Inspiratory Bursting Research Articles

Characterizing Muscarinic Receptor Subtype Roles in Inspiratory Burst Modulation of Hypoglossal Motoneurons in Mice

Loss of tongue muscle (innervated by hypoglossal (XII) motoneurons) tone is responsible for airway obstruction during sleep. Contributing factors include loss of noradrenergic drive and activation of muscarinic acetylcholine receptors (mAChRs). While noradrenergic effects are well studied, less is known about muscarinic effects. In mice, XII motoneurons express four mAChRs; M1, M3, and M5 are excitatory while M2 is inhibitory. We hypothesize that M2 is upregulated with postnatal maturation whereas M1, M3, and M5 are downregulated. We therefore expect that activation of M1, M3, and M5 receptors will potentiate inspiratory bursting early in postnatal development whereas activation of M2 receptors will inhibit inspiratory bursting later in postnatal development. To test this, we utilized rhythmic medullary slice preparations from mice at postnatal days 0-5 (P0-5) and P9-14, which contained preBötzinger Complex (site of inspiratory rhythm generation) and XII motoneurons. Blocking M1 receptors locally with pirenzepine (10 μM & 100 μM) decreased the muscarinic potentiation of inspiratory bursting in P0-5 mice at 100 μM (168±23% at 10 μM and 129±16% at 100 μM in muscarine [p<0.05] vs 183±34% muscarine (100 μM) control response, n=9). Activating M1 receptors locally with cevimeline (1 mM) for 30s or 60s did not mediate potentiation of inspiratory bursting as seen with muscarine (116±7% at 30s and 117±7% at 60s vs 181±40% control muscarine response [n=6, p<0.05]). Bath application of M2 antagonist methoctramine (MTH) in P0-5 mice at 1 μM had no effect on muscarine-potentiation of inspiratory bursting (207±109% at 1 μM MTH in muscarine vs 212±84% muscarine control response, n=9). Preliminary analysis of the P9-14 age group using 1 μM MTH suggests there may be an increase in muscarine-mediated potentiation of inspiratory bursting with M2 blockade (170±56% at 1 μM MTH in muscarine vs 152±56% muscarine control response, n=6, p>0.5). Bath application of M3 receptor antagonist 4-DAMP in P0-5 mice at 10 nM had no effect on muscarine-potentiated inspiratory bursting (172±12% vs 178±30% muscarine control), whereas application at 100 nM decreased muscarine-mediated inspiratory burst potentiation compared to control (133±12%, n=4, p<0.05). However, bath application with another M3 receptor antagonist, J Fumarate 104129, had no significant effect on muscarinic modulation of inspiratory bursting in P0-5 mice (188±46% at 0.6 nM [n=6], 185±32% at 6 nM [n=6], and 144±24% at 60 nM [n=4] vs 176±34% muscarine control [n=6]). Overall, our data partially supports a role of M1 receptors contributing to muscarinic potentiation of inspiratory bursting in XII motoneurons, with the contributions of M2 and M3 appearing insignificant early in postnatal life. Preliminary analysis suggests that M2 has a larger contribution later in maturation. Future research will evaluate the contribution of muscarinic receptor subtypes to inspiratory burst modulation later in postnatal life. NIH/NHLBI Funding: R15HL148870. 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.

Contribution of Ih to Postnatal Maturation of Muscarinic Modulation of Inspiratory Bursting at Hypoglossal Motoneurons in Neonatal Mice

Activation of hypoglossal motoneurons (XII MNs) contributes to stiffening of the upper airway during inspiration. The magnitude of this excitation is decreased during sleep, likely due to a combination of withdrawal of noradrenaline and acetylcholine release at XII MNs. Acetylcholine acts at muscarinic acetylcholine receptors (mAChRs) and surprisingly mAChR activation has an excitatory effect on inspiratory bursting in neonatal preparations, which becomes inhibitory in adult preparations. mAChRs modulate several ion channels and characterizing muscarinic effect changes at the XII nucleus will help to explain the mechanism of the inhibitory shift over postnatal maturation. The hyperpolarization activated cyclic nucleotide gated (HCN) channel is positively modulated by mAChRs. HCN channels give rise to Ih, a mixed cation current activated at hyperpolarized membrane potentials that contribute to membrane depolarization. Ihincreases dramatically with postnatal maturation in XII MNs. Thus, we hypothesize that 1) the effects of muscarinic modulation of Ih increases with postnatal maturation, and 2) that muscarinic activation of Ih contributes to the net inhibitory component of muscarinic modulation of inspiratory bursting at XII motoneurons. To test our hypotheses, we used the rhythmic medullary slice preparation to test the functional effects of muscarinic modulation of Ih on inspiratory bursting in neonatal CD1 mice (postnatal day, P0-P4) in combination with pharmacological block of Ih with ZD7288 (25 μM, 150s). Local application of muscarine (100μM, 30s) in the XII motor nucleus increased inspiratory burst amplitude (189 ± 58% of baseline, n = 9). Contrary to our hypothesis, there was no statistically significant difference between inspiratory burst amplitude elicited by muscarine in the presence of ZD7288 (192 ± 47% of baseline, n = 9) versus with the aCSF vehicle control (174 ± 43% of baseline, p = 0.098). We next tested a higher ZD7288 concentration (100 μM) and longer application times (150s, 330s) in neonatal preparations. Preliminary data (n=2) indicate that ZD7288 may increase muscarinic potentiation of inspiratory burst amplitude at XII MNs (muscarinic potentiation (100μM, 30s): control — 112 ±65 % of baseline, aCSF vehicle control - 131 ± 38% of baseline; ZD7288 100μM, 150s - 148 ± 44% of baseline; ZD7288 100μM, 330s - 107 ± 72% of baseline). Future research will elucidate whether the Ih contribution to muscarinic modulation of inspiratory bursting increases with postnatal maturation. 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.

