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.

Divergent receptor utilization is necessary for phrenic long-term facilitation over the course of motor neuron loss following CTB-SAP intrapleural injections.

Intrapleural injection of cholera toxin B conjugated to saporin (CTB-SAP) mimics respiratory motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Seven-day CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) primarily through TrkB and PI3K/Akt-dependent mechanisms [i.e., Gs-pathway, which can be initiated by adenosine 2A (A2A) receptors in naïve rats], whereas 28-day CTB-SAP rats elicit moderate pLTF though BDNF- and MEK-/ERK-dependent mechanisms [i.e., Gq-pathway, which is typically initiated by serotonin (5-HT) receptors in naïve rats]. Here, we tested the hypothesis that pLTF following CTB-SAP is 1) A2A receptor-dependent at 7 days and 2) 5-HT receptor-dependent at 28 days. Adult Sprague-Dawley male rats were anesthetized, paralyzed, ventilated, and exposed to acute intermittent hypoxia (AIH; 3-, 5-min bouts of 10.5% O2) following bilateral, intrapleural injections at 7 days and 28 days of 1) CTB-SAP (25 µg) or 2) unconjugated CTB and SAP (control). Intrathecal C4 delivery included either the 1) A2A receptor antagonist (MSX-3; 10 µM; 12 µL) or 2) 5-HT receptor antagonist (methysergide; 20 mM; 15 µL). pLTF was abolished with A2A receptor inhibition in 7-day, not 28-day, CTB-SAP rats versus controls (P < 0.05), whereas pLTF was abolished following 5-HT receptor inhibition in 28-day, not 7-day, CTB-SAP rats versus controls (P < 0.05). In addition, 5-HT2A receptor expression was unchanged in CTB-SAP rats versus controls, whereas 5-HT2B receptor expression was decreased in CTB-SAP rats versus controls (P < 0.05). This study furthers our understanding of the contribution of differential receptor activation to pLTF and its implications for breathing following respiratory motor neuron death.NEW & NOTEWORTHY The current study investigates underlying receptor-dependent mechanisms contributing to phrenic long-term facilitation (pLTF) following CTB-SAP-induced respiratory motor neuron death at 7 days and 28 days. We found that A2A receptors are required for enhanced pLTF in 7-day CTB-SAP rats, whereas 5-HT receptors are required for moderate pLTF in 28-day CTB-SAP rats. Targeting these time-dependent mechanisms have implications for breathing maintenance over the course of many neuromuscular diseases.

Glutamatergic input varies with phrenic motor neuron size.

Like all skeletal muscles, the diaphragm muscle accomplishes a range of motor behaviors by recruiting different motor unit types in an orderly fashion. Recruitment of phrenic motor neurons (PhMNs) is generally assumed to be based primarily on the intrinsic properties of PhMNs with an equal distribution of descending excitatory inputs to all PhMNs. However, differences in presynaptic excitatory input across PhMNs of varying sizes could also contribute to the orderly recruitment pattern. In the spinal cord of Sprague-Dawley rats, we retrogradely labeled PhMNs using cholera toxin B (CTB) and validated a robust confocal imaging-based technique that utilizes semiautomated processing to identify presynaptic glutamatergic (Glu) terminals within a defined distance around the somal membrane of PhMNs of varying size. Our results revealed an ~10% higher density of Glu terminals at PhMNs in the lower tertile of somal surface area. These smaller PhMNs are likely recruited first to accomplish lower force ventilatory behaviors of the diaphragm as compared with larger PhMNs in the upper tertile that are recruited to accomplish higher force expulsive behaviors. These results suggest that differences in excitatory synaptic input to PhMNs may also contribute to the orderly recruitment of diaphragm motor units.NEW & NOTEWORTHY The distribution of excitatory glutamatergic synaptic input to phrenic motor neurons differs across motor neurons of varying size. These findings support the size principle of motor unit recruitment that underlies graded force generation in a muscle, which is based on intrinsic electrophysiological properties of motor neurons resulting from differences in somal surface area. A higher density of glutamatergic inputs at smaller, more excitable motor neurons substantiates the earlier and more frequent recruitment of these units.

Teashirt 1 (Tshz1) is essential for the development, survival and function of hypoglossal and phrenic motor neurons in mouse.

