Increase In Phrenic Nerve Activity Research Articles

APOE Genotype Differentially Regulates Respiratory Motor Plasticity in the Active vs Rest phase

Acute intermittent hypoxia (AIH) elicits respiratory motor plasticity and is emerging as a promising strategy to improve breathing and other (non-respiratory) movements in clinical disorders that end life due to respiratory insuffciency ( e.g., spinal cord injury & ALS). AIH-induced phrenic long-term facilitation (pLTF) is the most commonly studied form of respiratory motor plasticity and presents as a persistent increase in phrenic nerve activity after AIH. Specific factors ( e.g. aging, inflammation, time of day) undermine AIH-induced pLTF. However, little information is available concerning the role of specific genes that regulate AIH-induced phrenic motor plasticity. Apolipoproteins (ApoE) are highly expressed in the central nervous system and function as lipid carriers. The APOE gene is tri-allelic ( E2, E3 and E4); ApoE2 and E3 proteins are associated with neuroprotective and neuro-neutral phenotypes, respectively. ApoE4 impairs synaptic plasticity and is implicated in neurodegeneration, particularly Alzheimer’s Disease. Indeed, we recently discovered that specific APOE alleles predict human expression of mAIH-induced plasticity in cortico-spinal pathways to the phrenic/diaphragm motor system. Initial experiments using human APOE4 knock-in rats demonstrate a causal effect in limiting phrenic motor plasticity (vs APOE3) during the active phase. However, the impact of APOE genotype on respiratory plasticity across the daily rest/active cycle is unknown. We tested the hypothesis that APOE4 undermines AIH-induced pLTF vsAPOE3 in a time-of-day-dependent manner. Anesthetized, vagotomized, paralyzed and ventilated adult (3-4 months) male Sprague-Dawley rats with knock-in humanized (h) APOE3 and h APOE4 were presented with AIH (15,1 min hypoxic episodes; arterial PO2 = 40-50mmHg). During the active phase, AIH-induced pLTF was significantly greater in h APOE3 (33±11%) vs h APOE4 rats (-26±7%; p<0.001; both n=6). In contrast, rest phase AIH-induced pLTF was similar in both h APOE3 and h APOE4 rats (121±12% and 122±5%, respectively; both n=7). These experiments demonstrate a causal relationship whereby APOE genotype and time-of-day interact to undermine AIH-induced pLTF in a manner that is unique to the active phase in rats. Since AIH has emerged as a promising neurotherapeutic to preserve/restore breathing in clinical disorders such as spinal cord injury and neuromuscular disease, understanding how genetic factors and time-of-day of AIH delivery impact phrenic motor plasticity will help identify individuals most/least likely to respond to treatment and reveal new targets for precision interventions of AIH. NIH R01HL148030 (GSM), NIH R01HL149800 (GSM), NIH T32HL134621-5 (ABM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Magnitude and Mechanism of Phrenic Long-term Facilitation Shift Between Daily Rest Versus Active Phase.

Plasticity is a fundamental property of the neural system controlling breathing. One key example of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic nerve activity elicited by acute intermittent hypoxia (AIH). pLTF can arise from distinct cell signaling cascades initiated by serotonin versus adenosine receptor activation, respectively, and interact via powerful cross-talk inhibition. Here, we demonstrate that the daily rest/active phase and the duration of hypoxic episodes within an AIH protocol have profound impact on the magnitude and mechanism of pLTF due to shifts in serotonin/adenosine balance. Using the historical "standard" AIH protocol (3, 5-min moderate hypoxic episodes), we demonstrate that pLTF magnitude is unaffected by exposure in the midactive versus midrest phase, yet the mechanism driving pLTF shifts from serotonin-dominant (midrest) to adenosine-dominant (midactive). This mechanistic "flip" results from combined influences of hypoxia-evoked adenosine release and daily fluctuations in basal spinal adenosine. Since AIH evokes less adenosine with shorter (15, 1-min) hypoxic episodes, midrest pLTF is amplified due to diminished adenosine constraint on serotonin-driven plasticity; in contrast, elevated background adenosine during the midactive phase suppresses serotonin-dominant pLTF. These findings demonstrate the importance of the serotonin/adenosine balance in regulating the amplitude and mechanism of AIH-induced pLTF. Since AIH is emerging as a promising therapeutic modality to restore respiratory and nonrespiratory movements in people with spinal cord injury or ALS, knowledge of how time-of-day and hypoxic episode duration impact the serotonin/adenosine balance and the magnitude and mechanism of pLTF has profound biological, experimental, and translational implications.