Ampakines increase diaphragm activation following mid-cervical contusion injury in rats

Ampakines are positive allosteric modulators of AMPA receptors. We hypothesized that low-dose ampakine treatment increases diaphragm electromyogram (EMG) activity after mid-cervical contusion injury in rats. Adult male and female Sprague Dawley rats were implanted with in-dwelling bilateral diaphragm EMG electrodes. Rats received a 150 kDyn C4 unilateral contusion (C4Ct). At 4- and 14-days following C4Ct, rats were given an intravenous bolus of ampakine CX717 (5 mg/kg, n = 10) or vehicle (2-hydroxypropyl-beta-cyclodextrin; HPCD; n = 10). Diaphragm EMG was recorded while breathing was assessed using whole-body plethysmography. At 4-days, ampakine administration caused an immediate and sustained increase in bilateral peak inspiratory diaphragm EMG bursting and ventilation. The vehicle had no impact on EMG bursting. CX717 treated rats were able to increase EMG activity during a respiratory challenge to a greater extent vs. vehicle treated. Rats showed a considerable degree of spontaneous recovery of EMG bursting by 14 days, and the impact of CX717 delivery was blunted as compared to 4-days. Direct recordings from the phrenic nerve at 21–24 days following C4Ct confirmed that ampakines stimulated bilateral phrenic neural output in injured rats. We conclude that low-dose intravenous treatment with a low-impact ampakine can enhance diaphragm activation shortly following mid-cervical contusion injury, when deficits in diaphragm activation are prominent.

A muscarinic, GIRK channel-mediated inhibition of inspiratory-related XII nerve motor output emerges in early postnatal development in mice.

In neonatal rhythmic medullary slices, muscarinic acetylcholine receptor (mAChR) activation of hypoglossal (XII) motoneurons that innervate the tongue has a net excitatory effect on XII inspiratory motor output. Conversely, during rapid eye movement sleep in adult rodents, XII motoneurons experience a loss of excitability partly due to activation of mAChRs. This may be mediated by activation of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Therefore, this study was designed to evaluate whether muscarinic modulation of XII inspiratory motor output in mouse rhythmic medullary slices includes GIRK channel-mediated inhibition and, if so, when this inhibitory mechanism emerges. Local pressure injection of the mAChR agonist muscarine potentiated inspiratory bursting by 150 ± 28% in postnatal day (P)0-P5 rhythmic medullary slice preparations. In the absence of muscarine, pharmacological GIRK channel block by Tertiapin-Q did not affect inspiratory burst parameters, whereas activation with ML297 decreased inspiratory burst area. Blocking GIRK channels by local preapplication of Tertiapin-Q revealed a developmental change in muscarinic modulation of inspiratory bursting. In P0-P2 rhythmic medullary slices, Tertiapin-Q preapplication had no significant effect on muscarinic potentiation of inspiratory bursting (a negligible 6% decrease). However, preapplication of Tertiapin-Q to P3-P5 rhythmic medullary slices caused a 19% increase in muscarinic potentiation of XII inspiratory burst amplitude. Immunofluorescence experiments revealed expression of GIRK 1 and 2 subunits and M1, M2, M3, and M5 mAChRs from P0 to P5. Overall, these data support that mechanisms underlying muscarinic modulation of inspiratory burst activity change postnatally and that potent GIRK-mediated inhibition described in adults emerges early in postnatal life.NEW & NOTEWORTHY Muscarinic modulation of inspiratory bursting at hypoglossal motoneurons has a net excitatory effect in neonatal rhythmic medullary slice preparations and a net inhibitory effect in adult animals. We demonstrate that muscarinic modulation of inspiratory bursting undergoes maturational changes from postnatal days 0 to 5 that include emergence of an inhibitory component mediated by G-protein-coupled inwardly rectifying potassium channels after postnatal day 3 in neonatal mouse rhythmic medullary slice preparations.

Modulatory effect of trans-spinal magnetic intermittent theta burst stimulation on diaphragmatic activity following cervical spinal cord contusion in the rat

Background CONTEXTMagnetic stimulation can noninvasively modulate the neuronal excitability through different stimulatory patterns. PurposeThe present study hypothesized that trans-spinal magnetic stimulation with intermittent theta burst stimulatory pattern can modulate respiratory motor outputs in a pre-clinical rat model of cervical spinal cord injury. Study designIn vivo animal study. MethodsThe effect of trans-spinal magnetic intermittent theta burst stimulation on diaphragmatic activity was assessed in adult rats with unilateral cervical spinal cord contusion at 2 weeks postinjury. ResultsThe results demonstrated that unilateral cervical spinal cord contusion significantly attenuated the inspiratory activity and motor evoked potential of the diaphragm. Trans-spinal magnetic intermittent theta burst stimulation significantly increased the inspiratory activity of the diaphragm in cervical spinal cord contused rats. Inspiratory bursting was also recruited by trans-spinal magnetic intermittent theta burst stimulation in the rats without diaphragmatic activity after cervical spinal cord injury. In addition, trans-spinal magnetic intermittent theta burst stimulation is associated with increases in oxygen consumption and carbon dioxide production. ConclusionsThese results suggest that trans-spinal magnetic intermittent theta burst stimulation can induce respiratory neuroplasticity. Clinical significanceWe propose that trans-spinal theta burst magnetic stimulation may be considered a potential rehabilitative strategy for improving the respiratory activity after cervical spinal cord injury. This will require future clinical study.

Arginine vasopressin potentiates inspiratory bursting in hypoglossal motoneurons of neonatal mice

Vasopressin (AVP) acts as a neurotransmitter and its activity can potentiate respiratory activity. Hypoglossal (XII) motoneurons that innervate the tongue express V1a vasopressin receptors, which are excitatory. Therefore, we hypothesized that V1a receptor activation at XII motoneurons would potentiate inspiratory bursting. We developed this study to determine whether AVP can potentiate inspiratory bursting in rhythmic medullary slice preparations in neonatal (postnatal, P0–5) mice. Bath or local application of AVP potentiated inspiratory bursting compared to baseline XII inspiratory burst amplitude. Antagonizing V1a receptors revealed significant attenuation of the AVP-mediated potentiation of inspiratory bursting, while antagonism of oxytocin receptors (at which AVP has similar binding affinity) revealed a trend to attenuate AVP-mediated potentiation of inspiratory bursting. Finally, we discovered that the AVP-mediated potentiation of inspiratory bursting increases significantly with postnatal maturation from P0–5. Overall, these data support that AVP potentiates inspiratory bursting directly at XII motoneurons.