Feeding and breathing are essential motor functions and rely on the activity of hypoglossal and phrenic motor neurons that innervate the tongue and diaphragm, respectively. Little is known about the genetic programs that control the development of these neuronal subtypes. The transcription factor Tshz1 is strongly and persistently expressed in developing hypoglossal and phrenic motor neurons. We used conditional mutation of Tshz1 in the progenitor zone of motor neurons (Tshz1MNΔ) to show that Tshz1 is essential for survival and function of hypoglossal and phrenic motor neurons. Hypoglossal and phrenic motor neurons are born in correct numbers, but many die between embryonic day 13.5 and 14.5 in Tshz1MNΔ mutant mice. In addition, innervation and electrophysiological properties of phrenic and hypoglossal motor neurons are altered. Severe feeding and breathing problems accompany this developmental deficit. Although motor neuron survival can be rescued by elimination of the pro-apoptotic factor Bax, innervation, feeding and breathing defects persist in Bax-/-; Tshz1MNΔ mutants. We conclude that Tshz1 is an essential transcription factor for the development and physiological function of phrenic and hypoglossal motor neurons.

Frequency-dependent lipid raft uptake at rat diaphragm muscle axon terminals.

INTRODUCTION: In motor neurons, cholera toxin B (CTB) binds to the cell-surface ganglioside GM1 and is internalized and transported via structurally unique components of plasma membranes (lipid rafts). METHODS: Lipid raft uptake by axon terminals adjoining type-identified rat diaphragm muscle fibers was investigated using CTB and confocal imaging. RESULTS: Lipid raft uptake increased significantly at higher frequency stimulation (80 Hz), compared with lower frequency (20 Hz) and unstimulated (0 Hz) conditions. The fraction of axon terminal occupied by CTB was ∼45% at 0- or 20-Hz stimulation, and increased to ∼65% at 80 Hz. Total CTB fluorescence intensity also increased (∼20%) after 80-Hz stimulation compared with 0 Hz. DISCUSSION: Evidence of increased lipid raft uptake at high stimulation frequencies supports an important role for lipid raft signaling at rat diaphragm muscle axon terminals, primarily for motor units physiologically activated at the higher frequencies. Muscle Nerve 59:611-611, 2019.

Bands of Fontana are caused exclusively by the sinusoidal path of axons in peripheral nerves and predict axon path; evidence from rodent nerves and physical models.

The precise cause of the bands of Fontana, striations on peripheral nerves visible to the naked eye, has been the subject of debate for hundreds of years. Some researchers have described them as reflecting the sinuous course of nerve fibres passing through nerves, and others have proposed that endoneurial collagen and sheaths surrounding nerves play a role in their appearance. We hypothesised that the bands are caused exclusively by reflection of light from the surfaces of nerve fibres travelling in phase in sinusoidal waveforms through peripheral nerves. We aligned images of obliquely illuminated nerves with confocal images of axons in those nerves, and the numbers and positions of the bands precisely matched the axonal waves. We also developed three-dimensional models of nerves with representations of the sinusoidal path of axons at their surface. We observed patterns resembling the bands of Fontana when these models were obliquely illuminated. This provides evidence that the bands of Fontana can be caused by light reflected sinusoidal path of axons alone. We subsequently describe a mechanism of band production based on our observations of both nerves and models. We report that smaller diameter nerves such as phrenic nerves and distal branches of sciatic nerves have shorter band intervals than larger nerves, such as proximal trunks of sciatic nerves, and that shorter band intervals correlate with longer axons per unit length of nerve, which suggests a greater tolerance to stretch. Inspection of banding patterns on peripheral nerves may permit prediction of axon length within nerves, and assist in the interpretation of nerve conduction data, especially in diseases where axon path has become altered.

Ultrasound-guided, open-source microneurography: Approaches to improve recordings from peripheral nerves in man.

OBJECTIVE: Microneurography is the only method for recording from single neurons in intact human nerves. It is challenging - requiring technical expertise, investment in specialised equipment and has sparse data yields. METHODS: We assessed whether ultrasound guidance in combination with an 'open access' amplifier and data capture system (Open-Ephys) would simplify and expand the scope of microneurographic recordings in humans. RESULTS: In 32 healthy consenting volunteers, ultrasound-guidance improved success rates for obtaining cutaneous C-fibres and reduced "Skin to Nerve" times from 28.5 min to 4.5 min for recordings of the peroneal nerve (P < 0.0001). We illustrate the potential utility of ultrasound-guided microneurography for difficult to access nerves with phrenic nerve recording during a Valsalva manoeuvre. We show that Open Ephys is a viable alternative to commercially available recording systems and offers advantages in terms of cost and software customisability. CONCLUSIONS: Ultrasound guidance for microneurography with Open Ephys facilitates cutaneous C nociceptor recordings and allows recordings to be made from nerves previously considered inaccessible. SIGNIFICANCE: We anticipate that the adoption of these techniques will improve microneurography experimental efficiency, adds an important visual learning aid and increases the generalisability of the approach.