Pattern of Acute Intermittent Hypoxia Protocol Impacts Phrenic Long-term Facilitation

An important model of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a prolonged increase in phrenic nerve activity following intermittent exposure to low oxygen (acute intermittent hypoxia, AIH). pLTF arises from distinct intracellular signaling cascades initiated by serotonin 2 and adenosine 2A (A 2A ) receptor activation, respectively, that interact via powerful crosstalk inhibition. Studies of AIH-induced motor plasticity typically use moderate AIH protocols consisting of 3, 5 min hypoxic episodes in rodent models, but 15, 1 min hypoxic episodes in humans. Since certain factors ( e.g. severity or duration of hypoxia) influence pLTF expression, it is essential to understand the influence of AIH protocol details on motor plasticity. Here we tested the hypothesis that serotonin-dominant plasticity is enhanced when the duration of hypoxic episodes in the AIH protocol is shorter due to less hypoxia-evoked adenosine release, eliminating adenosine constraint and revealing enhanced plasticity. Anesthetized, vagotomized, paralyzed and ventilated male Sprague-Dawley rats were exposed to different AIH protocols with the same cumulative duration (15 min) and severity (arterial Po2 = 40-55mmHg) of hypoxic episodes: 1) 3, 5 min episodes with 5 min intervals (3x5); and 2) 15, 1 min hypoxic episodes with 1 min intervals (15x1). Consistent with prior reports, 3x5 AIH elicits ~60% pLTF (56±5%; n=7). In contrast, 15x1 AIH elicits significantly greater pLTF (139±14%; n=7; p<0.001). To determine if this effect is due to differences in hypoxia evoked-adenosine release in the ventral C 4 spinal cord, tissue oxygen pressure and spinal adenosine concentration were measured with an oxygen optode, and adenosine probe, respectively. Although tissue oxygen pressure between protocols was not different, hypoxia-evoked adenosine release was substantially greater with the 3x5 vs. 15x1 AIH protocols (p<0.001; both n=3). To verify pLTF magnitude results from differential spinal A 2A receptor activation, rats were pretreated with a selective A 2A receptor antagonist, MSX-3 (10uM; 12 uL) delivered intrathecally at C 4 ; with the 3x5 AIH protocol, pLTF was enhanced as expected (141±15%; p<0.001; n=7). However, MSX-3 had no effect on pLTF elicited by the 15x1 AIH protocol (122±11%; n=7; p=0.931). Thus, the mechanism driving pLTF is serotonin-dominant and adenosine-constrained with 3x5 AIH, but adenosine- unconstrained with the 15x1 AIH protocol. We conclude that, by shortening hypoxic episodes from 5 to 1 min (with the same cumulative hypoxic duration), reduced hypoxia-evoked adenosine release reveals the full extent of serotonin-driven plasticity. AIH is emerging as a promising therapeutic modality to restore respiratory and non-respiratory movements in people with spinal injury and ALS. Thus, knowledge that the AIH protocol can be optimized by reducing the length of individual hypoxic episodes has important experimental, biological and translational implications. Supported by: NIH R01HL148030, R01HL149800 & T32HL134621 (ABM), and UF Brain Spinal Cord Research Trust Fund (BSCIRTF) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Pattern of Ampakine and Hypoxia Exposure Determines Phrenic Motor Facilitation