Neuronal muscarinic acetylcholine receptor subtype modulation of inspiratory bursting at hypoglossal motoneurons in developing neonatal mice in vitro

Muscarinic acetylcholine receptor modulation contributes to changes in hypoglossal motoneuron excitability through pre- and post-synaptic effects, an important mechanism for regulation of airway tone during sleep. Previous data indicate a net excitatory effect of muscarinic modulation of inspiratory bursting in neonatal mouse brainstem slices, yet a net inhibitory effect on inspiratory bursting in adult rats. We tested the hypothesis that a developmental shift in muscarinic modulation impacts hypoglossal motoneurons, using neuroanatomical and electrophysiological methods. Immunofluorescence confirmed hypoglossal motoneurons express M1, M2, M3, and M5 muscarinic subtypes. We observed maturational decreases in M1 and M5 labeling intensity, M3 labeling intensity generally decreased with postnatal (P) age, although there was a transient increase in expression at P17. M2 labeling intensity did not change, although distribution of M2 labeling with postnatal maturation may change. Next, using the in-vitro rhythmic slice preparation from CD1 mice of either sex, local application of muscarine (100 μM) into the hypoglossal nucleus was tested in mice P0-14. The net effect of applying muscarine results in excitatory output after local application into the rhythmic slice because each data point is a positive percent change from control. The magnitude of this potentiation appears to lessen as the mice age toward P14. To evaluate the mechanisms through which the magnitude of muscarinic potentiation of inspiratory bursting changes with postnatal maturation, we next assessed the role of excitatory and inhibitory muscarinic receptor subtypes. Blocking M1 locally (pirenzepine, 100 μM) significantly decreased muscarine-mediated potentiation of inspiratory bursting (to 72±14% of control muscarine response, n=9, P0-5). Preliminary data indicate bath application of 4-DAMP (M3 antagonist) attenuated muscarinic potentiation of inspiratory bursting (range: to 7%-25% of control burst response, n=3, P0-5). Modulating M5 locally with a positive allosteric modulator VU 0238429 (1000 μM) had little effect on burst amplitude (119±4% of control burst response, n=3, P0-6). Blocking M2 locally (methoctramine, 2μM) showed no effect on inspiratory burst potentiation after applying muscarine (181±65% vs 187±69%, n=7, P0-5). Bath application of methoctramine did not have a significant effect on inspiratory burst potentiation by muscarine (179±65% vs control 182±90%, n=10) in P0-5 mice while preliminary analysis suggests an increasing effect of blocking M2 with postnatal maturation (147±56% vs control 134±30%, n=4, P9-14). These data support excitatory muscarinic receptor subtypes contributing to the excitatory response early in postnatal maturation. Based on our neuroanatomical data, we speculate the increasing inhibitory response to muscarinic modulation with postnatal maturation is due to a decreased contribution of excitatory muscarinic receptor subtypes. NIH R15HL148870 (Revill) and NIH/NHLBI R25 HL126140 (Moreno, Garcia, Parthasarathy) 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.

Muscarinic modulation increases excitability inspiratory and non-inspiratory hypoglossal motoneurons in mice postnatal days 0-5

Hypoglossal motoneurons (XII MNs) innervate the tongue and their activity normally contributes to stiffening of the airway to maintain airway patency. During sleep, there is normal reduction in airway tone. The specific mechanisms that contribute to decreased airway tone during sleep remain to be completely resolved, although research from adult rodents suggests activation of muscarinic acetylcholine receptors (mAChRs) is an important contributor by inhibiting XII MNs. Conversely, data from neonatal rodents, have shown that muscarinic modulation leads to an overall excitatory effect in XII MNs. Therefore, the overarching objective of this research project is to uncover the mechanisms through which muscarinic modulation can influence the excitability of XII MNs across postnatal maturation. Rhythmic medullary slices from neonatal mice (postnatal day 0-5) were used for whole cell electrophysiology experiments to test the effects of activating mAChRs at XII MNs in both non-inspiratory and inspiratory XII MNs. Data are reported as mean±SD. We hypothesized that mAChR activation at XII MNs would increase excitability of non-inspiratory and inspiratory XII MNs, and potentiate inspiratory bursting in inspiratory XII MNs. Starting at -60 mV, muscarinic modulation depolarized non-inspiratory and inspiratory XII MNs similarly (3.6±2.6 vs 4.6±2.1 mV, n=12, p>0.05), and had no effect on input resistance (CTL vs Musc; non-insp: 120±23 vs 128±33 MΩ; inspiratory: 140±28 vs 137±32 MΩ; n =12; p>0.05). Muscarinic modulation can increase the hyperpolarization-activated cation current (I h ), but we observed no difference in the sag potential in response to -150 (CTL vs Musc; non-insp: 2.3±2.3 vs 3.8±3.0 mV; inspiratory: 2.7±2.8 vs 4.9±2.8 mV; n =12; p > 0.05), -300 (CTL vs Musc; non-insp: 9.0±7.8 vs 11.7±8.0 mV; inspiratory: 9.9±6.4 vs 12.9±7.5 mV; n=12; p>0.05), and -500 pA (CTL vs Musc; non-insp: 21.9±15.0 vs 23.6±14.2 mV; inspiratory: 22.0±9.5 vs 23.5±9.5 mV; n=12; p>0.05) current steps from -45 mV in non-inspiratory compared to inspiratory XII MNs. Muscarinic modulation significantly increased the firing frequency after muscarine application for inspiratory cells with the current step injections 100 pA (CTL vs Musc; 14.7±6.8 vs 21.1±6.4 Hz, n=9 and 10; p<0.05) and 175 pA (CTL vs Musc; 19.5±6.4 vs 25.0±9.4 Hz, n=11 and 8; p<0.05). However, muscarinic modulation did not affect the slope of firing rate/injected current relationship for non-inspiratory and inspiratory XII MNs (CTL vs Musc; non-insp: 0.16±0.12 vs 0.15±0.04 Hz/pA, n=9; inspiratory: 0.12±0.02 vs 0.16±0.07 Hz/pA, n=11 and 12; p>0.05). Finally, muscarinic modulation significantly increased inspiratory burst amplitude (CTL vs Musc; 15.2±3.1 vs 18.3±4.5 mV; n=3; p<0.05), and a trend to increase inspiratory burst area (CTL vs Musc; 4260±779 vs 5354±3063 mV ms, n=3; p=0.08). These data indicated that muscarinic modulation has similar effects on non-inspiratory and inspiratory XII MNs early in postnatal growth. Funding: NIH 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.

