OBJECTIVES/HYPOTHESIS Selective reinnervation of the posterior cricoarytenoid muscle with a single phrenic nerve rootlet has been shown to restore physiologic motion in animal models. However, clinical translation of this work is challenged by the limited knowledge of the cervical anatomy of the phrenic nerve. STUDY DESIGN Prospective collaborative study. METHODS Dissection of 111 cadaveric necks (88 embalmed and 23 unembalmed) from 56 cadavers. RESULTS The mean (standard deviation) lengths of unembalmed cadaver C3, C4, and C5 nerve rootlets were 3.9 (2.4), 3.6 (2.6), and 0.5 (0.8) cm, respectively. Embalmed cadavers had shorter C3 and C4 phrenic nerve rootlet lengths than unembalmed cadavers (P =.02 and P =.03, respectively). There was no difference in mean nerve rootlet length based on sex, body height or weight, or side of dissection. A total of eight unique phrenic nerve rootlet patterns were identified. The most common pattern consisted of phrenic with single C3 and C4 rootlets with an immeasurable C5 rootlet, which was present in 30 of 111 (26%) of the necks. The classic three branching pattern of single C3, C4, and C5 rootlets was found in 25 of 111 (22%) of the necks. Six of 111 (5%) of the dissections displayed accessory phrenic nerves arising from the C3, C4, or C5 anterior rami. A χ(2) analysis showed no difference between side or sex and frequency of pattern. CONCLUSIONS The present study demonstrates the wide variability within the cervical anatomy of the phrenic nerve.

Anterograde transport of tritiated amino acids (leucine, lysine, and proline) was used to examine the spinal projections of respiratory premotor neurons in the ventral respiratory group (VRG) of cats. This population of neurons corresponds anatomically with the nucleus ambiguus-retroambigualis. Small volumes (20 to 50 nl) of tritiated amino acids were pressure ejected into the middle of the VRG through a micropipette which permitted simultaneous recording of respiratory modulated activity. In two cats injections were made caudal to the obex in regions which contained expiratory modulated neurons. In five cats injections were made rostral to the obex in regions containing inspiratory neurons. After a 2-week survival period, cats were anesthetized and perfused. The entire neuraxis was removed and processed using standard autoradiographic techniques. Transport of tritiated amino acids revealed a marked bilateral projection to lamina IX of the spinal cord at the C4 to C6 level and a primarily contralateral projection to laminae VIII and IX in the thoracic spinal cord. Distinct descending pathways to the phrenic motor neurons were observed in the lateral funiculus and in the ventral funiculus; descending fibers to the intercostal motoneurons in the thoracic cord appeared to be restricted to the ventral funiculus. Labeling of axon terminals in both the cervical and thoracic cords was confined to ventral horn regions which contain motoneurons. These results suggest that monosynaptic projections from brainstem bulbo-spinal neurons to spinal motoneurons are important in controlling respiratory movements of the diaphragm and intercostal muscles.

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.

The morphology of 11 dorsal respiratory group (DRG) inspiratory neurons located in the ventrolateral nucleus of the solitary tract (vl-NTS) was studied using the technique of intracellular labeling with the enzyme horseradish peroxidase (HRP). Six of these cells were cut in the transverse plane and had a mean somal diameter of 30.4 micron, while five others sectioned in the horizontal plane had a mean of 38.2 micron. These neurons produced an average of 6.2 primary dendrites (range: 4-10), many of which projected rostrally or caudally up to 1.0 mm from the cell bodies. These dendrites were oriented along the longitudinal axis; they ran parallel and ventral to the tractus solitarius. In general, all dendrites possessed numerous spines and appendages. Many axons could be traced for considerable distances within the medulla (in one instance, up to 8 mm). These axons were last discerned in the contralateral ventral medulla rostral to the level of their cell bodies. The axons of three neurons bifurcated in the ipsilateral medulla; one branch remained ipsilateral and projected caudally, while the other crossed the midline. A small number of counterstained cells of size similar to or larger than the HRP-stained neurons formed a column that constituted the vl-NTS. Based upon our observations of stained and counterstained cells, we conclude that the inspiratory neurons of the vl-NTS are few in number and represent a morphologically homogeneous population. The primary orientation of the dendritic arbors of vl-NTS inspiratory neurons appears to optimize the surface area available to receive synaptic contacts from sensory afferents emerging from the tractus solitarius.