Phrenic motor facilitation (pMF) is a persistent increase in phrenic nerve activity upon the removal of a triggering stimuli. A variety of stimulation paradigms can induce pMF including electrical activation of the carotid sinus nerve, serotonin or adenosine receptor agonists, acute intermittent hypoxia (AIH) and inactivity. Recently, Wollman et al. (2020) demonstrated a new kind of pMF using a combination of a pharmacological agent (ampakine) and a single brief hypoxic episode. A subsequent study confirmed this finding and also showed that pairing ampakine with multiple hypoxic episodes did not increase magnitude of pMF (Thakre et al., 2021). The present study investigated if ampakine‐hypoxia induced pMF is sensitive to the pattern in which ampakine and hypoxia are administered. Phrenic nerve output was recorded from urethane‐anesthetized, mechanically ventilated and vagotomized adult male Sprague‐Dawley rats. Hypoxic exposures were 5 min in duration with moderate severity (arterial O2 partial pressure approximately 45 mmHg). The ampakine used was CX717, given intravenously (i.v.) at 15 mg/kg. We first verified the impact of a single administration of i.v. ampakine followed after 2 min by hypoxia. In agreement with prior work, this paradigm evoked a sustained increase in phrenic burst amplitude (60 min: 73±19% baseline, n=8). In the next experiments, the order of ampakine and hypoxia was reversed such that hypoxia was followed after 2 min by ampakine. This paradigm did not evoke a sustained increase in phrenic burst amplitude (60 min: 11±15% baseline, n=6). In the final series of experiments, ampakine was delivered during an ongoing episode of hypoxia (2 min after onset). This paradigm also did not evoke a sustained increase in phrenic burst amplitude (60 min: 12±14% baseline, n=7). Our results indicate that the mechanisms of pMF evoked by the ampakine‐hypoxia interaction are sensitive to the order in which these stimuli are presented. The presentation of ampakine followed by a single brief hypoxic episode appears to be ideal for producing pMF. These findings could have significant implications in planning hypoxia‐based neurorehabilitation strategies with ampakines as an adjunct therapy.

Daily acute intermittent hypoxia enhances phrenic motor output and stimulus-evoked phrenic responses in rats.

Plasticity is a hallmark of the respiratory neural control system. Phrenic long-term facilitation (pLTF) is one form of respiratory plasticity characterized by persistent increases in phrenic nerve activity following acute intermittent hypoxia (AIH). Although there is evidence that key steps in the cellular pathway giving rise to pLTF are localized within phrenic motor neurons (PMNs), the impact of AIH on the strength of breathing-related synaptic inputs to PMNs remains unclear. Furthermore, the functional impact of AIH is enhanced by repeated/daily exposure to AIH (dAIH). Here, we explored the effects of AIH versus 2 wk of dAIH preconditioning on spontaneous and evoked phrenic responses in anesthetized, paralyzed, and mechanically ventilated rats. Evoked phrenic potentials were elicited by respiratory cycle-triggered lateral funiculus stimulation at the C2 spinal level delivered before and 60 min post-AIH (or the equivalent in time controls). Charge-balanced biphasic pulses (100 μs/phase) of progressively increasing intensity (100-700 μA) were delivered during the inspiratory and expiratory phases of the respiratory cycle. Although robust pLTF (∼60% from baseline) was observed after a single exposure to moderate AIH (3 × 5 min; 5-min intervals), there was no effect on evoked phrenic responses, contrary to our initial hypothesis. However, in rats preconditioned with dAIH, baseline phrenic nerve activity and evoked responses were increased, suggesting that repeated exposure to AIH enhances functional synaptic strength when assessed using this technique. The impact of daily AIH preconditioning on synaptic inputs to PMNs raises interesting questions that require further exploration.NEW & NOTEWORTHY Two weeks of daily acute intermittent hypoxia (dAIH) preconditioning enhanced stimulus-evoked phrenic responses to lateral funiculus stimulation (targeting respiratory bulbospinal projection to phrenic motor neurons). Furthermore, dAIH preconditioning enhanced baseline phrenic motor output responses to maximal chemoreflex activation in intact rats.

A projection from the hypothalamic paraventricular nucleus (PVN) to nucleus tractus solitarii (nTS) is essential for cardiorespiratory responses to hypoxia

