Cells of origin of propriospinal fibers and of fibers ascending to supraspinal levels. A HRP study in cat and rhesus monkey

In the spinal cord of cat and rhesus monkey the cells of origin of long and short propriospinal fibers and those of the spinal fibers ascending to supraspinal levels were identified by means of retrograde HRP labeling after large unilateral HRP-injections, i.e. in the spinal white and grey matter at different levels, in the pons and in the dorsal column nuclei. The findings indicate the existence of the following arrangement. Long ascending supraspinal fibers arise mainly from neurons in the dorsal grey (laminae I-IV and the medial parts of laminae V and VI) as well as from neurons in the medial part of the ventral grey (lamina VIII), in Clarke's column and in the spinal border cell area. Some neurons in the dorsal grey projects to the dorsal column nuclei, which in turn distribute fibers back to the spinal cord. Long propriospinal fibers mainly derived from neurons in the medial part of the ventral grey (lamina VIII), while short propriospinal fibers are characteristically derived from neurons in the intermediate zone (lateral halves of laminae V and VI and lamina VII). The neurons located laterally in laminae V-VII distribute fibers mainly ipsilaterally, while those located medially in lamina VII distribute them to some degree bilaterally. The findings in cats with transections of either the dorsal or the ventral halves of the spinal white matter (both above and below the injected segment), show that the fibers from the dorsal grey and the lateral parts of laminae V-VII travel mainly through the dorsal half of the white matter, while those from the medial part of lamina VII and from lamina VIII travel mainly through the ventral half.     

The projection of the medial and posterior articular nerves of the cat's knee to the spinal cord

We studied the spinal projections of the medial and posterior articular nerves (MAN and PAN) of the knee joint in the cat with the aid of the transganglionic transport of horseradish peroxidase. The afferent fibers of the MAN entered the spinal cord via the lumbar dorsal roots L5 and L6 and those of the PAN entered via the dorsal roots L6 and L7. Within the dorsal root ganglia, most labeled neurons had small to medium diameters. A relatively higher number of medium-size cell bodies were labeled from the PAN than from the MAN. In the spinal cord labeled MAN afferent fibers and terminations were most dense in the L5 and L6 segments, and those of the PAN were most dense in L6 and L7, that is, in the respective segments of entry. Labeled afferent fibers from both nerves projected rostrally at least as far as L1 and caudally as far as S2. Labeled fibers were found in Lissauer's tract as well as in the dorsal column immediately adjacent to the dorsal horn. In the spinal gray matter, both nerves had two main projection fields, one in the cap of the dorsal horn in lamina I, the other in the deep dorsal horn in laminae V-VI and the dorsal part of lamina VII. Both nerves, but particularly the PAN, projected to the medial portion of Clarke's column. No projection was found to laminae II, III, and IV of the dorsal horn or to the ventral horn. Since these findings parallel observations on hindlimb muscle afferent fibers, the present data support the existence of a common pattern for the central distribution of deep somatic afferent fibers.     

Fine afferent fibers from viscera do not terminate in the substantia gelatinosa of the thoracic spinal cord

Transganglionic transport of horseradish peroxidase (HRP) has been used to study the anatomy of the central projection of somatic and visceral afferent fibers to the thoracic spinal cord of the cat. A dense concentration of somatic afferent fibers and terminals was found in laminae I and II of the dorsal horn and more scattered terminals were present in laminae III, IV and V and in Clarke's column. In contrast, visceral afferent fibers and terminals were found only in lamina I or reaching lamina V via a small bundle of fibers located in the lateral border of the dorsal horn. These results indicate that fine afferent fibers from viscera, unlike those of cutaneous origin, do not project to the substantia gelatinosa (lamina II) of the dorsal horn.     