Brainstem neurones which project to the phrenic nucleus were identified using retrogradely transported horseradish peroxidase (HRP) as a marker. Following iontophoretic injection of HRP into the phrenic nucleus, labelled cells were encountered throughout large areas of the medulla and pons, but occurred with characteristic high densities in those regions known to contain phasic respiratory neurones: namely, the ventrolateral solitary tract nucleus (vl-NTS), known as the dorsal respiratory group (DRG), the ambiguus complex or ventral respiratory group (VRG) and the parabrachial pontine nuclei (BCM-KF). In 12 cats a total of 1540 cells was identified within these regions, the relative contralateral and ipsilateral contributions were respectively 72%:28%(vl-NTS), 65%:35% for the ambiguus complex, and 5%:95%(BCM-KF). In addition, labelled cells, predominantly ipsilateral, were observed in the pontine and medullary reticular formation and the vestibular nuclei. The labelled cells of the DRG had round, oval or triangular perikarya. Their mean soma diameter was 18.3 micrometers. The HRP-positive cells of the VRG had slightly larger somas (mean 21.2 micrometers) and they were fusiform and triangular. The neurones labelled in the BCM-KF nuclei were more heterogeneous with a mean soma size of 14.9 micrometers. The bilateral projections to the phrenic nucleus from the DRG and the VRG, and the predominantly ipsilateral projection from the BCM-KF are discussed in relation to current electrophysiological and autoradiographic findings.

Despite extensive neurophysiological work carried out to characterize the crossed phrenic phenomenon, relatively little is known about the morphological substrate of this reflex which restores function to a hemidiaphragm paralyzed by spinal cord injury. In the present study WGA-HRP was injected into normal and functionally recovered hemidiaphragm muscle in rats during the crossed phrenic phenomenon. The retrograde transynaptic transport characteristics of WGA-HRP was utilized to delineate the source of the neurons which mediate the crossed phrenic phenomenon. The results indicated that the neurons which drive phrenic motoneurons in spinal hemisected rats during the crossed phrenic phenomenon are located bilaterally in the rostral ventral respiratory group (rVRG) of the medulla. No transneuronal labeling of propriospinal neurons was noted in either normal or spinal-hemisected rats. Thus, propriospinal neurons do not relay respiratory drive to phrenic motoneurons. The neurons of the rVRG project monosynaptically to phrenic motoneurons. The present results suggest that both crossed and uncrossed bulbospinal pathways from the rVRG collateralize to both the left and right phrenic nucleic and functional recovery of a hemidiaphragm paralyzed by ipsilateral spinal cord hemisection is mediated by supraspinal neurons from both sides of the brain stem. These results are important to our complete understanding of the mechanisms which govern motor recovery in mammals following spinal cord injury.

The hypothesis that excitatory drive is transmitted monosynaptically from bulbospinal medullary respiratory neurons to spinal respiratory motoneurons was tested by an ultrastructural analysis of the phrenic motoneuronal pool in the rat. Combined anterograde labeling of the principal inspiratory bulbospinal neuron population (ventral respiratory group) and retrograde labeling of the phrenic motoneuron pool demonstrated the presence of labeled synaptic profiles, indicating that at least some bulbospinal inspiratory neurons make monosynaptic contacts with phrenic motoneurons. The synaptic boutons of ventral respiratory group neurons that were labeled in the phrenic nucleus had asymmetrical membrane densities at sites of synaptic contact with labeled phrenic somal or dendritic profiles, supporting the notion that this bulbospinal pathway has excitatory contacts with phrenic motoneurons. The morphological types of labeled boutons included three of the eight previously identified bouton types in the phrenic nucleus (Goshgarian and Rafols: Journal of Neurocytology 13:85-109, 1984), including the "S"-terminal, the "NFs"-terminal, and the "F"-terminal. There was no conclusive evidence of labeled double synapses, indicating that this type of synaptic contact is not common in the intact bulbospinal pathway.