The PVN contributes to cardiorespiratory responses to peripheral chemoreflex activation. We have shown that hypoxia (Hx) activates PVN neurons that project to the nTS but the functional role of this nTS projection is not known. We hypothesized that selective inhibition/activation of the PVN to nTS pathway blunts/augments chemoreflex cardiorespiratory responses. Male SD rats received bilateral microinjections of retrograde AAV‐pgk‐Cre into the nTS. One week later, rats received bilateral PVN injections of Cre‐dependent AAV2‐DIO‐hSyn‐mCherry expressing inhibitory (Gi) or excitatory (Gq) DREADD, or control virus (mCh), and 3–5 weeks allowed for expression in nTS‐projecting PVN neurons. To evaluate the contribution of the PVN to nTS pathway to chemoreflex function, ventilatory responses (plethysmography) to progressive Hx (14–8% O2) were assessed in conscious animals before and after ip injection of saline or the synthetic selective DREADD ligand C21 (1mg/kg). We similarly evaluated Hx‐induced nTS neuronal activation (Fos immunohistochemistry); 60 min after ip saline or C21, conscious mCh (n=6) and Gi (n=4) rats were exposed to Hx (2 hr, 10% O2) and Gq rats were exposed to 12% O2. Saline had no effect on either ventilatory responses or nTS neuronal activation to Hx in any group. In Gi rats, selective inhibition of nTS‐projecting PVN neurons with C21 blunted hypoxic ventilatory responses to 10% and 8% O2 (n=8, p<0.05) and appeared associated with decreased Hx‐induced nTS neuronal activation (581 ± 33 vs. 375 ± 150 cells; saline vs. C21; n=2 each). In contrast, selective activation of the PVN to nTS pathway by C21 in Gq rats (n=8) enhanced Hx ventilatory responses to milder hypoxia (14 and 12% O2, p<0.05), and produced greater Hx‐induced Fos‐IR in the nTS (377 ± 45 vs. 550 ± 23 cells; saline vs. C21, n=3 ea). To confirm that altered cardiorespiratory chemoreflex responses were mediated by PVN terminals in the nTS, anesthetized rats were exposed to brief (45s) Hx episodes (10% O2, mCh and Gi rats; 12% O2, Gq rats) before and after bilateral nTS microinjection of C21 (0.1mM; 90nl/side). Mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and phrenic nerve activity (PhrNA)] were measured. Under control conditions, Hx decreased MAP and increased HR, sSNA and PhrNA in all groups. Peak responses to 10% O2 were similar in mCh and Gi rats and greater than control responses to 12% O2 in Gq rats. nTS microinjection of C21 had minor baseline effects, which were not different among groups. nTS C21 did not alter the Hx‐induced increase in PhrNA of mCh rats (n=2). In Gi rats (n=3), DREADD‐mediated inhibition of PVN terminals in the nTS blunted both PhrNA (−63 ± 10%; p<0.05) and sSNA (−32 ± 18%; p<0.05) responses to Hx. In contrast, activation of PVN terminals in the nTS enhanced both PhrNA (+51 ± 36%) and sSNA (+166 ± 41%) responses to Hx in Gq rats (n=3). Together, these results suggest that a PVN to nTS pathway directly enhances nTS neuronal activation and cardiorespiratory responses to hypoxia.Support or Funding InformationRO1‐HL‐98602 F31‐HL‐140858This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spinal Conversion of Testosterone to Estradiol for the Expression of Phrenic Long‐term Facilitation in Adult Male Rats

Estrogen modulates the expression of neuroplasticity, including phrenic long‐term facilitation (pLTF), a persistent increase in phrenic nerve activity observed after acute exposure to intermittent hypoxia (AIH; three 5 min episodes of 10.5% O2, delivered at 5‐minute intervals). Though robustly expressed in adult males, adult female rats express pLTF only in the presence of adequate circulating estradiol (E2); removal of the ovaries, the primary source of circulating E2, abolishes pLTF. Acute, spinal application of membrane‐impermeable E2 (E2‐BSA) restores plasticity in ovariectomized female rats through a fast‐acting cellular mechanism involving membrane‐associated estrogen receptors, suggesting spinal E2 signaling may be important for pLTF expression in females. Previous studies indicate that E2 may play an important role in the expression of AIH‐induced pLTF in adult male rats as well. Specifically, gonadectomy blunts pLTF expression by reducing circulating testosterone, and systemic testosterone replacement restores pLTF. However, this testosterone‐induced restoration of pLTF is only effective in the presence of aromatase, an enzyme that converts testosterone to E2. Here we tested the hypothesis that the relevant conversion of testosterone to E2 necessary for expression of pLTF in male rats occurs in the spinal cord near phrenic motor neurons. We recorded phrenic nerve activity and pLTF expression in anesthetized, paralyzed and ventilated male Sprague‐Dawley rats (3–4 mos) in the presence or absence of intrathecally delivered Letrozole, an aromatase inhibitor, over the cervical spinal cord. We combined these data with immunohistochemical characterization of aromatase expression in the ventral cervical spinal cord. Collectively, these data provide important information regarding the necessity for spinal E2 signaling in the expression of AIH‐induced pLTF in male rats, and underscore the utility of this model to dissect the role of E2 signaling in expression of spinal neuroplasticity.Support or Funding InformationFunding: This work was supported by the University of Minnesota Division of Physical Therapy with a generous contribution from the University of Minnesota Department of Rehabilitation Medicine.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phrenic motor neuron adenosine 2A receptors elicit phrenic motor facilitation.

