Nerve growth factor receptor immunoreactivity in the neuronal perikarya of human sensory and sympathetic nerve ganglia

We examined immunohistochemically the dorsal root ganglia, sympathetic ganglia, spinal cord, ventral and dorsal roots, and sciatic nerves obtained at autopsy from adult humans, using a monoclonal antibody against the human nerve growth factor receptor. We observed labelling in a granular pattern in the neuronal perikarya of dorsal root and sympathetic nerve ganglia. Ventral horn cells and axons were not labelled.     

Interaction of transplanted olfactory-ensheathing cells and host astrocytic processes provides a bridge for axons to regenerate across the dorsal root entry zone

A single fourth lumbar dorsal rootlet was transected at the entry point into the spinal cord. The nerve fibres were labelled with biotin dextran injected into the rootlet. An endogenous matrix containing olfactory-ensheathing cells (OECs) labelled with green fluorescent protein was applied to the opposing cut surfaces of the rootlet and the spinal cord, which were then brought into apposition and held in place by fibrin glue. Two weeks later, a ladderlike bridging structure has been formed by astrocytic processes growing out for about 200-300 microm from the spinal cord. The transplanted cells remained largely confined to this area. They were elongated along the nerve axis but did not enter the spinal cord itself. Labelled dorsal root axons crossed the repaired dorsal root entry zone in alignment with the bridging astrocytic processes and the transplanted cells and then proceeded beyond the transplant to enter the grey matter of the dorsal horn and send axons both rostrally and caudally for at least 10 mm in the white matter of the ascending dorsal columns.     

Expression of cholinergic phenotype by embryonic ventral horn neurons transplanted into the spinal cord in the rat

Solid grafts of E12 embryonic spinal ventral horn were transplanted into motoneuron-depleted adult lumbar spinal cord in the rat. A muscle was implanted parallel to the vertebral column with its nerve inserted into the lumbar cord at the site of transplantation so as to provide a target for innervation by the grafted neurons. Previous retrograde labelling studies have shown that modest numbers of grafted motoneuron-like cells participate in the muscle's reinnervation and these are often found outside the graft within the host spinal cord. However, Nissl stained sections show that larger numbers of neurons survive within tissue recognisable as being of graft origin. In this study we have examined the expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) by neurons within the graft. These enzymes are involved in cholinergic neurotransmission and are characteristic of motoneurons. Thirty-four to seventy days following transplantation the grafts contained numerous neurons with acetylcholinesterase (AChE) activity. Different patterns of AChE staining were observed which probably reflected the degree of differentiation and maturation within the graft. AChE positive neurons were found in isolation or in groups resembling developing motor pools. Most of the AChE-positive neurons appeared immature with scant cytoplasm. However, neurons could be found which appeared relatively mature with a regularly shaped nucleus, prominent nucleolus and Nissl bodies. The grafts contained few AChE-positive axons and no dense plexuses of varicose fibres around the neurons such as are found around motoneurons in the mature ventral horn. Comparisons between the size of AChE-positive neurons in the graft and the size of AChE-positive neurons in the developing ventral horn found that the size of grafted neurons to be intermediate between the sizes of spinal motoneurons at E19 and P0. Far fewer grafted neurons were found to be immunoreactive for choline acetyltransferase (ChAT) than histochemically reactive for AChE. This was consistent with our findings in the spinal cord during normal development where we found that fixation and staining procedures which labelled adult motoneurons failed to reliably demonstrate ChAT immunoreactivety in normal motoneurons prenatally, although AChE histochemical reactivity could be demonstrated as early as E16. We conclude that the grafts contain numbers of immature motoneurons which fail to proceed beyond a certain stage of development, perhaps because of a failure to form appropriate efferent and afferent connections.     