In vitro effects of Crotalus atrox snake venom on chick and mouse neuromuscular preparations.

The neuromuscular effect of venoms is not a major clinical manifestation shared between rattlesnakes native to the Americas, which showed two different venom phenotypes. Taking into account this dichotomy, nerve muscle preparations from mice and chicks were used to investigate the ability of Crotalus atrox venom to induce in vitro neurotoxicity and myotoxicity. Unlike crotalic venoms of South America, low concentrations of C. atrox venom did not result in significant effects on mouse neuromuscular preparations. The venom was more active on avian nerve-muscle, showing reduction of twitch heights after 120 min of incubation with 10, 30 and 100 µg/mL of venom with diminished responses to agonists and KCl. Histological analysis highlighted that C. atrox was myotoxic in both species of experimental animals; as evidenced by degenerative events, including edematous cells, delta lesions, hypercontracted fibers and muscle necrosis, which can lead to neurotoxic action. These results provide key insights into the myotoxicity and low neurotoxicity of C. atrox in two animal models, corroborating with previous genomic and proteomic findings and would be useful for a deeper understanding of venom evolution in snakes belonging to the genus Crotalus.

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

KEY POINTS: 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. ABSTRACT: 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.

Anatomy and physiology of phrenic afferent neurons.

Large-diameter myelinated phrenic afferents discharge in phase with diaphragm contraction, and smaller diameter fibers discharge across the respiratory cycle. In this article, we review the phrenic afferent literature and highlight areas in need of further study. We conclude that 1) activation of both myelinated and nonmyelinated phrenic sensory afferents can influence respiratory motor output on a breath-by-breath basis; 2) the relative impact of phrenic afferents substantially increases with diaphragm work and fatigue; 3) activation of phrenic afferents has a powerful impact on sympathetic motor outflow, and 4) phrenic afferents contribute to diaphragm somatosensation and the conscious perception of breathing. Much remains to be learned regarding the spinal and supraspinal distribution and synaptic contacts of myelinated and nonmyelinated phrenic afferents. Similarly, very little is known regarding the potential role of phrenic afferent neurons in triggering or modulating expression of respiratory neuroplasticity.

Impact of glutamatergic and serotonergic neurotransmission on diaphragm muscle activity after cervical spinal hemisection.

Incomplete cervical spinal cord hemisection at C2 (SH) disrupts descending excitatory drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle. Spontaneous recovery over time is associated with increased phrenic motoneuron expression of glutamatergic N-methyl-d-aspartate (NMDA) and serotonergic 5-HT2A receptors. We hypothesized that NMDA and 5-HT2A receptor-mediated neurotransmission play a role in ipsilateral diaphragm muscle activity post-SH. Adult male Sprague-Dawley rats were implanted with bilateral diaphragm EMG electrodes for chronic EMG recordings up to 28 days post-SH (SH 28D). The extent of recovery was calculated by peak root-mean-square (RMS) EMG amplitude. In all animals, absence of ipsilateral activity was verified at 3 days post-SH. Diaphragm EMG activity was also recorded during exposure to hypoxia-hypercapnia (10% O2-5% CO2). In SH animals displaying recovery of ipsilateral diaphragm EMG activity at SH 28D, cervical spinal cord segments containing the phrenic motor nucleus (C3-C5) were surgically exposed and either the NMDA receptor antagonist d-2-amino-5-phosphonovalerate (d-AP5; 100 mM, 30 µl) or 5-HT2A receptor antagonist ketanserin (40 mM, 30 µl) was instilled intrathecally. Following d-AP5, diaphragm EMG amplitude was reduced ipsilaterally, during both eupnea (42% of pre-d-AP5 value; P = 0.007) and hypoxia-hypercapnia (31% of pre-d-AP5 value; P = 0.015), with no effect on contralateral EMG activity or in uninjured controls. Treatment with ketanserin did not change ipsilateral or contralateral RMS EMG amplitude in SH animals displaying recovery at SH 28D. Our results suggest that spinal glutamatergic NMDA receptor-mediated neurotransmission plays an important role in ipsilateral diaphragm muscle activity after cervical spinal cord injury.NEW & NOTEWORTHY Spontaneous recovery following C2 spinal hemisection (SH) is associated with increased phrenic motoneuron expression of glutamatergic and serotonergic receptors. In this study, we show that pharmacological inhibition of glutamatergic N-methyl-d-aspartate (NMDA) receptors blunts ipsilateral diaphragm activity post-SH. In contrast, pharmacological inhibition of serotonergic 5-HT2A receptors does not change diaphragm EMG activity post-SH. Our results suggest that NMDA receptor-mediated glutamatergic neurotransmission plays an important role in enhancing rhythmic respiratory-related diaphragm activity after spinal cord injury.