Although adenosine 2A (A2A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A2A receptor siRNA injections. A2A receptor siRNA injections selectively knocked down A2A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced phrenic motor facilitation. Upregulation of A2A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A2A receptors for pMF are unknown. This is an important question since the physiological outcome of A2A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A2A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A2A receptor immunostaining significantly colocalized with NeuN-positive neurons (89±2%). Intrapleural siRNA injections were used to selectively knock down A2A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A2A receptor knock-down was verified by a ∼45% decrease in A2A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P<0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A2A receptor agonist CGS21680 was greatly attenuated in siA2A (21%) versus siNT treated rats (147%; P<0.01). There were no significant effects of siA2A on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced pMF.

Creatine and creatine pyruvate reduce hypoxia-induced effects on phrenic nerve activity in the juvenile mouse respiratory system

Adequate concentrations of ATP are required to preserve physiological cell functions and protect tissue from hypoxic damage. Decreased oxygen concentration results in ATP synthesis relying increasingly on the presence of phosphocreatine. The lack of ATP through hypoxic insult to neurons that generate or regulate respiratory function, would lead to the cessation of breathing (apnea). It is not clear whether creatine plays a role in maintaining respiratory phrenic nerve (PN) activity during hypoxic challenge.The aim of the study was to test the effects of exogenously applied creatine or creatine pyruvate in maintaining PN induced respiratory rhythm against the deleterious effects of severe hypoxic insult using Working Heart-Brainstem (WHB) preparations of juvenile Swiss type mice. WHB's were perfused with control perfusate or perfusate containing either creatine [100μM] or creatine pyruvate [100μM] prior to hypoxic challenge and PN activity recorded throughout. Results showed that severe hypoxic challenge resulted in an initial transient increase in PN activity, followed by a reduction in that activity leading to respiratory apnea. The results demonstrated that perfusing the WHB preparation with creatine or creatine pyruvate, significantly reduced the onset of apnea compared to control conditions, with creatine pyruvate being the more effective substance. Overall, creatine and creatine pyruvate each produced time-dependent degrees of protection against severe hypoxic-induced disturbances of PN activity. The underlying protective mechanisms are unknown and need further investigations.

Mammalian target of rapamycin is required for phrenic long-term facilitation following severe but not moderate acute intermittent hypoxia.

Phrenic long-term facilitation (pLTF) is a persistent increase in phrenic nerve activity after acute intermittent hypoxia (AIH). Distinct cell-signaling cascades give rise to pLTF depending on the severity of hypoxemia within hypoxic episodes. Moderate AIH (mAIH; three 5-min episodes, PaO2 ∼35-55 mmHG) elicits pLTF by a serotonin (5-HT)-dependent mechanism that requires new synthesis of brain-derived neurotrophic factor (BDNF), activation of its high-affinity receptor (TrkB), and ERK MAPK signaling. In contrast, severe AIH (sAIH; three 5-min episodes, PaO2 ∼25-30 mmHG) elicits pLTF by an adenosine-dependent mechanism that requires new TrkB synthesis and Akt signaling. Although both mechanisms require spinal protein synthesis, the newly synthesized proteins are distinct, as are the neurochemicals inducing plasticity (serotonin vs. adenosine). In many forms of neuroplasticity, new protein synthesis requires translational regulation via mammalian target of rapamycin (mTOR) signaling. Since Akt regulates mTOR activity, we hypothesized that mTOR activity is necessary for sAIH- but not mAIH-induced pLTF. Phrenic nerve activity in anesthetized, paralyzed, and ventilated rats was recorded before, during, and 60 min after mAIH or sAIH. Rats were pretreated with intrathecal injections of 20% DMSO (vehicle controls) or rapamycin (0.1 mM, 12 μl), a selective mTOR complex 1 inhibitor. Consistent with our hypothesis, rapamycin blocked sAIH- but not mAIH-induced pLTF. Thus spinal mTOR activity is required for adenosine-dependent (sAIH) but not serotonin-dependent (mAIH) pLTF, suggesting that distinct mechanisms regulate new protein synthesis in these forms of spinal neuroplasticity.