Cell types and synchronous-activity patterns of inspiratory neurons in the preBötzinger complex of mouse medullary slices during early postnatal development

To examine whether and how the inspiratory neuronal network in the preBötzinger complex (preBötC) develops during the early postnatal period, we quantified the composition of the population of inspiratory neurons between postnatal day 1 (p1) and p10 by applying calcium imaging to medullary transverse slices in double-transgenic mice expressing fluorescent marker proteins. We found that putative excitatory and glycinergic neurons formed a majority of the population of inspiratory neurons, and the composition rates of these two inspiratory neurons inverted at p5–6. We also found that the activity patterns of these two types of inspiratory neurons became significantly well-synchronized with the inspiratory rhythmic bursting pattern in the preBötC within the first postnatal week. GABAergic and GABA-glycine cotransmitting inspiratory neurons formed only a small population just after birth, which almost disappeared until p10. In conclusion, the inspiratory neuronal network in the preBötC matures at the level of both neuronal population and neuronal activities during early postnatal development.

Neuropathology of distinct diaphragm areas following mid-cervical spinal cord contusion in the rat

BACKGROUNDThe diaphragm is innervated by phrenic motoneurons distributed from the third to fifth cervical spinal cord. The rostral to caudal phrenic motoneuron pool segmentally innervates the ventral, medial, and dorsal diaphragm. PURPOSEThe present study was designed to investigate the physiological and transcriptomic mechanism of neuropathology of distinct diaphragm areas following mid-cervical spinal cord injury. STUDY DESIGNIn vivo animal study. METHODSElectromyograms and transcriptome of the ventral, medial, and dorsal diaphragm were examined in rats that received cervical laminectomy or mid-cervical spinal cord contusion in the acute (ie, 1–3 days) or subchronic (ie, ∼14 days) injury stages. RESULTSMid-cervical spinal cord contusion significantly attenuated the inspiratory bursting amplitude of the dorsal diaphragm but not the ventral or medial diaphragm. Moreover, the discharge onset of the dorsal diaphragm was significantly delayed compared with that of the ventral and medial diaphragm in contused rats. Transcriptomic analysis revealed a robust change in gene expression in the ventral diaphragm compared with that in the dorsal diaphragm. Specifically, enrichment analysis of differentially expressed genes demonstrated that the cell cycle and immune response were significantly upregulated, whereas several metabolic pathways were downregulated, in the ventral diaphragm of acutely contused rats. However, no significant Kyoto Encyclopedia of Genes and Genomes pathway was altered in the dorsal diaphragm. CONCLUSIONSThese results suggest that mid-cervical spinal cord injury has different impacts on the physiological and transcriptomic responses of distinct diaphragm areas. CLINICAL SIGNIFICANCEFuture therapeutic strategies can consider applying different therapies to distinct diaphragm areas following cervical spinal cord injury. Additionally, confirmation of activities across different diaphragm areas may provide a critical reference for the placement of diaphragmatic pacing electrodes.

Characterizing the Contribution of Muscarinic Receptor Subtypes to Inspiratory Bursting Activity at Hypoglossal Motor Neurons of CD1 Mice

Obstructive sleep apnea (OSA) is characterized by cessation of breathing during sleep, due to repetitive episodic collapse of the airway or increased airway resistance. This loss of airway patency during sleeping conditions is controlled in part by the tongue, innervated by the hypoglossal (XII) motor nucleus. Decreased excitability of XII motor neurons (MNs) that innervate the tongue may occur due to activation of muscarinic acetylcholine receptors acting through G‐protein coupled signal transduction pathways. The five types of muscarinic acetylcholine receptors can be broadly categorized as excitatory or inhibitory. M1, M3, and M5 receptors are excitatory receptors coupled to Gq, whereas M2 and M4 receptors are inhibitory receptors coupled to Gi. XII MNs express M1, M2, M3, and M5 subtypes of muscarinic receptors, but the specific mechanisms of how cholinergic signaling modulates excitability of XII MNs are not well understood. The objective of this study was to investigate how the muscarinic receptor subtypes individually contribute to inspiratory activity at XII MNs. We tested the hypothesis that activation of M1 and M5 receptors potentiate, whereas activation of M2 receptors inhibit inspiratory bursting in XII MNs. Using medullary rhythmic slice preparations from neonatal CD1 mice (postnatal day P0‐5) local application of muscarine (30s, 100 μM) to the XII motor nucleus increased inspiratory burst amplitude to 180 ± 7% [n = 23]. Blocking M1 receptors locally with pirenzepine (10 μM & 100 μM) decreased the muscarine‐mediated potentiation of inspiratory bursting to 94 ± 15% and 72 ± 14% of control muscarine response [n = 9]. Activating M1 receptors locally with cevimeline (1 mM) for 30s and 60s increased burst amplitude to 116 ± 7% and 117 ± 7% of control muscarine response [n = 6]. Blocking M2 receptors locally with AF‐DX 116 (1 μM & 10 μM), the muscarine‐mediated effect was 101 ± 10% [n = 3] and 95 ± 10% [n = 3] of control muscarine‐mediated excitation. M2 receptor blockade was also tested by application of the M2 receptor antagonist, methoctramine (MTH). Local application of MTH (2 μM) had no effect on muscarine‐mediated potentiation of inspiratory bursting (171 ± 15% compared to control muscarine response of 177 ± 14% [n = 10]). Similarly, blocking M2 receptors with bath application of MTH (200 nM) had no effect on muscarine‐mediated potentiation of inspiratory bursting (166 ± 20% compared to 161 ± 14% control muscarine response) or on inspiratory burst period (10 ± 0.3% compared to 10 ± 0.4% control muscarine response) [n = 5]. Modulating M5 receptors locally with VU 0238429 (20 μM and 1000 μM) increased burst amplitude to 116% ± 5% and 119 ± 4% of control muscarine‐mediated excitation [n = 3]. Our data partially support a role of M1 receptors contributing to the muscarinic potentiation of inspiratory bursting in XII MNs. Future research will evaluate other muscarinic receptor agonists and antagonists, as well as determine how the muscarinic modulation of inspiratory bursting changes with postnatal maturation.