Synaptic connections between spinal motoneurons and dorsal root ganglion cells in the cat

Light- and electron-microscopical horseradish peroxidase (HRP) studies have been employed in conjunction with a degeneration study in order to clarify the origin and axonal passage of afferent synaptic terminals in cat dorsal root ganglia. After injection of HRP into ganglia (C3) without involvement of the ventral roots and spinal nerves, a few ipsilateral spinal ventral horn neurons (C3) were retrogradely labeled with HRP. The labeled neurons were localized in the dorsomedial and the ventromedial nuclei. Following ventral rhizotomy of C3, the afferent terminals in the ganglia (C3) anterogradely degenerated and contained accumulated and disintegrated neurofilaments, depleted, aggregated and enlarged synaptic vesicles. Subsequent to an HRP and wheat germ agglutinin (WGA)-HRP-mixture injection into the dorsal neck or suboccipital muscles, many spinal motoneurons (C3) were labeled retrogradely with an HRP mixture. On the other hand, the afferent synaptic terminals in ganglia contained the membrane-bound and electron-dense bodies which were anterogradely labeled with an HRP mixture in addition to the normal synaptic elements. The present findings strongly suggest that some spinal motoneurons send their axon collaterals to the dorsal root ganglia, in which the terminals of the axon collaterals directly synapse with the dorsal root ganglion cells.     

Transplantation of embryonic noradrenergic neurons in two models of adult rat spinal cord injury: ultrastructural immunocytochemical study

The synaptic connections established by grafted noradrenergic (NA) neurons into the lesioned adult rat spinal cord were analysed using immunocytochemistry at the electron microscopic level. An embryonic cell suspension of the locus coeruleus region from E-13 rat embryos was transplanted into the spinal cord following either: (1) spinal cord transection or (2), partial selective denervation by 6-hydroxy dopamine (6-OH DA). One month after grafting, the NA-neurons established, in the two models, an innervation pattern similar to that found in the intact spinal cord. In both models, the transplanted NA-immunoreactive neurons formed extensive synaptic contacts with dendrites, spines and perikarya. The proportion of axodendritic and axospinous contacts was inverse in the two models. The first model thus reproduced more closely the normal synaptic pattern prefering dendritic targets, which could correspond to a better integration of the graft. In the second model, a partially NA-denervated spinal cord, there existed a competition between residual intrinsic and grafted neuron-derived fibres, which presumably affects synaptogenesis. In conclusion, the present study illustrate the complexity of cell interations conducting to the formation of a specific circuitry. Recognition phenomenon are likely modulated by space constraints, which ultimately shape-up the geometry of synaptic contacts.     

Growth of central substance P-containing neurons into superior cervical ganglia transplanted in the spinal cord of adult rats

Superior cervical ganglia (SCG) contain substance P-like immunoreactive (SP-IR) fibers but not SP-IR neurons. In the present study, SCG were excised from adult rats and transplanted into the same animal's spinal thoracic cord (Th10). One or two weeks after the operation, SP-IR fibers from the host spinal cord or a higher level had grown and entered the transplanted SCG where they formed direct contacts with SCG neurons. However, these phenomena could not be observed when dorsal root ganglia (L4), which contained numerous SP-IR cells, were transplanted into their own spinal cord (Th10). This suggests that the SP-IR neuron system in the adult is able to grow "new axons' to the grafted tissue to form a "new SP-IR' neuronal circuit when the grafted tissue has lost its own SP-IR input.     

Calcium-binding proteins, parvalbumin- and calbindin-D 28k-immunoreactive neurons in the rat spinal cord and dorsal root ganglia: a light and electron microscopic study

