Phrenic Neurons Research Articles

Genetic Basis of Aerobically Supported Voluntary Exercise: Results from a Selection Experiment with House Mice.

The biological basis of exercise behavior is increasingly relevant for maintaining healthy lifestyles. Various quantitative genetic studies and selection experiments have conclusively demonstrated substantial heritability for exercise behavior in both humans and laboratory rodents. In the "High Runner" selection experiment, four replicate lines of Mus domesticus were bred for high voluntary wheel running (HR), along with four nonselected control (C) lines. After 61 generations, the genomes of 79 mice (9-10 from each line) were fully sequenced and single nucleotide polymorphisms (SNPs) were identified. We used nested ANOVA with MIVQUE estimation and other approaches to compare allele frequencies between the HR and C lines for both SNPs and haplotypes. Approximately 61 genomic regions, across all somatic chromosomes, showed evidence of differentiation; 12 of these regions were differentiated by all methods of analysis. Gene function was inferred largely using Panther gene ontology terms and KO phenotypes associated with genes of interest. Some of the differentiated genes are known to be associated with behavior/motivational systems and/or athletic ability, including Sorl1, Dach1, and Cdh10 Sorl1 is a sorting protein associated with cholinergic neuron morphology, vascular wound healing, and metabolism. Dach1 is associated with limb bud development and neural differentiation. Cdh10 is a calcium ion binding protein associated with phrenic neurons. Overall, these results indicate that selective breeding for high voluntary exercise has resulted in changes in allele frequencies for multiple genes associated with both motivation and ability for endurance exercise, providing candidate genes that may explain phenotypic changes observed in previous studies.

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

There was an error published in Development141, 784-794.In the supplementary data originally published, the supplementary Materials and methods section was missing. The correct version now appears online.We apologise to the authors and readers for this mistake.

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.

The pattern and extent of retrograde transsynaptic transport of WGA-Alexa 488 in the phrenic motor system is dependent upon the site of application

The first aim of the study was to determine if WGA-Alexa 488 would undergo retrograde transsynaptic transport in the phrenic motor system as we have shown with WGA-HRP in a previous study. The advantage of using WGA-Alexa 488 is that labeled neurons could be isolated and analyzed for intracellular molecular mechanisms without exposing tissue sections to chemicals for histochemical staining. The second aim of the study was to investigate the pattern and extent of labeling that occurs when WGA-Alexa 488 is applied to the cervical phrenic nerve as compared to intradiaphragmatic injection. After injecting the hemidiaphragm ipsilateral to a C2 spinal cord hemisection, WGA-Alexa 488 presumably diffused to the contralateral hemidiaphragm and labeled the phrenic nuclei bilaterally. In all animals with hemidiaphragmatic injection, the rostral ventral respiratory group (rVRG) was also labeled bilaterally in the medulla. Thus, injection of WGA-Alexa 488 into the diaphragm results in retrograde transsynaptic transport in the phrenic motor system. After applying WGA-Alexa 488 to the ipsilateral intact cervical phrenic nerve in both C2 hemisected rats and rats with a sham hemisection, only ipsilateral phrenic neurons were labeled; there was no labeling of the rVRG or any other center in the medulla. These results suggest that WGA-Alexa 488 must be applied in the vicinity of the phrenic myoneural junction where there is a high concentration of WGA receptors in order for transsynaptic transport to occur. The present study provides investigators with a new tool to study plasticity in the respiratory system after spinal cord injury.

Neurokinin-1 receptor-expressing cells of the ventral respiratory group are functionally heterogeneous and predominantly glutamatergic.

According to a recent theory (Gray et al., 1999) the neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons of the ventral respiratory group (VRG) are confined to the pre-Bötzinger complex (pre-BötC) and might be glutamatergic interneurons that drive respiratory rhythmogenesis. In this study we tested whether the NK1R-ir neurons of the VRG are glutamatergic. We also examined whether different groups of NK1R-ir neurons coexist in the VRG on the basis of whether these cells contain preproenkephalin (PPE) mRNA or project to the spinal cord. NK1R immunoreactivity was found in two populations of VRG neurons that are both predominantly glutamatergic because most of them contained vesicular glutamate transporter 2 mRNA (77 +/- 9%; n = 3). A group of small fusiform neurons (somatic cross section: 91 +/- 3.6 microm2) that has neither PPE mRNA nor spinal projections is primarily restricted to the pre-BötC. These cells may be the interneurons the destruction of which produces massive disruptions of the respiratory rhythm (Gray et al., 2001). The rest of the NK1R-ir neurons of the VRG are multipolar, are larger (somatic cross section: 252 +/- 15 microm2), and express high levels of PPE mRNA. Some of these cells located in the rostral half of the rostral VRG project to the spinal cord (C4 or T3). Using electrophysiological methods, we showed that these bulbospinal NK1R-ir neurons are slowly discharging inspiratory-augmenting neurons, suggesting that they may control phrenic or intercostal motor neurons. In summary, NK1R-expressing cells of the VRG are a heterogeneous group of predominantly glutamatergic neurons that include subpopulations of respiratory premotor neurons.