Chemogenetic Activation of Hypoglossal Motoneurons in Neuromuscular Disease

The tongue is essential for breathing, swallowing, mastication, speech, coughing, and suckling. Many conditions and neuromuscular diseases are associated with lingual dysfunction, making it a priority to develop treatments which can improve tongue motor function. Our laboratory established that intralingual delivery of adeno‐associated virus serotype 9 (AAV9) encoding a particular gene can drive expression of that gene in hypoglossal motoneurons (XII MNs). This method can also drive expression of designer receptors exclusively activated by designer drugs (DREADDs) in XII MNs (Fleury‐Curado et al, 2021). Here we tested if intralingual delivery of AAV9‐hSyn‐hM3(Gq)‐mCherry could drive functional DREADD expression in XII MNs in a mouse model exhibiting tongue neuromuscular pathology. Specifically, we studied a mouse model of Pompe disease (Gaa‐/‐). The prevalence of Pompe disease is estimated to be 1 per 8,700 live births and is associated with macroglossia, dysphagia, dysarthria, sleep disordered breathing, and XII MN pathology. Intralingual injections of AAV9‐hSyn‐hM3(Gq)‐mCherry (2.4 x 1011 vg) or saline (sham) were administered to the base of tongue in anesthetized Pompe mice (AAV9‐DREADD n=11, saline n=4) or wild type (C57BL//6J mice (AAV9‐DREADD, n=5) at 4‐6 weeks of age. Lingual EMG activity was measured during spontaneous breathing under isoflurane anesthesia 12 weeks later. Tonic and phasic inspiratory EMG responses to intraperitoneal injection of saline (i.e., vehicle) or the DREADD ligand JHU37160‐dihydrochloride (J60) were assessed. Saline produced no significant changes in lingual EMG measures. All Pompe mice studied responded to J60 with an increase in tonic lingual EMG activity (time to response, TTR = 219±68 seconds; 1034±350% baseline at 30 min). Only 1 of 5 mice showed discernable increase in tonic EMG output after J60. The DREADD ligand produced an increase in phasic inspiratory bursting in all Pompe mice (TTR = 197±78 seconds; 506±248% baseline at 30 min). The increase in phasic bursting also occurred in all WT mice (TTR = 161±22 seconds; 512±151% baseline at 30 min). The DREADD ligand also caused a decrease in respiratory rate (bursts per minute) that was more pronounced in Pompe mice (79±5% baseline at 30 min post‐J60) compared to WT mice (91±12% baseline at 30 min post‐J60. Histological evaluation of the medulla confirmed expression of the mCherry fluorophore in XII MNs in all animals that received intralingual injection of AAV9‐hSyn‐hM3(Gq)‐mCherry. We conclude that intralingual delivery of an AAV9 vector encoding a DREADD can drive functional expression of DREADDs in XII MNs in a mouse model of Pompe disease that displays lingual myofiber atrophy and XII MN pathology. Thus, the ongoing neuromuscular pathology did not prevent the efficacy of chemogenetic tongue muscle activation. Indeed, the DREADD ligand caused a robust increase in both tonic and phasic lingual EMG activity in Pompe mice. The tonic EMG response appears to be more consistent in Pompe vs WT mice.

Optogenetic activation of the diaphragm

Impaired diaphragm activation is common in many neuromuscular diseases. We hypothesized that expressing photoreceptors in diaphragm myofibers would enable light stimulation to evoke functional diaphragm activity, similar to endogenous bursts. In a mouse model, adeno-associated virus (AAV) encoding channelrhodopsin-2 (AAV9-CAG-ChR2-mVenus, 6.12 × 1011 vg dose) was delivered to the diaphragm using a minimally invasive method of microinjection to the intrapleural space. At 8–18 weeks following AAV injection, mice were anesthetized and studied during spontaneous breathing. We first showed that diaphragm electromyographic (EMG) potentials could be evoked with brief presentations of light, using a 473 nm high intensity LED. Evoked potential amplitude increased with intensity or duration of the light pulse. We next showed that in a paralyzed diaphragm, trains of light pulses evoked diaphragm EMG activity which resembled endogenous bursting, and this was sufficient to generate respiratory airflow. Light-evoked diaphragm EMG bursts showed no diminution after up to one hour of stimulation. Histological evaluation confirmed transgene expression in diaphragm myofibers. We conclude that intrapleural delivery of AAV9 can drive expression of ChR2 in the diaphragm and subsequent photostimulation can evoke graded compound diaphragm EMG activity similar to endogenous inspiratory bursting.