The distribution of two calcium-binding proteins, parvalbumin (PV) and calbindin-D 28K (CaBP), was studied by the peroxidase-anti-peroxidase immunohistochemical method at the light and electron microscopic level in the rat spinal cord and dorsal root ganglia. The possible coexistence of these two proteins was also investigated. PV-positive neurons were revealed in all layers of the spinal cord, except lamina I, which was devoid of labelling. Most of the PV-positive cells were found in the inner layer of lamina II, lamina III, internal basilar nucleus, central gray region, and at the dorsomedial and ventromedial aspects of the lateral motor column in the ventral horn. Neuronal processes intensely stained for PV sharply delineated inner lamina II. With the electron microscope most of them appeared to be dendrites, but vesicle containing profiles were also found in a smaller number. CaBP-positive neurons appeared to be dispersed all over the spinal gray matter. The great majority of them were found in laminae I, II, IV; the central gray region; the intermediolateral nucleus; and in the ventral horn just medial to the lateral motor column. Laminae I and II were densely packed with CaBP-positive punctate profiles that proved to be dendrites and axons in the electron microscope. A portion of labelled neurons in lamina IV and on the ventromedial aspect of the lateral motor column in the ventral horn disclosed both PV- and CaBP-immunoreactivity. All of the funiculi of the spinal white matter contained a large number of fibres immunopositive for both PV and CaBP. The highest density of CaBP-positive fibres was found in the dorsolateral funiculus, which was also densely packed with PV-positive fibres. PV-positive fibres were even more numerous in the dorsal part of the dorsal funiculus. The territory of the gracile funiculus in the brachial cord and that of the pyramidal tract in its whole extent were devoid of labelled fibres. In the thoracic cord, the dorsal nucleus of Clarke received a large number of PV-positive fibres. Dorsal root ganglia displayed both PV- and CaBP-immunopositivity. The cell diameter distribution histogram of PV-positive neurons disclosed two peaks--one at 35 microns and the other at 50 microns. CaBP-positive cells in the dorsal root ganglia corresponded to subgroups of small and large neurons with mean diameters of 25 microns and 45 microns, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fate of B1-B2 and B3 rhombencephalic cells transplanted into the transected spinal cord of adult rats: light and electron microscopic studies.

Embryonic cell suspensions (14-day embryos) containing either B3 or B1-B2 serotonergic cell groups were obtained by microdissection of specific rhombencephalic regions and transplanted into the transected spinal cord of adult male Sprague-Dawley rats. After 3 months of survival, the animals were sacrificed and the spinal cords processed for the immunocytochemical detection of serotonin (5-HT). 5-HT-immunoreactive fibers from B1-B2-grafted cells were selectively distributed in the ventral horn and the intermediolateral cell column (IML) where they established conventional synaptic contacts. However, B3 5-HT cells grew and extended their processes into the dorsal horn where in addition we observed scarce synaptic contacts as in the normal spinal cord. These results suggest that the specificity of the 5-HT innervation of the spinal cord by grafted neurons is due, at least partly, to the presence of local mechanisms mediating guidance and cell recognition, possibly operating in conjunction with preexisting substrate pathways.     

Regeneration of adult dorsal root axons into transplants of embryonic spinal cord

Transplants of the embryonic rat spinal cord survive and differentiate in the spinal cords of adult and newborn host rats. Very little is known about the extent to which these homotopic transplants can provide an environment for regeneration of adult host axons that normally terminate in the spinal cord. We have used horseradish peroxidase injury filling and transganglionic transport methods to determine whether transected dorsal roots regenerate into fetal spinal cord tissue grafted into the spinal cords of adult rats. Additional transplants were examined for the presence of calcitonin gene-related peptide-like immunoreactivity, which in the normal dorsal horn is derived exclusively from primary afferent axons. Host animals had one side of the L4-5 spinal cord resected and replaced by a transplant of E14 or E15 spinal cord. Adjacent dorsal roots were sectioned and juxtaposed to the graft. The dorsal roots and their projections into the transplants were then labeled 2-9 months later. The tracing methods that used transport or diffusion of horseradish peroxidase demonstrated that severed host dorsal root axons had regenerated and grown into the transplants. In addition, some donor and host neurons had extended their axons into the periphery to at least the midthigh level as indicated by retrograde labeling following application of tracer to the sciatic nerve. Primary afferent axons immunoreactive for calcitonin gene-related peptide were among those that regenerated into transplants, and the projections shown by this immunocytochemical method exceeded those demonstrated by the horseradish peroxidase tracing techniques. Growth of the host dorsal roots into transplants indicates that fetal spinal cord tissue permits regeneration of adult axotomized neurons that would otherwise be aborted at the dorsal root/spinal cord junction. This transplantation model should therefore prove useful in studying the enhancement and specificity of the regrowth of axons that normally terminate in the spinal cord.     