Differential Roles of Mammalian Target of Rapamycin (mTOR) in Phrenic Long‐Term Facilitation

Phrenic long‐term facilitation (pLTF) is a persistent increase in phrenic nerve activity after acute intermittent hypoxia (AIH). This form of spinal plasticity is expressed via distinct cell signaling cascades that depend on the severity of hypoxia during intermittent exposures. Each mechanism requires spinal protein synthesis, though the newly synthesized proteins differ (e.g. BDNF vs immature TrkB). Translational regulation of new protein synthesis is often regulated by mammalian Target of Rapamycin (mTOR)‐dependent translation. Here we tested the hypothesis that mTOR is necessary for severe AIH (sAIH; PaO2 25‐30mmHG), but not moderate AIH (mAIH; PaO2 35‐45mmHG) induced spinal protein synthesis. Phrenic nerve activity was recorded in anesthetized, paralyzed and ventilated male Sprague‐Dawley rats before, during and 60 min after mAIH or sAIH. Rats were pre‐treated with intrathecal injections of vehicle or Rapamycin (0.1mM, 12µl), a highly selective inhibitor of mTOR complex 1. Consistent with our hypothesis, Rapamycin blunted sAIH, but not mAIH induced pLTF. Thus, spinal activation of mTOR and/or its down‐stream targets are necessary for sAIH‐, but not mAIH‐induced pLTF. These results provide further evidence that mAIH and sAIH elicit pLTF via distinct cellular cascades despite differing only in the severity of hypoxemia within hypoxic episodes.

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.

Microinjection of methysergide into the raphe nucleus attenuated phrenic long-term facilitation in rats

Exposure to acute intermittent hypoxia (AIH) evokes persistent increase in respiratory activity that lasts up to 60 min after hypoxic episodes have ceased. This persistent increase in phrenic nerve activity (PNA) is known as phrenic long-term facilitation (LTF). AIH-induced phrenic LTF in anesthetized rats is serotonin dependant. The present study was performed to determine whether microinjection of methysergide (4 mM, 20 +/- 5 nl), a broad spectrum 5-HT receptor antagonist, into the caudal raphe nuclei influences phrenic LTF. Peak integrated PNA and respiratory frequency were recorded at 15, 30, and 60 min after five 3-min episodes of normocapnic hypoxia in urethane-anesthetized, vagotomized, paralyzed and ventilated male Sprague-Dawley rats. In control animals, phrenic nerve amplitude was elevated 66.7 +/- 8.6% from baseline 1 h after episodic hypoxia, indicating phrenic LTF. Experimental microinjections of methysergide prior to AIH exposure attenuated phrenic LTF (amplitude increase 2.62 +/- 2.9% over baseline). We conclude that methysergide microinjections into the caudal raphe region attenuated phrenic LTF induced by AIH, indicating involvement of 5-HT receptor activation at a supraspinal level.

Photostimulation of Retrotrapezoid Nucleus Phox2b-Expressing NeuronsIn VivoProduces Long-Lasting Activation of Breathing in Rats

The retrotrapezoid "nucleus" (RTN), located in the rostral ventrolateral medullary reticular formation, contains a bilateral cluster of approximately 1000 glutamatergic noncatecholaminergic Phox2b-expressing propriobulbar neurons that are activated by CO(2) in vivo and by acidification in vitro. These cells are thought to function as central respiratory chemoreceptors, but this theory still lacks a crucial piece of evidence, namely that stimulating these particular neurons selectively in vivo increases breathing. The present study performed in anesthetized rats seeks to test whether this expectation is correct. We injected into the left RTN a lentivirus that expresses the light-activated cationic channel ChR2 (channelrhodopsin-2) (H134R mutation; fused to the fluorescent protein mCherry) under the control of the Phox2-responsive promoter PRSx8. Transgene expression was restricted to 423 +/- 38 Phox2b-expressing neurons per rat consisting of noncatecholaminergic and C1 adrenergic neurons (3:2 ratio). Photostimulation delivered to the RTN region in vivo via a fiberoptic activated the CO(2)-sensitive neurons vigorously, produced a long-lasting (t(1/2) = 11 s) increase in phrenic nerve activity, and caused a small and short-lasting cardiovascular stimulation. Selective lesions of the C1 cells eliminated the cardiovascular response but left the respiratory stimulation intact. In rats with C1 cell lesions, the mCherry-labeled axon terminals originating from the transfected noncatecholaminergic neurons were present exclusively in the lower brainstem regions that contain the respiratory pattern generator. These results provide strong evidence that the Phox2b-expressing noncatecholaminergic neurons of the RTN region function as central respiratory chemoreceptors.