The Role of Arginine Vasopressin in Modulating Inspiratory Bursting Behavior in the Hypoglossal Motor Nucleus in Neonatal Mice

Arginine vasopressin (AVP) is classically known as a hormone, but it also acts as a neurotransmitter that binds V1a receptors in the central nervous system. V1a receptors are expressed at high levels early in postnatal life in cranial and spinal motoneurons. While AVP receptor expression levels decrease in some cranial motoneuron populations with increasing developmental age, AVP receptor mRNA levels are expressed at high levels in the hypoglossal (XII) motor nucleus in adult mice. Recordings from individual XII motoneurons demonstrated that AVP has excitatory effects on young XII motoneurons, although the signaling pathways and effector ion channels remain to be fully elucidated. Additionally, previous research demonstrated that GABAergic disinhibition within the paraventricular nucleus of adult male rats, which has the effect of promoting AVP release, led to stimulation of breathing and increased diaphragm and genioglossus (innervated by XII, primary tongue protruder) muscle activity. Whether AVP acts directly at XII motoneurons to potentiate inspiratory bursting is unknown. Our aim was to characterize the effects of AVP on inspiratory bursting behavior at XII motoneurons. To test our hypothesis that AVP will potentiate inspiratory bursting behavior, we utilized an in vitro medullary brainstem slice (600 µM) preparation of neonatal (postnatal day, P0-5) CD1 mouse brains of either sex that contains the nuclei for respiratory movement of the tongue. By superfusing the slice with warmed artificial cerebral spinal fluid, we were able to record inspiratory bursting activity using a suction electrode under baseline conditions and in response to local drug application. Local injections of AVP at 0.01 µM had little effect on inspiratory bursting amplitude (111% ± 3% of baseline, n=6), but at 0.1 µM or 1 µM, initial results suggest it increased inspiratory burst amplitude (134% ± 5% of baseline, n=13 and 130% ± 8% of baseline, n=7 respectively, p = 0.0889). The excitatory effect of AVP was significantly diminished using a V1a receptor antagonist, d(CH2)51Tyr(Me)2Arg8) Vasopressin (500nM) (Initial AVP increase in burst amplitude: 141% ± 8%; AVP after antagonist: 113% ± 4%; AVP after washout: 120% ± 3%, n=10, p = 0.0437). Vasopressin and oxytocin are closely related nonapeptides, and since the XII motor nucleus also expresses oxytocin receptors, we next tested whether the AVP effects could be attributed to activation of the oxytocin receptor. Our preliminary results suggest that the oxytocin receptor antagonist, L-371,257 (50nM), did attenuate the response of AVP (Initial AVP increase in burst amplitude: 144% ± 11%; AVP after antagonist: 114% ± 5%; AVP after Washout: 122% ± 6%, n=8, p = 0.0901). Lastly, we discovered that vasopressin-related potentiation of inspiratory burst amplitude has an age-dependent increase from P0-5 (regression fit: y=13.933x+12.401, R2=0.4411, p=0.000402). Our results indicate that vasopressin, acting at V1a, and possibly oxytocin, receptors, can potentiate XII inspiratory bursting behavior in neonatal mice. Whether vasopressin has a role in maintaining airway patency in vivo in adult animals will need to be addressed in future studies.

Cholinergic Modulation by Activation of Muscarinic Acetylcholine Receptor Subtypes Modulates Inspiratory Bursting in Hypoglossal Motoneurons in Mice in Vitro

During wakefulness, tonic and respiratory input provides drive to upper airway dilatory muscles, and in particular genioglossus, the main tongue protruder, which stiffens the airway and keeps airway resistance low. These respiratory muscles experience decrements in activity during rapid eye movement sleep. Disruption in motor output and insufficient muscle tone of the tongue due to inhibition, or loss of excitation, of hypoglossal motoneurons (which innervate tongue muscles) contributes to sleep-disordered breathing. Recent data from adult rats in vivo suggest that muscarinic inhibition of hypoglossal motoneurons may be a major mechanism leading to loss of tongue muscle tone during sleep. However, available data indicate that there are contrasting effects of muscarinic modulation: in neonatal rhythmic slice preparations, muscarine applied within the hypoglossal nucleus has an excitatory effect on inspiratory burst amplitude. Muscarinic receptor subtypes can be broadly divided into those that are coupled to excitatory G-protein coupled receptors (M1, M3, M5) and those that are coupled to inhibitory G-protein coupled receptors (M2, M4). Hypoglossal motoneurons express M1, M2, M5, and possibly M3 receptor subtypes. Many modulatory systems undergo postnatal maturation, which we hypothesize underlies the switch in muscarinic modulation from net excitation to net inhibition of inspiratory bursting behavior at hypoglossal motoneurons. As an initial test of our hypothesis, our goal was to determine the contribution of excitatory and inhibitory muscarine receptor subtypes to the muscarinic modulation of inspiratory burst output from hypoglossal motoneurons in neonatal mice. Using the in vitro rhythmic slice preparation, we first investigated inspiratory behavior by observing the net effects of muscarine locally applied into the hypoglossal nucleus (100 mM, 30 s) on inspiratory burst amplitude in neonatal CD-1 mice (postnatal day, P0-P5). Our data confirm that in mice ages P0-5, local application of muscarine increased inspiratory burst amplitude (range: 53-180% potentiation, n = 9) compared to control inspiratory burst activity. Preliminary results suggest that the local application of M2 antagonist methoctramine in addition to muscarine (range: 20%-135% potentiation, n=4) had little effect on inspiratory behavior compared to baseline effects of muscarine (range: 4%-143% potentiation). Next, we tested the contribution of M3 receptors to muscarinic modulation on inspiratory burst amplitude. At baseline, locally applied muscarine increased inspiratory burst amplitude (range: 20%-63% potentiation); the muscarinic potentiation was attenuated during bath application of the M3 receptor-preferring antagonist 4-DAMP (range: 17%-25% potentiation) and recovered upon washout (range: 13%-117% potentiation, n=3). These preliminary results indicate muscarinic excitation is mediated in part by the M3-receptor subtype. Future experiments will determine how the M2 and M3 receptor contributions change with postnatal maturation.