Synaptic Evoked Potentials from Regenerating Dorsal Root Axons within Fetal Spinal Cord Tissue Transplants

Previously injured dorsal roots were electrically stimulated to determine if regenerating sensory axons can form physiologically active synaptic contacts with neurons within fetal spinal cord tissue transplants. Dorsal rootlets, sectioned at their spinal cord entry zone, were apposed to intraspinal transplants of fetal spinal cord tissue grafted along each side of a nerve growth factor-treated nitrocellulose implant. Two to six months later, the rootlets were transected between the spinal cord and their respective ganglia and electrically stimulated. Evoked potentials were recorded from the dorsal surface of the transplant, but were absent from adjacent ipsilateral and contralateral spinal cord regions. A glass micropipette was advanced through the transplant and used to record intramedullary field potentials evoked by dorsal root stimulation. Maximal negative potentials occurred 400–700 μm below the dorsal surface of the transplant, shifting to positive potentials deeper into the transplant. Additionally, both spontaneous and electrically evoked single neuronal action potentials were observed along the microelectrode track. Evoked potentials were abolished following transection of the rootlets between the stimulation site and the transplant. Immunocytochemical evidence of the production of fos protein following electrical stimulation of the regenerated dorsal rootlets was demonstrated within transplant neurons and some ventrally located host neurons, providing an anatomical correlate to the electrophysiological recordings of synaptic activation. These results provide evidence of the structural and functional integration of regenerated sensory axons with both transplant and host neurons.     

NT-3 delivered by an adenoviral vector induces injured dorsal root axons to regenerate into the spinal cord of adult rats

Sensory axons interrupted in the dorsal roots of adult mammals are normally unable to regenerate into the spinal cord. We have investigated whether the introduction of a neurotrophin gene into the spinal cord might offer an approach to otherwise intractable spinal root injuries. The dorsal roots of the 4th, 5th, and 6th lumbar spinal nerves of adult rats were severed and reanastomosed. Fourteen to nineteen days later, adenoviral vectors containing either the LacZ or NT-3 genes were injected into the ventral horn of the lumbar spinal cord, resulting in strong expression of the transgenes in glial cells and motor neurons between 4 and 40 days after injection. When dorsal root axons were transganglionically labelled with HRP conjugated to cholera toxin subunit B, 16 to 37 days after dorsal root injury, large numbers of labelled axons could be seen to have regenerated into the cord, but only in those animals injected with vector carrying the NT-3 gene. The regenerated axons were found at the injection site, mainly in the grey matter, and had penetrated as deep as lamina V. Gene therapy with adenoviral vectors encoding a neurotrophin has therefore been shown to be capable of enhancing and directing the regeneration of a subpopulation of dorsal root axons (probably myelinated A fibres), into and through the CNS environment. J. Neurosci. Res. 54:554–562, 1998. © 1998 Wiley-Liss, Inc.     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Human and Rat Primary C-Fibre Afferents Store and Release Secretoneurin, a Novel Neuropeptide

