore, we tested the hypothesis that during recovery from the acute effects of CBD in goats, there is an increase in Ca2+ permeable GluA1R and GluN1R and/or a decrease in Ca2+ impermeable GluA2R expression at brainstem sites that contribute to respiratory control. We tested this hypothesis via optical densitometric (OD) quantification of medullary neurons immunostained with antibodies targeting GluA1R, GluA2R, and GluN1R at 5‐13 days or 30 days after bilateral CBD. CBD resulted in hypoventilation and a reduced CO2 chemoreflex, which both recovered significantly over 30 days post‐CBD. At 5‐13 days post‐CBD, GluA1 OD was unchanged from control goats, but GluA2 and GluN1 OD were reduced 15‐30% within 6 of medullary respiratory nuclei measured. However, at 30 days post‐CBD GluA1 OD remained unchanged, but GluA2 and GluN1 OD returned to control levels. We conclude that a shift toward Ca2+ permeable glutamate receptors 5‐13 days post‐CBD may initiate the recovery of ventilation after CBD. Furthermore, at 30 days post‐CBD the partial recovery of ventilation is maintained by relatively normal glutamate receptor expression.Grant Funding Source: Supported by National Heart, Lung, and Blood Institute Grants HL25739 and HL007852 and by VA

Degeneration of Brainstem Respiratory Neurons in Dementia with Lewy Bodies

Respiratory dysfunction, including sleep disordered breathing, is characteristic of multiple system atrophy (MSA) and may reflect degeneration of brainstem respiratory nuclei involved in respiratory rhythmogenesis and chemosensitivity, including the pre-Bötzinger complex (preBötC), nucleus raphe pallidus (RPa), and nucleus raphe obscurus (ROb). However, impaired ventilatory responses to hypercapnia have also been reported in dementia with Lewy bodies (DLB), suggesting that these nuclei may also be affected in DLB. To determine whether there is involvement of the preBötC, RPa, and ROb in DLB. We applied stereological methods to analyze sections immunostained for neurokinin-1 receptor and tryptophan hydroxylase in neuropathologically confirmed cases of DLB, MSA, and controls. Reduction of neuronal density occurred in all three nuclei in DLB, as well as in MSA. The magnitude of neuronal depletion in ROb was similar in DLB and MSA (49% versus 56% respectively, compared to controls, P < 0.05), but neuronal loss in the preBötC and RPa was less severe in DLB than in MSA (40% loss in preBötC of DLB, P < 0.05 and 68% loss in MSA, P < 0.0001, compared to controls; 46% loss in RPa of DLB, P < 0.05 and 73% loss in MSA P < 0.0001, compared to controls). Medullary respiratory nuclei are affected in dementia with Lewy bodies but less severely than in multiple system atrophy. This may help explain differences in the frequency of sleep disordered breathing in these two disorders.

Chronic intermittent hypoxia promotes expression of 3-mercaptopyruvate sulfurtransferase in adult rat medulla oblongata

The present experiments were carried out to investigate the expression of 3-mercaptopyruvate sulfurtransferase (3MST) in medulla oblongata of rats and effects of chronic intermittent hypoxia (CIH) on its expression. Sprague Dawley adult rats were randomly divided into two groups, including control (Con) group and CIH group. The endogenous production of hydrogen sulfide (H2S) in medulla oblongata tissue homogenates was measured using the methylene blue assay method, 3MST mRNA and protein expression were analyzed by RT-PCR and Western blotting, respectively, and the expression of 3MST in the neurons of respiratory-related nuclei in medulla oblongata of rats was investigated with immunohistochemical technique. CIH elevated the endogenous H2S production in rat medulla oblongata (P<0.01). The RT-PCR and Western blotting analyses showed that 3MST mRNA and protein were expressed in the medulla oblongata of rats and CIH promoted their expression (P<0.01). Immunohistochemical staining indicated that 3MST existed in the neurons of pre-Bötzinger complex (pre-BötC), hypoglossal nucleus (12N), ambiguous nucleus (Amb), facial nucleus (FN) and nucleus tractus solitarius (NTS) in the animals and the mean optical densities of 3MST-positive neurons in the pre-BötC, 12N and Amb, but not in FN and NTS, were significantly increased in CIH group (P<0.05). In conclusion, 3MST exists in the neurons of medullary respiratory nuclei and its expression can be up-regulated by CIH in adult rat, suggesting that 3MST-H2S pathway may be involved in regulation of respiration and protection on medullary respiratory centers from injury induced by CIH.