The involvement of spinal bovine adrenal medulla 22-like peptide, the proenkephalin derivative, in modulation of nociceptive processing

Bovine adrenal medulla 22 (BAM22), one of the cleavage products of proenkephalin A, possesses high affinity for opioid receptors and sensory neuron‐specific receptor (SNSR). The present study was designed to examine the expression of BAM22 in the spinal cord and dorsal root ganglion (DRG) of naive rats as well as in a model of inflammation. BAM22‐like immunoreactivity (BAM22‐IR) was expressed in fibers in the spinal cord, with high density seen in lamina I in naïve rats. The expression of BAM22‐IR in the superficial laminae was greatly reduced following dorsal rhizotomy. BAM22‐IR was also located in 19% of DRG cells, mainly in the small‐ and medium‐sized subpopulations. Following injection of complete Freund's adjuvant (CFA) in the hindpaw, the expression of BAM22‐IR in the superficial laminae of the spinal cord and small‐sized DRG neurons on the ipsilateral side was markedly increased. Double labeling showed that the Fos‐positive nucleus was surrounded by BAM22‐IR cytoplasm in the spinal dorsal horn neurons or closely associated with BAM22‐IR fibers in the superficial laminae. Furthermore, CFA‐induced mechanical allodynia in the inflamed paw was potentiated by intrathecal administration of anti‐BAM22 antibody. Together, these results demonstrate for the first time that BAM22‐like peptide is mainly located in the superficial laminae of the spinal cord and mostly originates from nociceptive DRG neurons. BAM22 could thus act as a ligand for presynaptic opioid receptors and SNSR. Our study also provides evidence suggesting that BAM22 plays a role in the modulation of nociceptive processing at the spinal level under normal and inflammatory conditions.     

Trajectory of group Ia and Ib fibers from the hind-limb muscles at the L3 and L4 segments of the spinal cord of the cat

Nineteen physiologically identified group Ia and five group Ib fibers at the L3 and L4 levels of the spinal cord originating from various hind-limb muscles were intraaxonally injected with horseradish peroxidase (HRP). The trajectories of the stained axons were reconstructed. They extended for distances of 8.6 mm-18.0 mm rostrocaudally. Ascending axons ran in various regions of the dorsal funiculus: The ascending axon from a toe muscle (3 microns in diameter) ran in the ventral most part of the paramedian region; those from shank muscles (3.0-5.0 microns) in both dorsal and ventral paramedian regions; those from thigh muscles (5.0-7.0 microns) in both the paramedian and the more lateral regions; and those from hip muscles (6.0-7.0 microns) in the lateral region. Main collaterals arising from the parent fiber were given off at intervals of 0.5-6.2 mm (mean 2.4 mm). Collaterals of a fiber from a toe muscle (1.0 micron in diameter) entered Clarke's column from the dorsomedial side and ramified mostly in the dorsomedial one-third of the column. Collaterals of fibers from shank muscles (1.0-2.0 microns) entered Clarke's column from the dorsal side and terminated in its middle parts as well as in laminae V-VII. Collaterals of fibers from thigh muscles (1.0-2.5 microns) passed lateral to or through the lateral part of Clarke's column and terminated in its ventrolateral part and in laminae V-VIII. Collaterals of fibers from hip muscles (1.5-2.5 microns) passed lateral to Clarke's column and ramified mostly in laminae VII-IX. As the muscle of origin became more proximal, the proportion of termination outside of Clarke's column progressively increased. Thus, the trajectory of group I fibers was somatotopically organized both in the dorsal funiculus and in the gray matter. The long axis of boutons ranged from 0.5 to 17 microns in Ia fibers and from 0.5 to 8 microns in Ib fibers. "Giant" Ia boutons (above 7 microns) were found both in and outside Clarke's column.     

Analysis of calcitonin gene-related peptide-like immunoreactivity in the cat dorsal spinal cord and dorsal root ganglia provide evidence for a multisegmental projection of nociceptive C-fiber primary afferents.