Phrenic paresis—a possible additional spinal cord dysfunction induced by neck manipulation in cervical spondylotic myelopathy (CSM): A report of two cases with anatomical and clinical considerations

The clinical records of two male subjects with severe cervical spondylotic myelopathy (CSM) who developed respiratory insufficiency after the cervical manipulation involved in preoperative anesthetic intubation were examined. Their cervical imaging was analyzed with respect to the known anatomic relationships of the spinal phrenic nerve nuclei to the spondylotic compressive lesions in an attempt to provide the anatomic and pathologic rationales that may explain this phrenic paresis as a possible traumatic complication of severe CSM. Perusal of extant literature revealed extensive descriptions of CSM symptoms, but none had previously reported an associated neuromuscular weakness of the diaphragm. Magnetic resonance imaging analyses indicated that the existing degree of upper cervical cord compression, when reinforced by the additional posterior and anterior pressures consequent to cervical spinal extension and flexion, could readily account for the functional impairment of phrenic nerve neuron cells and/or their efferent fibers. Thus, the anatomic relations of the phrenic nerve nuclear columns and their efferent tracts predispose them to interference by compressive lesions found in CSM, and undue manipulation of the cervical spine when advanced stenosis is known to be present should be recognized as a possible cause of cervical spondylotic myelopathic-phrenic paresis.

GABA receptors in the phrenic nucleus of the rat.

The phrenic nucleus was identified by microinjections of N-methyl-D-aspartic acid in urethan-anesthetized adult male Wistar rats. Microinjections of GABAA and GABAB receptor agonists (muscimol and baclofen, respectively) at the same site decreased the phrenic nerve burst amplitude. Microinjections of GABAA and GABAB receptor antagonists (bicuculline and 2-hydroxysaclofen, respectively) blocked as well as reversed the effects of their respective agonists. These results were confirmed by recording extracellular action potentials from single phrenic neurons. Micropressure applications of muscimol and baclofen decreased the activity of single neurons in the phrenic nucleus; this effect was blocked as well as reversed by micropressure applications of bicuculline and 2-hydroxysaclofen, respectively. These results demonstrated the presence of GABA receptors on the neurons in the phrenic nucleus and suggested that their activation results in the decrease of the phrenic nerve burst amplitude. The importance of these results in the identification of neural circuits mediating inhibition of phrenic neurons is discussed.

Branching patterns of the rat phrenic nerve during development and reinnervation

Previous studies have shown that the phrenic motor nucleus in the rat projects onto the diaphragm muscle, forming an orderly topographic map. Moreover, this topography is partially restored upon reinnervation. This orderly map is expressed prior to birth, suggesting that early contacts between nerve and muscle are topographically appropriate. The phrenic divides during embryonic development into rostral and caudal branches, and motor axons preferentially enter the appropriate branch. In an effort to understand the mechanisms that underlie the choices growing phrenic neurons make in selecting their appropriate muscle targets, we examined the patterns of branching displayed by the phrenic nerve during development and reinnervation. In all muscles studied the phrenic nerve splits into three primary branches, rostral, caudal, and crural. At a coarse level the pattern of branching of the phrenic is remarkably consistent from animal to animal and at all ages of development. At a finer level of resolution, however, there is an asymmetry between right and left hemidiaphragms. Moreover, the precise emergence of any particular branch is unpredictable, resulting in an overall incongruence in branching architecture from animal to animal. The hemidiaphragm muscle grows unevenly, particularly on the right side, resulting in greater muscle fiber elongation medially. Upon reinnervation, the same coarse pattern of branching is reestablished, but the higher order pattern is much simpler and muscle growth is slower than in controls. These results suggest that very early in development primary branches of the phrenic funnel axons into three well-defined zones in the muscle. These observations taken together with previous physiological studies indicate that an orderly topographic map can emerge in spite of considerable variation in the pattern of branches.