Changes in neurochemicals within the ventrolateral medullary respiratory column in awake goats after carotid body denervation

A current and major unanswered question is why the highly sensitive central CO2/H(+) chemoreceptors do not prevent hypoventilation-induced hypercapnia following carotid body denervation (CBD). Because perturbations involving the carotid bodies affect central neuromodulator and/or neurotransmitter levels within the respiratory network, we tested the hypothesis that after CBD there is an increase in inhibitory and/or a decrease in excitatory neurochemicals within the ventrolateral medullary column (VMC) in awake goats. Microtubules for chronic use were implanted bilaterally in the VMC within or near the pre-Bötzinger Complex (preBötC) through which mock cerebrospinal fluid (mCSF) was dialyzed. Effluent mCSF was collected and analyzed for neurochemical content. The goats hypoventilated (peak +22.3 ± 3.4 mmHg PaCO2) and exhibited a reduced CO2 chemoreflex (nadir, 34.8 ± 7.4% of control ΔVE/ΔPaCO2) after CBD with significant but limited recovery over 30 days post-CBD. After CBD, GABA and glycine were above pre-CBD levels (266 ± 29% and 189 ± 25% of pre-CBD; P < 0.05), and glutamine and dopamine were significantly below pre-CBD levels (P < 0.05). Serotonin, substance P, and epinephrine were variable but not significantly (P > 0.05) different from control after CBD. Analyses of brainstem tissues collected 30 days after CBD exhibited 1) a midline raphe-specific reduction (P < 0.05) in the percentage of tryptophan hydroxylase-expressing neurons, and 2) a reduction (P < 0.05) in serotonin transporter density in five medullary respiratory nuclei. We conclude that after CBD, an increase in inhibitory neurotransmitters and a decrease in excitatory neuromodulation within the VMC/preBötC likely contribute to the hypoventilation and attenuated ventilatory CO2 chemoreflex.

Nitric oxide synthase expression in the medullary respiratory related nuclei and its involvement in CO-mediated central respiratory effects in neonatal rats

The present study was conducted in order to observe the potential participation of the nitric oxide synthase–NO pathway in CO-mediated regulation of respiration of neonatal rats. An immunofluorescent histochemical technique was used to examine the existence of the neuronal nitric oxide synthase, a key enzyme of synthesizing NO, in medullary respiratory nuclei. The rhythmic respiratory-like discharges of hypoglossal rootlets of medullary slices were recorded to test the role of the nitric oxide synthase in CO-mediated respiratory effects. We observed neuronal nitric oxide synthase expressed in the medullary respiratory nuclei in conjunction with CO lengthened expiratory duration, decreased respiratory frequency, and increased inspiratory amplitude. These CO-mediated respiratory effects could be partially eliminated by prior treatment of the slices with Nω-nitro- l-arginine methyl ester, an inhibitor of nitric oxide synthase. The results suggest that nitric oxide synthase–NO pathway might be involved in the CO-mediated central regulation of respiration at the level of medulla oblongata in neonatal rats.

Differential modulation of inspiratory motoneurons and respiratory rhythm generating circuits by ATP

Adenosine triphosphate (ATP) gates a diverse family of P2 (P2X1-7 and P2Y1-11) receptors that are expressed throughout the CNS, including respiratory regions of the brainstem [1]. Activation of ATP receptors potentiates activity of inspiratory motoneurons (MN) [2], modulates respiratory behaviour in vivo when applied to medullary respiratory nuclei and, via activation of pH-sensitive P2X2 receptors, may contribute to central respiratory chemosensitivity [3]. Aims of this study were to: i) determine the effects of ATP receptor activation within the pre-Botzinger complex (preBotC, proposed site of rhythm generation) on respiratory behaviour; ii) test the hypothesis that respiratory networks are endogenously modulated by ATP; and iii) compare the sensitivity of inspiratory MN pools and rhythm generating networks to purinergic modulation. To determine the effects of ATP on respiratory rhythm and inspiratory motor output, ATP was pressure-injected into the pre-BotC and XII nuclei of rhythmically-active medullary slice preparations from neonatal rats, while monitoring XII nerve and MN output. Effects on phrenic MNs were determined by locally applying drugs over the phrenic MN pool of brainstem spinal cord preparations. ATP (10 s, 0.01–1 mM) caused up to a 4-fold, suramin-sensitive (0.05–1.0 mM), increase in frequency, that was followed by a brief (22 ± 5%) reduction. To test whether this post-ATP inhibition was due to hydrolysis of ATP to adenosine and activation of adenosine receptors, we applied ATPγs, a non-hydrolyzable ATP analogue. Peak potentiation of frequency by 0.1 mM ATPγs (3.20 ± 0.3 fold increase, n = 7) was similar to that evoked by ATP (3.30 ± 0.3, n = 7), but the effects of ATPγs were longer lasting (102.9 s ± 10.64 vs 29.3 s ± 2.06 for ATP). The secondary reduction in frequency was also absent following ATPγs. Since P2 receptor antagonists also antagonize glutamate receptors which are essential for rhythm generation, the role of endogenous ATP in modulating respiratory rhythm was investigated via bath application of ectoATPase inhibitors (DEPC, pCMPS) and an allosteric modulator of P2X2 recptors (Cu2+). DEPC (100 μM, n = 4) and pCMPS (30 μM, n = 3) increased respiratory frequency 1.30–1.40 fold, while Cu2+ (10–50 μM, n = 6) increased frequency ~1.5-fold. Local application of ATP (1–10 mM) over XII and phrenic nuclei produced a biphasic response comprising an initial potentiation of burst amplitude (1.40 ± 0.20 of control and 1.22 ± 0.7 respectively) followed by a decrease in burst amplitude (to 0.82 ± 0.05 and 0.90 ± 0.05 of control respectively) that was theophylline-sensitive and absent following application of ATP-γ-s. The doses of ATP required to potentiate burst amplitude by 1.20–1.40 of control [2] were ~100 times higher than required to increase rhythm 2.5- to 4-fold. Results show that exogenous ATP potently increases respiratory frequency and that rhythm generating networks are significantly more sensitive to ATP than respiratory motoneurons. In addition, effects of ectoATPase inhibitors and allosteric modulators of ATP receptors suggest that respiratory networks are endogenously modulated by ATP. The differential sensitivity of rhythm generating elements and motoneurons provides an opportunity to explore the physiological significance of P2 receptor diversity to respiratory control.