Recent studies have suggested that calcitonin gene-related peptide (CGRP) can be used as a marker for a subpopulation of nociceptive primary afferents. Consequently, CGRP-immunoreactive (CGRP-IR) primary afferents have been reported to project many segments rostral to their segment of entry and to send collaterals into the superficial and deep laminae of the dorsal horn. This study reports that some CGRP-IR primary afferents of sacral origin project rostral through the ipsilateral lumbar enlargement in the cat. The ultrastructure of these multisegmentally projecting primary afferent axons and terminals identified in a partially denervated cat was examined and compared to the ultrastructure of CGRP-IR afferents from an intact cat. Retrograde transport of wheatgerm agglutinin-colloidal gold injected into the cat L4 spinal cord resulted in labeling of primary afferent cell bodies in the ipsilateral L4-S1 dorsal root ganglia (DRG). Analysis of every fourth section through the ipsilateral S1 DRG revealed as many as 1,000 retrogradely labeled neuronal cell bodies. One third of these cell bodies were double labeled for CGRP-like immunoreactivity. The number of single- and double-labeled cells increased in ganglia closer to the injection site (L4-L7). At the ultrastructural level, in the lumbosacral dorsal spinal cord of a normal cat, most CGRP-IR axons were unmyelinated, while the rest were small myelinated axons. In both the superficial dorsal horn and lamina V, CGRP-IR varicosities were dome shaped, scallop shaped, or elongated. The CGRP-IR varicosities contained small agranular vesicles and frequently a few dense core vesicles. These labeled varicosities formed asymmetric synapses on unlabeled dendritic spines, shafts, or neuronal somata. One cat received multiple unilateral dorsal rhizotomies (S1-L4) and an ipsilateral hemisection (mid L4). CGRP-IR axons and terminals were found within each of the rhizotomized segments, although their density was greatly reduced compared to that in the intact animals. In Lissauer's tract the majority (greater than 90%) of CGRP-IR fibers were unmyelinated. In laminae I and V, the remaining CGRP-IR varicosities were mainly the dome-shaped varicosities morphologically similar to those observed in the normal spinal cords. They contained small agranular vesicles and a few dense core vesicles and formed asymmetric synapses on unlabeled dendritic shafts and spines. These data demonstrate that unmyelinated, presumably C-fiber nociceptive primary afferents and some small myelinated A-delta nociceptive primary afferents of sacral origin project rostral through the cat lumbar enlargement and make synaptic connections in both the superficial and deep laminae of the cat dorsal spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Projections to the spinal cord from medullary somatosensory relay nuclei.

Descending projections to the spinal card from the dorsal column nuclei were studied in the cat, rat and monkey with the retrograde horseradish peroxidase (HRP) technique, and in the cat with the autoradiographic anterograde axonal transport technique. Retrogradely labeled neurons were seen in the dorsal column nuclei after HRP injections at all levels of the spinal cord and additionally in the magnocellular division of the spinal caudalis nucleus of the trigeminal nerve after injections into cervical spinal segments in all three species. HRP-positive neurons were predominantly located along the middle of the rostro-caudal axis of the dorsal column nuclei and amongst the fusiform, triangular and polygonal cells that surround, especially ventrally, the cell nest zone containing thalamic relay neurons. The labeled neurons are densely concentrated in those portions of the dorsal column nuclei where most corticofugal and non-primary afferent projections terminate and where the terminal distribution of primary afferent fibers is overlapping and diffuse. Previous studies have shown that most neurons in this middle and ventral region do not project to the thalamus or cerebellum. The majority of the cells in the dorsal column nuclei with descending axons or axon collaterals project by way of the ipsilateral dorsal columns, but some fibers project into the dorsolateral funiculus; the descending trigeminal fibers course in the dorsolateral funiculus. The terminal fields for these fibers in the cervical spinal cord include the lateral cervical nucleus, laminae IV and V, and possibly lamina I. These results indicate that the dorsal column nuclei may contribute to a feedback mechanism regulating the flow of sensory information ascending along other somatosensory spinal pathways.     

Phylogenetic changes in the expression of delta opioid receptors in spinal cord and dorsal root ganglia

To assess the validity of rodent models for investigating the role of delta opioid receptors (DOR) in analgesia, the distribution of DOR binding and mRNA were compared between rodent and primate spinal cord and dorsal root ganglia (DRG), using receptor autoradiography and in situ hybridization, respectively. In mouse and rat spinal cord, [(125)I]-deltorphin-labeled DOR binding sites were detected throughout the gray matter. In contrast, in primate and particularly in human spinal cord, DOR binding was mainly present in laminae I-II, with little to no binding in deeper layers. Accordingly, in rodent spinal cord, DOR mRNA was expressed by a large number of neurons distributed throughout the ventral and dorsal horns, whereas in the primate, DOR expression was significantly lower, as evidenced by a moderate number of labeled cells throughout the gray matter in monkey and by only few labeled cells in human, mainly in Clarke's column and lamina IX. Major species differences in DOR expression were also observed in primary afferent cells bodies. In rat DRG, intense DOR mRNA hybridization was primarily observed over large ganglion cells immunopositive for neurofilament 200. In contrast, in monkey and human DRG, DOR mRNA was primarily detected over small and medium-sized ganglion cells. These results demonstrate major differences in the expression and distribution of DOR in the spinal cord and DRG between mammalian species. Specifically, they point to a progressive specialization of DOR toward the regulation of primary somatosensory, namely nociceptive, inputs during phylogeny and suggest that the effects of DOR agonists in rodents may not be entirely predictive of their action in humans.     