Spinal NMDA receptor activation is necessary for de novo, but not the maintenance of, A2a receptor-mediated phrenic motor facilitation

Adenosine 2a (A2a) receptor agonists elicit persistent increases in phrenic nerve activity by transactivating the neurotrophin receptor, TrkB, near phrenic motoneurons. Our working model proposes that A2a receptor-mediated TrkB receptor activation strengthens glutamatergic synapses onto phrenic motoneurons. Activation of glutamate N-methyl d-aspartate (NMDA) receptors has been implicated in other models of phrenic motor plasticity. Thus we hypothesized that NMDA receptor activation also would contribute to A2a receptor-mediated phrenic motor facilitation. Adult male Sprague-Dawley rats were anesthetized with urethane, mechanically ventilated, neuromuscularly paralyzed, and bilaterally vagotomized. The A2a receptor agonist CGS-21680 and the NMDA receptor-channel blocker MK-801 were administered intrathecally over the C4 spinal segment. Phrenic nerve activity was recorded before, during, and after drug administration. MK-801 (concentration range 0.1, 1.0, 10.0, and 100 microM) was administered 30 min before CGS-21680 (50 microM). MK-801 dose-dependently blocked A2a receptor-mediated phrenic motor facilitation. When administered at 60 min post-CGS-21680, MK-801 prevented further increases in phrenic nerve activity compared with the CGS-21680 alone (CGS-21680 alone at 120 min: 114 +/- 19%; CGS-21680 and MK-801 at 60 min post-CGS-21680: 61 +/- 11%, above baseline, P < 0.05) but did not return phrenic motor output to baseline values. Our data suggest that NMDA receptor activation is necessary for de novo A2a receptor-mediated phrenic motor facilitation and that the maintenance of preexisting phrenic motor facilitation does not involve NMDA receptor-dependent mechanisms.

Benzodiazepine-sensitive GABA A receptors in the commissural subnucleus of the NTS are involved in the carotid chemoreceptor reflex in rats

We studied the role of benzodiazepine (BDZ) receptors in the commissural subnucleus of the nucleus tractus solitarius (commNTS) in chemoreceptor reflex in urethane-anesthetized, pancronium-immobilized, artificially ventilated and bilaterally vagotomized rats. A BDZ agonist, diazepam (1–4 μmol/kg), administered intravenously reduced resting phrenic nerve activity (PNA) and blood pressure (BP). Stimulation of carotid chemoreceptors induced an increase in PNA and an increase in BP. Diazepam inhibited this chemoreceptor reflex. The effects of intravenous injection of diazepam (4 μmol/kg) on the chemoreceptor reflex were antagonized by microinjection of the BDZ antagonist flumazenil (100 pmol) into the commNTS. Microinjection of flumazenil (100 pmol) alone had no effect on the basal PNA and BP, and the chemoreceptor reflex. These results suggest that BDZ receptors are present in the carotid chemoreceptor reflex pathway in the commNTS and potentiate GABA A transmission.

ATP is a key mediator of central and peripheral chemosensory transduction.