Postnatal development of small and large dorsal root ganglion neurons in the cat. A study on cervical levels (C 5–C 6) and on phrenic afferents

During feline postnatal development, the size of phrenic afferent neurons labelled by horseradish peroxidase was evaluated in comparison to that of the bulk of counterstained neurons located in the same cervical dorsal root ganglia (DRG) (C 5–C 6). From age 1 week to maturity, small and large cell components were individualized from experimental size distributions using a mathematical approach. The analysis of data in adult indicated a close correspondence between small cells and unmyelinated afferents and between large cells and myelinated afferents, respectively. From age 1 week to adulthood, mean increases in cell diameter ranged between 10 μm (small cells from phrenic afferents) and 29.5 μm (large counterstained cells). In each population, the ratio of small/large cells remained constant during growth. In contrast to data in adults, at 1 week, large phrenic neurons were bigger than the counterstained ones. At 19 weeks, the cat DRG cells had not yet reached their adult size.

Neuronal and glial changes in the rat phrenic nucleus occurring within hours after spinal cord injury.

The present study describes specific morphological changes in the normal ultrastructure of the rat phrenic nucleus which occur within 4 hours after an ipsilateral spinal cord hemisection rostral to the nucleus. Phrenic neurons were identified at EM levels by retrograde HRP labeling. Ultrastructural features of the phrenic nucleus in uninjured animals and at 4 hours and 1, 2, and 4 days after injury were qualitatively analyzed and then quantitated with a computerized morphometric system. Our results indicated that by 4 hours posthemisection, there was a significant increase in the number of double synapses. Furthermore, the number of double synapses remained significantly higher than normal at all the other posthemisection periods. A significant increase in the length of dendrodendritic membrane appositions was also noted as early as 4 hours posthemisection. The mean normal appositional length of 1.42 +/- 0.09 microns increased to 1.89 +/- 0.12 microns at 4 hours and further increased to 2.20 +/- 0.20 microns by 1 day posthemisection. The increase in the length of membrane appositions was most likely due to an active retraction of astroglial processes from their normal position in between the dendrites. Although there was an increase in the mean length of the dendrodendritic appositions, the mean percentage of the appositions (expressed as the total number of appositions divided by the total number of dendrites in each sample) was not increased significantly over normal values during the early posthemisection periods. By 2 and 4 days posthemisection, however, the percentage of dendrodendritic appositions increased to significantly higher values than normal. Normally, 4.68 +/- 0.69% of the dendrites in the phrenic nucleus were found to be in apposition, and this number increased significantly to 7.27 +/- 1.06% by 2 days and 7.46 +/- 0.79% by 4 days posthemisection. At these later posthemisection periods, the mean length of the appositions decreased to levels which were no longer significantly higher than normal. A distribution analysis of the length of each dendrodendritic apposition in both the normal and spinal hemisected rats showed that there were more dendrodendritic appositions in the phrenic nucleus at the later posthemisection periods. It also showed that their mean length was decreased because many of the new appositions were relatively short. The above neuronal and glial alterations of the phrenic nucleus have never before been described as a response to injury of the mammalian spinal cord. Furthermore, the possibility that the above changes could represent the morphological substrate for the unmasking of functionally ineffective synapses in ou

Comparison of the response of diaphragm and upper airway dilating muscle activity in sleeping cats

The steady state and transient effects of hyperoxic hypercapnia on the electromyographic activities of the genioglossus (GG), posterior cricoarytenoid (PCA), and diaphragm (D) were studied in cats instrumented with chronically implanted electrodes during non-rapid eye movement sleep. Hypercapnia (inhalation of 3.4 and 7.4% CO 2 in O 2) increased the phasic electrical activity occurring during inspiration of all three muscles. Activities of the PCA and D increased in a parallel fashion with increasing CO 2. The GG responded to steady state CO 2 inhalation alinearly, with larger increases in activity occurring when CO 2 was raised from 3.4 to 7.4% than when CO 2 was increased from 0 to 3.4%. When gas mixtures containing CO 2 were given, the GG reached its new steady state level more slowly than the D or PCA, and when CO 2 was rapidly removed from the inspired gas mixture, the GG attained its steady state sooner than the D. These results suggest that in sleeping cats, hypercapnia does not affect either transient or steady state responses of the GG in the same way as the D or the PCA. These differences seem to be explained largely by different threshold characteristics of hypoglossal and phrenic neurons.