Vanilloid receptor VR1 is both presynaptic and postsynaptic in the superficial laminae of the rat dorsal horn.

Terminals in the rat spinal cord that express the vanilloid receptor VR1 are from small and medium dorsal root ganglion (DRG) neurons and appear prominent in lamina I and inner lamina II. Because primary afferents from these neurons can be myelinated or unmyelinated and their terminals in these laminae can be of various morphological and functional types, we undertook this study to identify the type(s) of VR1-positive afferent fibers and terminals. In the DRG, many small and medium-sized neurons are immunopositive. Under electron microscopy, dorsal root afferents that are immunopositive for VR1 are predominantly unmyelinated. Large numbers of VR1-positive terminals in lamina I are of the nonglomerular type and may contain dense core vesicles. VR1 immunoreactivity in terminals in lamina I is in good agreement with data on noxious, heat-sensitive neurons in the dorsal horn. Two types of glomerular afferent terminals in lamina II also are immunopositive for VR1. In both laminae, most VR1-positive terminals are distinct from substance P-positive terminals. However, the immunoreactivity in lamina II also is prominent in dendrites that are contacted by primary afferent endings. Because we also observed patchy immunostaining in cell bodies in lamina II, this unexpected result may reflect synthesis of VR1 by neurons in this lamina. However, because dorsal rhizotomy abolishes VR1 staining in both laminae I and II, it is suggested that the expression and intracellular dynamics of VR1 in lamina II neurons are controlled by presynaptic input.     

Projections from the red nucleus to the parvicellular reticular formation and the cervical spinal cord in the rat, with special reference to innervation by branching axons

After biotinylated dextranamine injection into the dorsal part of the red nucleus (RN) in the rat, labeled axons were distributed contralaterally in the lateral tegmental field including the parvicellular reticular formation (RFp), and ipsilaterally in the medial reticular formation. In the cervical spinal cord, labeled axons were present bilaterally with a contralateral dominance mainly in laminae V–VI and the dorsal part of laminae VII. After ipsilateral injections of rhodamine dextranamine, Fluoro-ruby (FR) into the RFp and Fluoro-gold (FG) into the upper cervical spinal cord, a population of FR-labeled neurons was found in the dorsal part of the contralateral RN, whereas the majority of FG-labeled neurons were located more ventrally. However, some of them were intermingled with FR-labeled neurons, and as many as one-third of FR-labeled neurons were labeled with FG. After combined injections of FR into the RFp and FG into the lower cervical spinal cord, RN neurons labeled with FG existed more ventrally than those retrogradely labeled from the upper cervical spinal cord, and less than 10% of FR-labeled neurons were labeled with FG. The present data suggest that axon collateral innervation of the RFp and the upper cervical spinal cord by single RN neurons may be responsible for coordinating head and orofacial movements.     

Immunohistochemical distribution of serotonin in spinal autonomic nuclei: I. Fiber patterns in the adult rat

The differential distribution of serotonin (5HT) fibers in spinal laminae VII and X is described for the adult rat. The results indicate that descending 5HT fibers preferentially innervate those regions of lamina VII that contain sympathetic and parasympathetic neurons. In lamina X, especially the dorsal commissural nucleus, large numbers of 5HT fibers are observed throughout the spinal cord. Moreover, sympathetic nuclei are more richly innervated with 5HT than the spinal parasympathetic nuclei. Spinal cord hemisections reveal that spinal autonomic nuclei are differentially innervated: ipsilateral serotoninergic projections to the intermediolateral cell column are preferentially interrupted. In addition, a large crossed 5HT projection exists throughout the length of the spinal cord that decussates five to six spinal segments rostral to its termination. Both crossed and uncrossed 5HT fibers span many spinal segments and have large numbers of collaterals. Spinal cord transections show that the vast majority of spinal 5HT descends from the brainstem but that some 5HT fibers are of intrinsic origin.     