The role of glycinergic inhibition in respiratory pattern formation and cardio-respiratory coupling in rats.

Cardio-respiratory coupling is reflected as respiratory sinus arrhythmia (RSA) and inspiratory-related bursting of sympathetic nerve activity. Inspiratory-related inhibitory and/or postinspiratory-related excitatory drive of cardiac vagal motoneurons (CVMs) can generate RSA. Since respiratory oscillations may depend on synaptic inhibition, we investigated the effects of blocking glycinergic neurotransmission (systemic and local application of the glycine receptor (GlyR) antagonist, strychnine) on the expression of the respiratory motor pattern, RSA and sympatho-respiratory coupling. We recorded heart-rate, phrenic, recurrent laryngeal and thoracic sympathetic nerve activities (PNA, RLNA, t-SNA) in a working-heart-brainstem preparation of rats, and show that systemic strychnine (50–200 ​nM) abolished RSA and triggered a shift of postinspiratory RLNA into inspiration, while t-SNA remained unchanged. Bilateral strychnine microinjection into the ventrolateral medullary area containing CVMs and laryngeal motoneurons (LMNs) of the nucleus ambiguus (NA/CVLM), the nucleus tractus solitarii, pre-Bötzinger Complex, Bötzinger Complex or Kölliker-Fuse nuclei revealed that only NA/CVLM strychnine microinjections mimicked the effects of systemic application. In all other target nuclei, except the Bötzinger Complex, GlyR-blockade attenuated the inspiratory-tachycardia of the RSA to a similar degree while evoking only a modest change in respiratory motor patterning, without changing the timing of postinspiratory-RLNA, or t-SNA. Thus, glycinergic inhibition at the motoneuronal level is involved in the generation of RSA and the separation of inspiratory and postinspiratory bursting of LMNs. Within the distributed ponto-medullary respiratory pre-motor network, local glycinergic inhibition contribute to the modulation of RSA tachycardia, respiratory frequency and phase duration but, surprisingly it had no major role in the mediation of respiratory-sympathetic coupling.

Combination of a Gellan Gum-Based Hydrogel With Cell Therapy for the Treatment of Cervical Spinal Cord Injury.

Cervical spinal cord trauma represents more than half of the spinal cord injury (SCI) cases worldwide. Respiratory compromise, as well as severe limb motor deficits, are among the main consequences of cervical lesions. In the present work, a Gellan Gum (GG)-based hydrogel modified with GRGDS peptide, together with adipose tissue-derived stem/stromal cells (ASCs) and olfactory ensheathing cells (OECs), was used as a therapeutic strategy after a C2 hemisection SCI in rats. Hydrogel or cells alone, and a group without treatment, were also tested. Four weeks after injury, compound muscle action potentials (CMAPs) were performed to assess functional phrenic motor neuron (PhMN) innervation of the diaphragm; no differences were observed amongst groups, confirming that the PhMN pool located between C3 and C5 was not affected by the C2 injury or by the treatments. In the same line, the vast majority of diaphragmatic neuromuscular junctions remained intact. Five weeks post-injury, inspiratory bursting of the affected ipsilateral hemidiaphragm was evaluated through EMG recordings of dorsal, medial and ventral subregions of the muscle. All treatments significantly increased EMG amplitude at the ventral portion in comparison to untreated animals, but only the combinatorial group presented increased EMG amplitude at the medial portion of the hemidiaphragm. No differences were observed in forelimb motor function, neither in markers for axonal regrowth (neuronal tracers), astrogliosis (GFAP) and inflammatory cells (CD68). Moreover, using Von Frey testing of mechanical allodynia, it was possible to find a significant effect of the group combining hydrogel and cells on hypersensitivity; rats with a SCI displayed an increased response of the contralateral forelimb to a normally innocuous mechanical stimulus, but after treatment with the combinatorial therapy this behavior was reverted almost to the levels of uninjured controls. These results suggest that our therapeutic approach may have beneficial effects on both diaphragmatic recovery and sensory function.

Midbrain control of breathing and blood pressure: The role of periaqueductal gray matter and mesencephalic collicular neuronal microcircuit oscillators.

Neural circuitry residing within the medullary ventral respiratory column nuclei and dorsal respiratory group interact with the Kölliker-Fuse and medial parabrachial nuclei to generate the corebreathing rhythm and patternduring resting conditions. Triphasic eupnea consists of inspiratory [I], post-inspiratory [post-I], and late-expiratory [E2] phases. Mesencephalic zones exert modulatory influences upon respiratory rhythm-generating circuitry, sympathetic oscillators, and parasympathetic nuclei. The earliest evidence supporting the existence of midbrain control of breathing derives from studies conducted by Martin and Booker in 1878. These authors demonstrated electrical stimulation of the deep layers of themesencephalic colliculi in the rabbitaugmented ventilation and sequentially elicited chest wall tremors and tetany. Investigations performed during the past several decadeswould demonstrate stimlation of distributed zones within the midbrain reticular formation elicits starkly disparate effects upon respiratory phase switching. Schmid, Böhmer, and Fallert demonstrated electrical stimulation of the nucleus rubreand emanating axon bundles alternately elicits or inhibits the activity of medullary expiratory- or inspiratory-related units and phrenic nerve discharge with differential latency. A series of studies would later indicate the red nucleus mediates hypoxic ventilatory depression. Periaqueductal gray matter neurons exhibit extensive afferent and efferent interconnectivity with suprabulbar, brainstem, and spinal cord zones aptly positioning these units to modulate breathing, autonomic outflow, nociception locomotion, micturtion, and sexual behavior.Experimental stimulatory activation of the tectal colliculi and periaqueductal gray matter via electrical current or glutamate, D,L-homocysteinic acid, or bicuculline microinjections coordinately modulates neuromotor inspiratory bursting frequency and amplitude and discharge of pre-Bötzinger complex, ventrolateral medullary late-I and post-I, and ventrolateral nucleus tractus solitarius decrementing early-I and augmenting and decrementing late-I neurons, elicits expiratory outflow and vocalization, and blunt the Hering-Breuer reflex in unanesthetzed decerebrate and anesthetized preprations of the cat and rat. Stimulation of the mesencephalic colliuli or dorsal divisions of the PAG potently amplifes renal sympathetic neural efferent activity, dynamic arterial pressure magnitude, and myocardial contraction frequency and elicits various behavioral defense responses. Elicited physiological effects exhibit extensive locoregional heterogeneity and variably enlist requisite contributions from the dorsomedial hypothalamus and/or lateral parabrachial nuclei. Stimulation of the dorsal mesencephalon occasionally elicits dynamic increases of arterial pressure magnitude exhibiting prominent oscillatory variability coherent with phrenic nerve discharge, perhaps by generating intra-neuraxial hysteresis, serving to intermittently deliver blood to organ vascular beds under high pressure in order to prevent organ edema, microcirculatory dysfunction, and downregulation of vascular smooth muscle alpha adrenergic receptors. Chemosensitive mesencephalic caudal raphéunits and projections of hypoxia-sensitive units in the caudal hypothalamus to the periaqueductal gray matter may imply the existence of a diencephalo-smesencephalic chemosensitive network modulating breathing and sympathetic discharge.