Regeneration of Respiratory Pathways within Spinal Peripheral Nerve Grafts

Central respiratory neurons exhibit normal activity after axonal regeneration within blind-ended peripheral nerve grafts (PNGs) inserted near the corresponding cell bodies in the medullary respiratory centers. Part of these medullary respiratory neurons project toward the spinal cord and contribute to descending respiratory pathways that control respiratory motoneurons. The present work investigates to what extent cervical respiratory pathways could be directed out of the central nervous system within PNGs inserted distant to the medullary respiratory nuclei. In adult rats (n=13), autologous segments of the peroneal nerve were implanted into the ventrolateral part of the C2 spinal cord at the level of the descending respiratory pathways. Two to four months after grafting, electrophysiological recording of teased graft filaments (n=562) revealed the presence of regenerated nerve fibers with unitary impulse traffic (n=164) in all tested PNGs (n=6). Respiratory discharges (n=52) corresponded to efferent and afferent activity. Efferent respiratory discharges (n=32) originated from central respiratory neurons which remained functional and preserved afferent connections. Retrograde horseradish peroxidase labeling applied to the distal cut end of PNGs (n=7) revealed stained (42/1997) neurons in areas where respiratory cells have been described. Afferent respiratory discharges (n=20) were synchronized with lung inflation but their origin (stretch pulmonary receptors and/or respiratory muscle receptors) was not determined. On the basis of additional data from light and electron microscopy of PNGs, comparison was made between anatomical, retrograde labeling, and electrophysiological data. The main conclusion is that spinal PNGs appear to be able to promote axonal regeneration of functional respiratory efferent and afferent pathways.

Somatostatin induced apnoea: prevention by central and peripheral administration of the opiate receptor blocking agent naloxone.

Somatostatin (SRIF) (6 nmol) given intracisternally (i.c.) into the alpha-chloralose anaesthetized rat has recently been shown to cause apnoea with a latency of 5-10 minutes (Kalia et al. 1984a). The apnoea produced by SRIF is very rapid, irreversible and leads to the death of the animal. In view of the existence of SRIF nerve cell bodies and terminals in medullary respiratory nuclei such as the ventral and ventrolateral subnuclei of the nucleus of the tractus solitarius (nTS) (Kalia et al. 1984b, Johanson et al. 1984), we have proposed the existence of somatostatinergic mechanisms in the respiratory nuclei of the medulla oblongata involved in mediating apnoeic conditions (Kalia et al. 1984a). In the present study, we have analysed whether the SRIF induced apnoea could be counteracted by a previous i.c. administration of the highly selective alpha 2-adreno-receptor blocking agent RX 781094 (2-(2-(I,4 benzodioxanyl]2-imi-dazoline HCl) (Doxey et al. 1983), or an opiate receptor blocking agent such as naloxone. Thus, both alpha 2-adrenoreceptor agonists and opiates induce respiratory depression, and opiates in high doses cause apnoea (Bolme et al. 1974, Hassen et al. 1982, Sitsen et al. 1982). In addition, catecholamine (CA) and enkephalin immuno-reactive nerve terminal networks exist in high densities within the nucleus tractus solitarius (nTS) of the medulla oblongata and may therefore interact with somatostatin nerve terminals in regulation of respiratory activity (Kalia et al. 1984b).