Anatomical organization of the spinocerebellar system in the cat,as studied by retrograde transport of horseradish peroxidase

The distribution of spinocerebellar tract (SCT) neurons has been studied in the entire length of the spinal cord of the cat following injections of horseradish peroxidase into the cerebellum, and whether or not the axons of the labeled neurons crossed within the spinal cord was determined in cases with injections preceded by hemisections at the cervical levels. The SCTs were classified into the following corssed and uncrossed tracts according to the cell origin and the fiber course; The crossed SCTs originate from (1) the central cervical nucleus (the CCN-SCT), (2) lamina VIII neurons of the cervical to the lumbar cord (the lamina VIII-SCT), (3) spinal border cells (the border cell-SCT), (4) neurons in the medial lamina VII of the lumbar to the caudal spinal segments (the medial lamina VII-SCT), (5) ventral horn neurons (laminae VII and VIII) of the sacral and caudal segments (the ventral horn-SCT) and (6) dorsal horn neurons (lamina V) of the sacral and the caudal segments (the dorsal horn-SCT). The uncorssed tracts originate from (1) neurons of the medial lamina VI of C2 to T1 (the medial lamina VI-SCT of the cervical cord), (2) neurons in the central part of lamina VII of C6 to T1 (the central lamina VII-SCT of the cervical enlargement), (3) lamina V neurons of the lower cervical to the lumbar cord (the lamina V-SCT), (4) Clarke's column (the Clarke's column-SCT) and (5) neurons in the medial lamina VI of L5 and L6 (the medial lamina VI-SCT of the lumbar cord). The present study suggests that the spinocerebellar system originates from more diverse laminae than has previously been known, and further refined studies on the topographic projections of each tract will yield more important and valuable information in this field.     

Immunohistochemical localization of calbindin-D28k and calretinin in the spinal cord of lungfishes

A common pattern of distribution of neurons and fibers containing the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the spinal cord of terrestrial vertebrates has been recently demonstrated. Lungfishes are considered the closest living relatives of tetrapods, but practically no experimental data exist on the organization of their spinal cord. By means of immunohistochemical techniques, the localization of CB and CR was investigated in the spinal cord of the African (Protopterus dolloi) and Australian (Neoceratodus forsteri) lungfishes. Abundant cell bodies and fibers immunoreactive for either CB or CR were widely distributed throughout the spinal cord. A large population of immunoreactive cells was found in the dorsal column of the gray matter in both species, and abundant cells were distributed in the lateral and ventral columns. Ventrolateral motoneurons and multipolar cells were only intensely CB and CR immunoreactive in Neoceratodus. For the most part, separate cell populations contained either CB or CR, but a small subset of dorsally located neurons contained both in the two lungfishes. Colocalization was found in motoneurons and in ventrolaterally located cells only in Neoceratodus. Fiber labeling showed a predominance of CR-containing axons in the lateral and ventral funiculi of presumed supraspinal origin. These results show that lung-fishes and tetrapods have many features in common, suggesting that primitive anatomical, and likely functional, organization of the spinal cord of tetrapods is present in lungfishes.     

The location of spinal neurons with long descending axons (long descending propriospinal tract neurons) in the cat: a study with the horseradish peroxidase technique.

The distribution spinal neurons with long descending axons was studied in the cat by means of retrograde transport of horseradish peroxidase. Labeled neurons appeared bilaterally in the cervical and the thoracic cord following injections in the lumbosacral cord. In some cases hemisections were made rostrally and contralaterally to the injections in an attempt to determine whether or not the axons crossed. Neurons with uncrossed descending axons were located in laminae I, V, VII and VIII. Lamina I neurons were present in all the spinal segments. In lamina V labeled neurons were distributed mainly laterally in the cervical cord but medially and laterally in the thoracic cord. In the upper cervical and the thoracic cord laminae VII and VIII neurons were distributed very densely along the lateral cord, accounting for 30 and 40 of the total labeled neurons, respectively. In the cervical enlargement they were located in the middle part of lamina VII and in lamina VIII, accounting for about 25% of the total labeled neurons. Neurons with crossed descending axons were found in laminae V, VII and VIII, in the medial part of lamina VII including the intermediomedial nucleus of the thoracic levels and close to the central canal. Lamina V neurons were very small in number. The largest collections of labeled neurons were present in the medial part of laminae VII and VIII. They accounted for about 45% to 55% and 37% of the total in the cervical and the thoracic cord. These neurons may function as the long spinal reflex paths for forelimb-hindlimb synergies and the intercalated paths between the supraspinal descending tracts and the spinal motor centers.     