Characterizing the Action of Arginine Vasopressin at Hypoglossal Motoneurons In Neonatal Mice

Vasopressin, traditionally considered a hormone, also acts as an excitatory neurotransmitter. The effects of vasopressin have been characterized in brainstem motoneurons, including hypoglossal motoneurons (XII MNs). The XII motor nucleus receives inspiratory rhythm from the preBötzinger Complex where it is relayed to the muscles of the tongue via the XII nerve. With some variation across postnatal maturation, the vasopressin V1a receptor gene is expressed from birth to adulthood in the XII motor nucleus, and is enriched in the XII nucleus in adults compared to other cranial motoneuron populations. Previous research demonstrated that vasopressin, acting via V1a receptors on preBötzinger Complex, can stimulate breathing in adult rats by increasing breathing frequency as well as diaphragm and genioglossus (primary tongue protruder) muscle activity. Whether vasopressin can also act directly at XII MNs to potentiate inspiratory bursting has not been well characterized. Thus, the aim of this research was to characterize vasopressin effects on XII MN inspiratory bursting behavior. We generated rhythmic, transverse medullary slices (600μM) from CD1 mice of either sex (postnatal days 0–5), and recorded inspiratory activity from XII nerve rootlets using a suction electrode. We then bath applied arginine vasopressin (0.01μM and 0.1μM, AVP, V1a receptor agonist), or locally injected AVP (0.1μM and 1.0μM). In doing so, we were able to assess the effects of arginine vasopressin on XII inspiratory burst amplitude. Bath applied AVP increased inspiratory burst amplitude by 43.4%±12.5% (n=9) when applied at 0.01μM, and 56.4%±18.1% at 0.1μM. When locally applied (n=7), AVP at 0.1μM increased inspiratory burst amplitude 38.8%±6.2%, and AVP at 1.0μM caused a 26.5%±6.3% increase in inspiratory burst amplitude with a notable increase in tonic activity. In a separate set of experiments (n=8) to test whether AVP acts via V1a receptors, local AVP increased inspiratory burst amplitude by 40.2%±9.3%. Local pre‐application of the V1a receptor antagonist ((d(CH2)51Tyr(Me)2Arg8) Vasopressin, 500nM) for 60 s attenuated the response to AVP (6.7%±14.7% decrease in inspiratory burst amplitude), but the AVP response was recovered after 15 minutes with an increase in XII inspiratory burst amplitude of 22.3%±4.1%. Overall, our results support that AVP can increase XII inspiratory burst amplitude via V1a receptor activation. Further research will need to determine whether AVP plays a role in maintaining airway patency in vivo.Support or Funding InformationKenneth A. Suarez Research Fellowship, Midwestern University

Ampakine pretreatment enables a single brief hypoxic episode to evoke phrenic motor facilitation.

Phrenic long-term facilitation (LTF) is a sustained increase in phrenic motor output occurring after exposure to multiple (but not single) hypoxic episodes. Ampakines are a class of drugs that enhance AMPA receptor function. Ampakines can enhance expression of neuroplasticity, and the phrenic motor system is fundamentally dependent on excitatory glutamatergic currents. Accordingly, we tested the hypothesis that combining ampakine pretreatment with a single brief hypoxic exposure would result in phrenic motor facilitation lasting well beyond the period of hypoxia. Phrenic nerve output was recorded in urethane-anesthetized, ventilated, and vagotomized adult Sprague-Dawley rats. Ampakine CX717 (15 mg/kg iv; n = 8) produced a small increase in phrenic inspiratory burst amplitude and frequency, but values quickly returned to predrug baseline. When CX717 was followed 2 min later by a 5-min exposure to hypoxia (n = 8; ~45 mmHg), a persistent increase in phrenic inspiratory burst amplitude (i.e., phrenic motor facilitation) was observed up to 60 min posthypoxia (103 ± 53% increase from baseline). In contrast, when hypoxia was preceded by vehicle injection (10% 2-hydroxypropyl-β-cyclodextrin; n = 8), inspiratory phrenic bursting was similar to baseline values at 60 min. Additional experiments with another ampakine (CX1739, 15 mg/kg) produced comparable results. We conclude that pairing low-dose ampakine treatment with a single brief hypoxic exposure can evoke sustained phrenic motor facilitation. This targeted approach for enhancing respiratory neuroplasticity may have value in the context of hypoxia-based neurorehabilitation strategies.NEW & NOTEWORTHY A single brief episode of hypoxia (e.g., 3-5 min) does not evoke long-lasting increases in respiratory motor output after the hypoxia is concluded. Ampakines are a class of drugs that enhance AMPA receptor function. We show that pairing low-dose ampakine treatment with a single brief hypoxic exposure can evoke sustained phrenic motor facilitation after the acute hypoxic episode.