Comparison of the response of diaphragm and upper airway dilating muscle activity in sleeping cats

The steady state and transient effects of hyperoxic hypercapnia on the electromyographic activities of the genioglossus (GG), posterior cricoarytenoid (PCA), and diaphragm (D) were studied in cats instrumented with chronically implanted electrodes during non-rapid eye movement sleep. Hypercapnia (inhalation of 3.4 and 7.4% CO 2 in O 2) increased the phasic electrical activity occurring during inspiration of all three muscles. Activities of the PCA and D increased in a parallel fashion with increasing CO 2. The GG responded to steady state CO 2 inhalation alinearly, with larger increases in activity occurring when CO 2 was raised from 3.4 to 7.4% than when CO 2 was increased from 0 to 3.4%. When gas mixtures containing CO 2 were given, the GG reached its new steady state level more slowly than the D or PCA, and when CO 2 was rapidly removed from the inspired gas mixture, the GG attained its steady state sooner than the D. These results suggest that in sleeping cats, hypercapnia does not affect either transient or steady state responses of the GG in the same way as the D or the PCA. These differences seem to be explained largely by different threshold characteristics of hypoglossal and phrenic neurons.

Changes in antidromic latencies of medullary respiratory neurons in hypercapnia and hypoxia.

We evaluated mechanisms underlying changes in discharge frequencies of medullary respiratory neurons. This evaluation was made by determining variations in antidromic latencies; these variations reflect changes in membrane potentials. In decerebrate, vagotomized, paralyzed, and ventilated cats, activities of the phrenic nerve and single respiratory neurons were monitored in hyperoxic normocapnia, hyperoxic hypercapnia, and/or normocapnic hypoxia. Axonal projections were defined as bulbospinal or laryngeal by antidromic activation. At normocapnic hyperoxia, antidromic latencies fell to minima during periods of spontaneous neuronal activity, with maxima occurring between neuronal bursts. In hypercapnia or hypoxia, these minima were not altered, whereas maximum latencies typically rose for neurons whose discharge frequencies increased. However, the increased frequencies most strongly correlated with increases in the difference between maximum and minimum latencies. No such correlation was evident for neurons whose discharge frequencies declined. We conclude that the overall change of membrane potential primarily defines neuronal discharge frequencies. Changes in membrane potentials induced by peripheral and central chemoreceptor afferents and by direct actions of hypercapnia and hypoxia are discussed.

Responses of early and late onset phrenic motoneurons to lung inflation

In anesthetized or decerebrate cats that were paralyzed and ventilated with a cycle-triggered pump, we produced changes in activity of the whole phrenic nerve and of individual phrenic motoneurons (fibers or cell in the spinal cord) by withholding lung inflation during the inspiratory (I) phase. The neurons were classified into early- and late-onset types (discharge onset less or greater than 80 msec, respectively, after whole phrenic onset). Both unit and whole phrenic activity exhibited a variety of responses to inflation (excitation, depression, or no effect); but there were no consistent differences between responses of early- and late-onset neurons. The distribution of responses was quite different from that of dorsal respiratory group (DRG) I neurons (Cohen and Feldman, 1984); in particular there was no group of phrenic neurons corresponding to the late-onset I-beta neurons (I neurons excited by inflation). We conclude that the inputs to late-onset phrenic neurons are not predominantly or exclusively from late-onset DRG neurons.

Responses to chemical stimulation of upper airway muscles diaphragm in awake cats.

The steady-state and transient effects of hyperoxic hypercapnia on the electromyographic activities of the genioglossus (GG), posterior cricoarytenoid (PCA), and diaphragm (D) were studied in conscious unsedated cats with chronically implanted electrodes. Hypercapnia (inhalation of 3.4 and 7.4% CO2 in O2) increased the phasic electrical activity occurring during inspiration in all three muscles and also increased tonic activity of the GG. The GG responded to steady-state CO2 inhalation alinearly and with larger increases in activity than the PCA and D. Phasic GG activity was present in only 4 of 10 cats breathing 100% O2, whereas phasic PCA and D activity could be observed in all animals studied. When gas mixtures containing CO2 were given, the GG reached its new steady-state level more slowly than the D or PCA, and when CO2 was rapidly removed from the inspired gas mixture, the GG attained its steady state sooner than either the PCA or D. These results suggest that in awake unsedated animals, chemical stimuli do not affect either transient or steady-state responses of the GG in the same way as the D. These differences seem to be explained mainly by different threshold characteristics of hypoglossal and phrenic neurons but also in part by dissimilarities in their steady-state responses.