Spinal cord neuron inputs to the cuneate nucleus that partially survive dorsal column lesions: A pathway that could contribute to recovery after spinal cord injury

Dorsal column lesions at a high cervical level deprive the cuneate nucleus and much of the somatosensory system of its major cutaneous inputs. Over weeks of recovery, much of the hand representations in the contralateral cortex are reactivated. One possibility for such cortical reactivation by hand afferents is that preserved second‐order spinal cord neurons reach the cuneate nucleus through pathways that circumvent the dorsal column lesions, contributing to cortical reactivation in an increasingly effective manner over time. To evaluate this possibility, we first injected anatomical tracers into the cuneate nucleus and plotted the distributions of labeled spinal cord neurons and fibers in control monkeys. Large numbers of neurons in the dorsal horn of the cervical spinal cord were labeled, especially ipsilaterally in lamina IV. Labeled fibers were distributed in the cuneate fasciculus and lateral funiculus. In three other squirrel monkeys, unilateral dorsal column lesions were placed at the cervical segment 4 level and tracers were injected into the ipsilateral cuneate nucleus. Two weeks later, a largely unresponsive hand representation in contralateral somatosensory cortex confirmed the effectiveness of the dorsal column lesion. However, tracer injections in the cuneate nucleus labeled only about 5% of the normal number of dorsal horn neurons, mainly in lamina IV, below the level of lesions. Our results revealed a small second‐order pathway to the cuneate nucleus that survives high cervical dorsal column lesions by traveling in the lateral funiculus. This could be important for cortical reactivation by hand afferents, and recovery of hand use. J. Comp. Neurol. 523:2138–2160, 2015. © 2015 Wiley Periodicals, Inc.     

Alteration of PACAP distribution and PACAP receptor binding in the rat sensory nervous system following sciatic nerve transection

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widely expressed neuropeptide that has been involved in nerve regeneration, neurone survival and nociception. In this study, the distribution of PACAP and PACAP-receptors were investigated in rat dorsal root ganglia (DRG), spinal cord and medulla oblongata at 3, 7 or 14 days following unilateral sciatic nerve transection using immunohistochemistry, 125I-PACAP-binding and in situ hybridisation. In control (contralateral side) DRG, about 30% of the nerve cell bodies (92% being small) were PACAP-immunoreactive (PACAP-IR). In the spinal cord, PACAP-IR fibres were seen in laminae I-II but not in the gracile nuclei. Following sciatic nerve transection, PACAP-IR fibres appeared in the gracile nuclei and occasionally in the deeper laminae of the dorsal horn consistent with the relative increase in larger PACAP-IR DRG neurones. However, the relative number of small PACAR-IR neurones was significantly lower on the transected side as compared to the control side suggesting a dual reaction for PACAP in the DRG following nerve injury. 125I-PACAP-binding was found in laminae I-II, around the central canal and in the gracile nuclei but not in the DRG. At 14 days after transection, 125I-PACAP-binding density was significantly reduced in the ipsilateral dorsal horn. PACAP-receptor (PAC(1)) mRNA was detected in neurones of the dorsal and ventral horn and in the gracile nuclei with no overt changes observed after transection. Very few DRG nerve cell bodies contained PAC(1) mRNA. The findings are consistent with a role for PACAP both in nociception and regeneration.     

Immunoreactive TRPV-2 (VRL-1), a capsaicin receptor homolog, in the spinal cord of the rat