Anatomical Recruitment of Spinal V2a Interneurons into Phrenic Motor Circuitry after High Cervical Spinal Cord Injury.

More than half of all spinal cord injuries (SCIs) occur at the cervical level, often resulting in impaired respiration. Despite this devastating outcome, there is substantial evidence for endogenous neuroplasticity after cervical SCI. Spinal interneurons are widely recognized as being an essential anatomical component of this plasticity by contributing to novel neuronal pathways that can result in functional improvement. The identity of spinal interneurons involved with respiratory plasticity post-SCI, however, has remained largely unknown. Using a transgenic Chx10-eGFP mouse line (Strain 011391-UCD), the present study is the first to demonstrate the recruitment of excitatory interneurons into injured phrenic circuitry after a high cervical SCI. Diaphragm electromyography and anatomical analysis were used to confirm lesion-induced functional deficits and document extent of the lesion, respectively. Transneuronal tracing with pseudorabies virus (PRV) was used to identify interneurons within the phrenic circuitry. There was a robust increase in the number of PRV-labeled V2a interneurons ipsilateral to the C2 hemisection, demonstrating that significant numbers of these excitatory spinal interneurons were anatomically recruited into the phrenic motor pathway two weeks after injury, a time known to correspond with functional phrenic plasticity. Understanding this anatomical spinal plasticity and the neural substrates associated with functional compensation or recovery post-SCI in a controlled, experimental setting may help shed light onto possible cellular therapeutic candidates that can be targeted to enhance spontaneous recovery.

Developmental plasticity of phrenic motoneuron and diaphragm properties with the inception of inspiratory drive transmission in utero.

The review outlines data consistent with the hypothesis that inspiratory drive transmission that generates fetal breathing movements (FBMs) is essential for the developmental plasticity of phrenic motoneurons (PMNs) and diaphragm musculature prior to birth. A systematic examination during the perinatal period demonstrated a very marked transformation of PMN and diaphragm properties coinciding with the onset and strengthening of inspiratory drive and FBMs in utero. This included studies of age-dependent changes of: i) morphology, neuronal coupling, passive and electrophysiological properties of PMNs; ii) rhythmic inspiratory activity in vitro; iii) FBMs generated in vivo detected by ultrasonography; iv) contractile and end-plate potential properties of diaphragm musculature. We also propose how the hypothesis can be further evaluated with studies of perinatal hypoglossal motoneuron-tongue musculature and the use of Dbx1 null mice that provide an experimental model lacking descending inspiratory drive transmission in utero.

Phrenic motor neuron TrkB expression is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation.

Phrenic long-term facilitation (pLTF) is a form of hypoxia-induced spinal respiratory motor plasticity that requires new synthesis of brain derived neurotrophic factor (BDNF) and activation of its high-affinity receptor, tropomyosin receptor kinase B (TrkB). Since the cellular location of relevant TrkB receptors is not known, we utilized intrapleural siRNA injections to selectively knock down TrkB receptor protein within phrenic motor neurons. TrkB receptors within phrenic motor neurons are necessary for BDNF-dependent acute intermittent hypoxia-induced pLTF, demonstrating that phrenic motor neurons are a critical site of respiratory motor plasticity.

Coupling multielectrode array recordings with silver labeling of recording sites to study cervical spinal network connectivity.