Genesis of rhythmic respiratory activity in pons independent of medulla.

We hypothesized that rhythmic respiratory-related activity could be generated in pons independent of medullary mechanisms. In decerebrate, cerebellectomized, vagotomized, paralyzed, and ventilated cats, we recorded efferent activities of the phrenic nerve and mylohyoid branch of the trigeminal nerve. Following transections of the brain stem at the pontomedullary junction, the phrenic and trigeminal nerves discharged with independent rhythms. Spontaneous trigeminal discharges eventually ceased but were reestablished after strychnine, doxapram, and/or protriptyline were administered. In some animals having no spontaneous trigeminal discharges after transection, these discharges appeared, with a rhythm different from the phrenic, following administration of these agents. In other cats having no transections between pons and medulla, these pharmacological agents induced trigeminal and phrenic discharges after kainic acid had been injected into the entire dorsal and ventral medullary respiratory nuclei. Phrenic and trigeminal discharges were linked, indicating survival of bulbospinal neurons or presence of pontospinal units. We conclude that rhythms, similar to respiratory rhythm, can occur by mechanisms in isolated pons. Such mechanisms are hypothesized to be within the pneumotaxic center and may underlie the neurogenesis of eupnea.

A cross-correlation study of interactions among respiratory neurons of dorsal, ventral and retrofacial groups in cat medulla

In anesthetized or decerebrated cats, extracellular activities of pairs of respiratory neurons located in the regions of the dorsal (DRN), ventral (VRN) and retrofacial (RFN) medullary respiratory nuclei were recorded using two separate microelectrodes. Neurons were classified as bulbospinal or laryngeal if stimulation of the spinal cord or vagus nerve elicited antidromic action potentials, or as propriobulbar if they were not antidromically activated. Of 163 pairs of single unit activities, either inspiratory (143 pairs) or expiratory (20 pairs), cross-correlation analyses indicated that 23% had short latency peaks, either broad (12%) or sharp (1%) in their cross-correlograms, 3% had short latency troughs and 74% had flat cross-correlograms. When the two neurons were located in the DRN (68 pairs) the probability of obtaining a positive cross-correlogram was high for inspiratory bulbospinal neurons, indicating shared inputs and excitatory relationships. When one neuron of the pair was located in the RFN and the other in either the DRN or VRN (95 pairs), cross-correlation analysis revealed shared inputs, excitatory and inhibitory relationships. Among expiratory neurons interactions were only inhibitory with a more frequent incidence ( 320) than between inspiratory neurons ( 2143. Our results indicate that: (i) short time scale synchrony due to shared inputs (broad peaks) are largely distributed in the respiratory neuronal network and operate over long distance (i.e. RFN, caudal medulla); (ii) excitatory coupling may exist between remote neurons but is more frequent between inspiratory bulbospinal neurons located in the DRN.

Somatostatin produces apnea and is localized in medullary respiratory nuclei: a possible role in apneic syndromes

Immunocytochemical studies on the nucleus of the tractus solitarius and adjacent areas of the dorsal medulla of the rat demonstrate the existence of somatostatin immunoreactive nerve cell bodies and nerve terminals within the ventrolateral and ventral subnuclei of the nucleus of the tractus solitarius. Injections of somatostatin (6 nmol in μl) into the cisterna magna of chloralose-anesthetized rats produced an appea with a latency of 5–7 min. This apnea was preceded by slow deep brething, a reduction in heart rate and fall of arterial blood pressure. The apnea was usuallly irreversible leading to death of the animal. These respiratory and cardiovascular effects of somatostatin were not abolished either by bilateral vagotomy or by low decerebration (below the inferior colliculus). It is suggested that activation of somatostatin receptors linked to neurons in medullary respiratory nuclei might be responsible for the inhibition of respiratory neuronal activity and thus may mediate apneic conditions.

Respiratory neurons in the region of the retrofacial nucleus: pontile, medullary, spinal and vagal projections

Activities were recorded from single respiratory neurons in the area of the retrofacial nucleus. Stimuli were delivered in the dorsal (DRN) and ventral (VRN) medullary respiratory nuclei, the rostral pons, the cervical spinal cord and the vagus nerve to elicit antidromic potentials for these neurons and, hence, establish their axonal projections. The great majority of the antidromically activated retrofacial inspiratory neurons sent their axons to the contralateral VRN. A majority of expiratory neurons had their axons in the contralateral DRN or in both the DRN and spinal cord. For the phase-spanning neurons, the projections were found mainly in the VRN, spinal cord or pons.