The vanilloid receptor-like 1 protein (VRL-1, also called TRPV2) is a member of the TRPV family of proteins and is a homolog of the capsaicin/vanilloid receptor (VR1, or TRPV1). Although VRL-1 does not bind capsaicin, like VR1 it is activated by noxious heat (>52 degrees C). Unlike VR1, however, VRL-1 is primarily expressed by medium- and large-diameter primary afferents, which suggests that nociceptive processing is but one of the functions to which VRL-1 contributes. To provide information on the diverse spinal circuits that are engaged by these VRL-1-expressing primary afferents, we completed a detailed immunocytochemical map of VRL-1 in rat spinal cord, including light and electron microscopic analysis, and generated a more comprehensive neurochemical characterization of VRL-1-expressing primary afferents. Consistent with previous reports, we found that VRL-1 and VR1 are expressed in different dorsal root ganglion (DRG) cell bodies. Almost all VRL-1-expressing cells labeled for N52 (a marker of myelinated afferents), consistent with VRL-1 expression in Adelta and Abeta fibers. EM analysis of the DRG and dorsal roots confirmed this and revealed two categories of neurons based on the intensity of immunolabeling. The densest VRL-1 immunoreactivity in the spinal cord was found in lamina I, inner lamina II, and laminae III/IV. This is consistent with the expression of VRL-1 by myelinated nociceptors that target laminae I and IIi and in nonnociceptive Abeta fibers that target laminae III/IV. Dorsal rhizotomy reduced, but did not eliminate, the immunostaining in all dorsal horn laminae, which indicates that VRL-1 expression derives from both DRG cells and from neurons intrinsic to the brain or spinal cord. Spinal hemisection reduced immunostaining of the ipsilateral dorsal columns in segments rostral to the lesion and in the dorsal column nuclei, presumably from the loss of ascending Abeta afferents, but there was no change caudal to the lesion. Thus, supraspinal sources of dorsal horn VRL-1 immunoreactivity are likely not significant. Although we never observed VRL-1 immunostaining in cell bodies in the superficial dorsal horn, there was extensive labeling of motoneurons and ventral root efferents-in particular, in an extremely densely labeled population at the lumbosacral junction. Finally, many ependymal cells surrounding the central canal were intensely labeled. These results emphasize that VRL-1, in contrast to VR1, is present in a diverse population of neurons and undoubtedly contributes to numerous functions in addition to nociceptive processing.     

The spinal course and medullary termination of myelinated renal afferents in the rat

Myelinated afferent fibers, recorded in the left renal nerve of rats, were antidromically activated by discrete electrical stimulation of the cervical spinal cord and the caudal medulla. The lowest thresholds for activation of these fibers were found in the most medial portion of the ipsilateral fasciculus gracilis. This region of minimum threshold continued rostrad through the nucleus commissuralis. Based on threshold vs depth contours, fibers appeared to terminate in the ipsilateral nucleus gracilis and nucleus solitarius. Myelinated fibers could be activated by punctate pressure on the renal hilus. Action potentials generated by hilar pressure collided with antidromically-conducted action potentials elicited by electrical stimulation at cervical levels. We conclude that myelinated renal afferents carry information from intrarenal receptors, via the dorsal column system, to both visceral afferent and dorsal column nuclei.     

Regeneration into the Spinal Cord of Transected Dorsal Root Axons Is Promoted by Ensheathing Glia Transplants

The permissivity of adult olfactory bulb to the ingrowth of olfactory axons could be due to the unique properties of ensheathing glia. To test whether these glial cells could be used to promote axonal regeneration in a spontaneously nonregenerating system, we transplanted suspensions of pure ensheathing cells into a rhizotomized spinal cord segment. Ensheathing cells were purified away from other cell types by immunoaffinity, using anti-p75 nerve growth factor receptor. After laminectomy at the lower thoracic level, the spinal cord was exposed and one dorsal root (T10) was completely transected at the cord entry point. The root stump was microsurgically anastomosed to the cord and a suspension of ensheathing cells was transplanted in the spinal cord at the dorsal root entry zone. Three weeks after transplantation, numerous regenerating dorsal root axons were observed reentering the spinal cord. Ingrowth of dorsal root axons was observed using Dil and antibodies against calcitonin gene-related peptide and growth-associated protein. Primary sensory afferents invaded laminae 1, 2, and 3, grew through laminae 4 and 5, and reached the dorsal grey commissure and lamina 4 of the contralateral side. We did not observe regenerating axons within the ipsilateral ventral horn and dorsal column. Transplanted ensheathing cells reached the same laminae as axons. Neither ensheathing cells nor regenerating axons invaded those laminae they did not inervate under normal circumstances. In conclusion, the regeneration of injured dorsal root axons into the adult spinal cord was possible after ensheathing glia transplantation. The use of ensheathing cells as stimulators of axonal growth might be generalized to other central nervous system injuries.