Past studies determined that there is a critical period at approximately embryonic day (E)17 during which phrenic motoneurons (PMNs) undergo a number of pivotal developmental events, including the inception of functional recruitment via synaptic drive from medullary respiratory centers, contact with spinal afferent terminals, the completion of diaphragm innervation, and a major transformation of PMN morphology. The objective of this study was to test the hypothesis that there would be a marked maturation of motoneuron electrophysiological properties occurring in conjunction with these developmental processes. PMN properties were measured via whole cell patch recordings with a cervical slice-phrenic nerve preparation isolated from perinatal rats. From E16 to postnatal day 1, there was a considerable transformation in a number of motoneuron properties, including 1) 10-mV increase in the hyperpolarization of the resting membrane potential, 2) threefold reduction in the input resistance, 3) 12-mV increase in amplitude and 50% decrease duration of action potential, 4) major changes in the shapes of potassium- and calcium-mediated afterpotentials, 5) decline in the prominence of calcium-dependent rebound depolarizations, and 6) increases in rheobase current and steady-state firing rates. Electrical coupling among PMNs was detected in 15-25% of recordings at all ages studied. Collectively, these data and those from parallel studies of PMN-diaphragm ontogeny describe how a multitude of regulatory mechanisms operate in concert during the embryonic development of a single mammalian neuromuscular system.

Brainstem neurones which project to the immediate vicinity of the spinal motoneurones which supply the intercostal and abdominal respiratory muscles were identified by means of the retrograde transport of horseradish peroxidase (HRP). A combined electrophysiological and histological technique was used in which recording of phasic inspiratory or expiratory motoneurone activity within upper (T3-T4) or lower (T8-T9) thoracic segments was followed by the ion-tophoretic injection of HRP at these recording sites. HRP labelled cells were concentrated in those brainstem regions known to contain phasic respiratory neurones, namely the ventrolateral nucleus of the solitary tract (vl-NTS) or dorsal respiratory group (DRG), the ambiguus complex or ventral respiratory group (VRG) and the parabrachial pontine (PB) nuclei. In 18 cats, 248 cells were labelled in these three respiratory regions of the brainstem while 668 were much more diffusely distributed in other regions of the medulla and pons. The ipsilateral and contralateral contributions within the respiratory regions were respectively; 23%:77%(DRG), 33%:67%(VRG), 95%:5%(PB). These results are considered in the general context of previous electrophysiological and histological findings, but also with particular reference to a related study of the projections from brainstem neurones to the phrenic nucleus [32].

BACKGROUND: Phrenic nerve injury is a recognized complication following cardiac intervention or surgery. With increasing use of transcatheter procedures to treat drug-refractory arrhythmias, clarification of the spatial relationships between the phrenic nerves and important cardiac structures is essential to reduce risks. METHODS AND RESULTS: We examined by gross dissection the courses of the right and left phrenic nerves in 19 cadavers. Measurements were made of the minimal and maximal distances of the nerves to the superior caval vein, superior cavoatrial junction, right pulmonary veins, and coronary veins. Histologic studies were carried out on tissues from six cavaders. Tracing the course of the right phrenic nerve revealed its close proximity to the superior caval vein (minimum 0.3 +/- 0.5 mm) and the right superior pulmonary vein (minimum 2.1 +/- 0.4 mm). The anterior wall of the right superior pulmonary vein was <2 mm from the right phrenic nerve in 32% of specimens. The left phrenic nerve passed over the obtuse cardiac margin and the left obtuse marginal vein and artery in 79% of specimens. In the remaining specimens, its course was anterosuperior, passing over the main stem of the left coronary artery or the anterior descending artery and great cardiac vein. CONCLUSIONS: The right phrenic nerve is at risk when ablations are carried out in the superior caval vein and the right superior pulmonary vein. The left phrenic nerve is vulnerable during lead implantation into the great cardiac and left obtuse marginal veins.