The phrenic nucleus of th albino rat: a correlative HRP and Golgi study.

The phrenic nucleus of the adult albino rat was studied by utilizing the O-dianisidine method for the retrograde transport of horseradish peroxidase in conjunction with the zinc chromate modification of the Golgi technique. Application of HRP to the transected phrenic nerve in the neck labeled a column of phrenic motor neurons from C3 to C5 in the ipsilateral spinal cord. However, when HRP was applied to the phrenic nerve intrathoracically, labeled neurons were found from C3 to C6. The long axis of the column of phrenic neurons was oriented tangentially from rostral to caudal poles. There was a gradual shift of the column from posterior to anterior and from lateral to medial positions in the ventral horn. The peroxidase material was also used to localize impregnated phrenic motor neurons in the Golgi sections and to provide quantitative data on phrenic motor neurons. In Golgi-impregnated material two types of phrenic neurons were distinguished on the basis of dendritic morphology and orientation. These neurons were designated (1) large neurons with smooth, radially oriented dendrites, and (2) smaller neurons with varicose, tangentially oriented dendrites. Both types of neurons had a small number of spines and bulbous appendages issuing from the dendritic trunks and branches. The dendritic fields of adjacent phrenic neurons overlapped extensively with one another and with dendrites of more distally placed ventral horn motor neurons. In peroxidase-labeled sagittal sections the dendrites of phrenic neurons were primarily oriented in the rostrocaudal plane. The mean total number of peroxidase-labeled neurons in the phrenic nucleus was 415.75 +/- 18.36 cells. In sagittal sections the mean long axis diameter of phrenic cell bodies was 34.5 micrometers. In frontal sections the mean long axis diameter of phrenic cell bodies was 22.5 micrometers. Thus, from direct measurement, the phrenic neurons were 34% longer in the sagittal plane than in the frontal plane. In the present study each phrenic nucleus contributed fibers only to the ipsilateral phrenic nerve, and no evidence for peripheral crossing of fibers was found.

Effect of stimulation of the medial and lateral zones of the respiratory center on electrical activity of the diaphragm, intercostal muscles, and phrenic neurons

Experiments on cats showed that the nucleus of the solitary tract displayed zones whose stimulation provoked separately stimulation or inhibition of the electrical activity of the phrenic neurons and the diaphragm. Stimulation in the nucleus ambiguus of such zones caused stimulation and inhibition of electrical activity of the intercostal inspiratory muscles. In stimulation of the corresponding zone in the giant cell nucleus the electrical activity of both groups of the inspiratory muscles proved to change. It is suggested that the action of stimulation of the giant cell nucleus zones on both groups of inspiratory muscles is mediated through the neurons of the solitary tract and the nucleus ambiguus.

Demonstration of functionally ineffective synapses in the guinea pig spinal cord

Hemisection of the spinal cord rostral to the nucleus of the phrenic nerve results in a permanent paralysis of the ipsilateral hemidiaphragm. In several mammalian species, functional recovery of the paretic hemidiaphragm can be achieved by transecting the contralateral phrenic nerve. The phrenicotomy results in an increased medullary respiratory output which activates a functionally latent, crossed medullospinal respiratory pathway. The nerve fibers of the crossed pathway trasmit respiratory impulses across the spinal cord at the level of the phrenic nuclei, thereby generating action potetentials in the phrenic neurons ipsilateral to the paretic hemidiaphragm. This response has been termed the “crossed phrenic phenomenon.” Previous studies in guinea pigs showed that the crossed phrenic phenomenon did not occur if the spinal hemisection was followed immediately by contralateral phrenicotomy, but it did occur if several months elapsed between the two operations. In the present experiment we sought to determine the shortest time interval that would permit development of a crossed phrenic phenomenon in order to decide whether functional recovery in guinea pigs results from the establishment of a new synaptic pathway by collateral sprouting or from the activation of a previously ineffective pathway. The former would require several weeks to occur, whereas the latter might be expected to occur within hours or days. In the present study the crossed phrenic phenomenon occurred within 3.5 h after spinal hemisection. We therefore conclude that, within several hours after spinal hemisection, functionally ineffective synapses become converted to functionally latent ones. The stress of contralateral phrenicotomy is sufficient to activate this functionally latent pathway. The mechanism by which the ineffective pathway is transformed to a latent one is unknown.