Midcervical spinal interneurons form a complex and diffuse network and may be involved in modulating phrenic motor output. The intent of the current work was to enable a better understanding of midcervical "network-level" connectivity by pairing the neurophysiological multielectrode array (MEA) data with histological verification of the recording locations. We first developed a method to deliver 100-nA currents to electroplate silver onto and subsequently deposit silver from electrode tips after obtaining midcervical (C3-C5) recordings using an MEA in anesthetized and ventilated adult rats. Spinal tissue was then fixed, harvested, and histologically processed to "develop" the deposited silver. Histological studies verified that the silver deposition method discretely labeled (50-µm resolution) spinal recording locations between laminae IV and X in cervical segments C3-C5. Using correlative techniques, we next tested the hypothesis that midcervical neuronal discharge patterns are temporally linked. Cross-correlation histograms produced few positive peaks (5.3%) in the range of 0-0.4 ms, but 21.4% of neuronal pairs had correlogram peaks with a lag of ≥0.6 ms. These results are consistent with synchronous discharge involving mono- and polysynaptic connections among midcervical neurons. We conclude that there is a high degree of synaptic connectivity in the midcervical spinal cord and that the silver-labeling method can reliably mark metal electrode recording sites and "map" interneuron populations, thereby providing a low-cost and effective tool for use in MEA experiments. We suggest that this method will be useful for further exploration of midcervical network connectivity.NEW & NOTEWORTHY We describe a method that reliably identifies the locations of multielectrode array (MEA) recording sites while preserving the surrounding tissue for immunohistochemistry. To our knowledge, this is the first cost-effective method to identify the anatomic locations of neuronal ensembles recorded with a MEA during acute preparations without the requirement of specialized array electrodes. In addition, evaluation of activity recorded from silver-labeled sites revealed a previously unappreciated degree of connectivity between midcervical interneurons.

Functional and morphological assessment of diaphragm innervation by phrenic motor neurons.

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Semi-automated assessment of transdiaphragmatic pressure variability across motor behaviors.

We developed and tested a semi-automated algorithm to generate large data sets of ventilatory information (amplitude, premotor drive and timing) across a range of motor behaviors. Adult spontaneously breathing, anesthetized mice (n = 27) underwent measurements of transdiaphragmatic pressure (Pdi) during eupnea, hypoxia-hypercapnia, and tracheal occlusion with values ranging from 8 ± 1 to 9 ± 2 to 44 ± 3 cmH2O, respectively. Premotor drive to phrenic motor neurons (estimated by the rate of rise during initial 60 ms) was ∼ 5-fold greater during tracheal occlusion compared to other behaviors. Variability in Pdi amplitude (normalized to spontaneously occurring sighs for each animal) displayed minimal evidence of complex temporal structure or dynamic clustering across the entire period of examination. Using a deterministic model to evaluate predictor variables for Pdi amplitude between successive inspiratory events, there was a large correlation for premotor drive and preceding Pdi amplitude vs. Pdi amplitude (r = 0.52). These findings highlight substantial variability in Pdi amplitude that primarily reflects linear components rather than complex, dynamic effects over time.

Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.

Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

Arterial chemoreceptor activation reduces the activity of parapyramidal serotonergic neurons in rats.

The parapyramidal (ppy) region targets primarily the intermediolateral cell column and is probably involved in breathing and thermoregulation. In the present study, we tested whether ppy serotonergic neurons respond to activation of central and peripheral chemoreceptors. Bulbospinal ppy neurons (n=30) were recorded extracellularly along with the phrenic nerve activity in urethane/α-chloralose-anesthetized, paralyzed, intact (n=7) or carotid body denervated (n=6) male Wistar rats. In intact animals, most of the ppy neurons were inhibited by hypoxia (n=14 of 19) (8% O2, 30s) (1.5 ± 0.03 vs. control: 2.4 ± 0.2 Hz) or hypercapnia (n=15 of 19) (10% CO2) (1.7 ± 0.1 vs. control: 2.2 ± 0.2 Hz), although some neurons were insensitive to hypoxia (n=3 of 19) or hypercapnia (n=4 of 19). Very few neurons (n=2 of 19) were activated after hypoxia, but not after hypercapnia. In carotid body denervated rats, all the 5HT-ppy neurons (n=11) were insensitive to hypercapnia (2.1 ± 0.1 vs. control: 2.3 ± 0.09 Hz). Biotinamide-labeled cells that were recovered after histochemistry were located in the ppy region. Most labeled cells (90%) showed strong tryptophan hydroxylase immunocytochemical reactivity, indicating that they were serotonergic. The present data reveal that peripheral chemoreceptors reduce the activity of the serotonergic premotor neurons located in the ppy region. It is plausible that the serotonergic neurons of the ppy region could conceivably regulate breathing automaticity and be involved in autonomic regulation.