Recent evidence suggests that ATP is a mediator of central (within the ventral surface of the medulla) and peripheral (within the carotid body) chemosensory transduction. This short review discusses the data obtained in experiments in vivo and in vitro supporting this hypothesis. P2 receptors for ATP are expressed within the ventrolateral medulla as well as by the peripheral chemosensory afferent neurones. Blockade of P2 receptors in the ventrolateral medulla attenuates the CO2-induced increase in respiration while blockade of purinergic signalling impairs carotid body function and diminishes the ventilatory response to hypoxia. Furthermore, ATP is released from the ventral surface of the medulla during hypercapnia and from the carotid body during hypoxia. Finally, exogenous ATP applied on the ventral surface of the medulla evokes rapid increase in phrenic nerve activity, while ATP applied to the carotid body evokes marked excitation of the carotid sinus nerve afferents. We suggest that in the ventrolateral medulla ATP is produced following CO2/H(+)-induced activation of central chemosensory elements (neuronal and/or glial) and acts within the respiratory network to produce physiologically relevant changes in ventilation. In the carotid body, ATP contributes in a significant manner to the transmission of the sensitivity of the carotid body to changes in arterial PO2 and may be considered as a key transmitter released by chemoreceptor cells to activate endings of the sinus nerve afferent fibres.

CO 2-induced prolongation of expiratory time during early development

In these studies, we determined the contribution of central mechanisms and the role of GABA A-receptor signal transduction pathways in mediating hypercapnia-induced slowing of breathing frequency. Experiments were performed in decerebrate, vagotomized, paralyzed and mechanically ventilated piglets of 3–5 days and 2–3 weeks of age ( n=19). Repeated exposure to progressive hyperoxic hypercapnia induced a reproducible increase in phrenic nerve activity, accompanied by a CO 2 concentration-dependent increase in expiratory duration. No differences were observed in piglets with intact or cut carotid sinus nerves. Intravenous administration of bicuculline (2 mg/kg; n=7), a γ-aminobutyric acid (GABA A) receptor antagonist, significantly reduced the CO 2-induced prolongation of Te. These data demonstrate for the first time that in early postnatal life, hypercapnia induced increase in phrenic activity is associated with centrally mediated prolongation of expiratory duration. Furthermore, the results suggest that brainstem GABAergic mechanisms play an important role in CO 2-induced prolongation of expiratory time during early development.

This Month in the Journal

This Month in the Journal

Changes in outflow to respiratory pump muscles produced by natural vestibular stimulation.

1. Activity was recorded from abdominal (expiratory) and phrenic (inspiratory) nerves during natural vestibular stimulation in multiple vertical planes and the horizontal plane in decerebrate cats. Vestibular stimulation was produced by rotating the head in animals whose upper cervical dorsal roots were transected to remove inputs from neck receptors; the upper airway and carotid sinus were denervated, and the vagus nerves were transected to assure that the head rotations did not elicit visceral or pulmonary inputs. 2. The plane of head rotation that produced maximal modulation of respiratory nerve activity (response vector orientation) was measured at one or more frequencies between 0.05 and 0.5 Hz. The dynamics of the response were then studied with sinusoidal (0.05-2 Hz) stimuli aligned with this orientation. In some animals, sinusoidal horizontal rotations of the head at 0.5 and 1 Hz or static head tilts in the pitch and roll planes were also delivered. 3. Typically, maximal modulation of abdominal nerve outflow was elicited by head rotations in a plane near pitch; nose-up rotations produced increased outflow, and nose-down rotations reduced nerve discharges. The gains of the responses (relative to stimulus position) remained relatively constant across stimulus frequencies, and the phases were consistently near stimulus position, like regularly firing otolith afferents. Static nose-up tilt produced elevated abdominal nerve activity throughout the stimulus period, providing further evidence that pitch-sensitive otolith receptors contribute to the response. Horizontal head rotations had little influence on abdominal nerve discharges. 4. The abdominal nerve responses to head rotation were abolished by chemical or aspiration lesions of the medial and inferior vestibular nuclei, which is concordant with the responses resulting from activation of vestibular receptors. Transections of axons arising from bulbospinal neurons in the ventral respiratory group, which are known to be the predominant source of expiratory signals to the spinal cord, reduced but did not abolish the vestibuloabdominal reflex. Thus it is likely that nonrespiratory neurons also participate in generating this response. 5. Nose-up pitch of the head; and in particular large (50 degrees) static tilts, produced small increases in phrenic nerve activity. Ear-down tilt and horizontal rotation of the head produced no responses in the phrenic nerve. 6. The existence of vestibular inputs to some respiratory motoneurons suggests that the vestibular system has influences on muscles in addition to those typically considered to have antigravity roles, and participates globally in adjusting muscle activity during movement and changes in posture.