Secretoneurin is a recently discovered neuropeptide derived from secretogranin II (SgII). Since this peptide could be detected in the dorsal horn of the spinal cord we studied whether it is localized in and released from primary afferent neurons. Secretoneurin was investigated with immunocytochemistry and radioimmunoassay in spinal cord, dorsal root ganglia and peripheral organs. SgII mRNA was determined in dorsal root ganglia. Normal rats and rats pre-treated neonatally with capsaicin to destroy selectively polymodal nociceptive (C-) fibres were used. Slices of dorsal spinal cord were perfused in vitro for release experiments. Immunocytochemistry showed a distinct distribution of secretoneurin-immunoreactivity (IR) in the spinal cord and lower brainstem. A particularly high density of fibres was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord. This distribution was qualitatively identical in rat and human post-mortem tissue. Numerous small diameter and some large dorsal root ganglia neurons were found to contain SgII mRNA. Capsaicin treatment led to a marked depletion of secretoneurin-IR in the substantia gelatinosa, but not in other immunopositive areas of the spinal cord and to a substantial loss of small (<25 μm) SgII-mRNA-containing dorsal root ganglia neurons. Radioimmunoassay revealed a significant decrease of secretoneurin-IR in the dorsal spinal cord, the trachea, heart and urinary bladder of capsaicin-treated rats. Perfusion of spinal cord slices with capsaicin as well as with 60 mM potassium led to a release of secretoneurin-IR. In conclusion, secretoneurin is a neuropeptide which is stored in and released from capsaicin-sensitive, primary afferent (C-fibre) neurons. It may, therefore, be a novel peptidergic modulator of pain transmission or of C-fibre mediated non-nociceptive information.     

Regional distribution of substance P, neurokinin alpha and neurokinin beta in rat spinal cord, nerve roots and dorsal root ganglia, and the effects of dorsal root section or spinal transection

The regional distribution of 3 mammalian tachykinins (substance P, neurokinin alpha and neurokinin beta) in the rat spinal cord and related structures was investigated using a method of radioimmunoassay combined with high performance liquid chromatography. Substance P and neurokinin alpha were found to be distributed in a very similar manner with fairly constant molar ratios i.e. ratios of substance P to neurokinin alpha were 3.69 in the dorsal root ganglia, 3.49 in the dorsal root and 3.09 in the dorsal horn of the cervical spinal cord. On the other hand, the distribution of neurokinin beta was different from other tachykinins; although concentrated in the dorsal horn, neurokinin beta in the dorsal root ganglia or in the dorsal roots was negligibly small in amount. When the cervical dorsal roots were sectioned unilaterally, substance P and neurokinin alpha were decreased in a parallel fashion in the dorsal horn, whereas neurokinin beta was not. In addition neurokinin alpha was selectively and significantly decreased in the dorsal horn of the intact side when compared to that in the unoperated control rat. Since the magnitude of a decrease of neurokinin alpha in molar basis was approximately the same as a decrease of substance P, these findings suggest that the neurokinin alpha and substance P-containing primary afferent fibres could project partly to the contralateral dorsal horn as well. When the thoracic spinal cord was transected, substance P (and neurokinin alpha) was decreased in the ventral part of the lumbar spinal cord, suggesting the presence of tachykinin(s)-containing descending fibres.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-localization of endomorphin-2 and substance P in primary afferent nociceptors and effects of injury: a light and electron microscopic study in the rat

Endomorphin-2 (EM2) is a tetrapeptide with remarkable affinity and selectivity for the mu-opioid receptor. In the present study, we used double-fluorescence and electron microscopic immunocytochemistry to identify subsets of EM2-expressing neurons in dorsal root ganglia and spinal cord dorsal horn of adult rats. Within the lumbar dorsal root ganglia, we found EM2 immunoreactivity mainly in small-to-medium size neurons, most of which co-expressed the neuropeptide substance P (SP). In adult rat L4 dorsal root ganglia, 23.9% of neuronal profiles contained EM2 immunoreactivity and ranged in size from 15 to 36 microM in diameter (mean 24.3 +/- 4.3 microM). Double-labelling experiments with cytochemical markers of dorsal root ganglia neurons showed that approximately 95% of EM2-immunoreactive cell bodies also label with SP antisera, 83% co-express vanilloid receptor subtype 1/capsaicin receptor, and 17% label with isolectin B4, a marker of non-peptide nociceptors. Importantly, EM2 immunostaining persisted in mice with a deletion of the preprotachykinin-A gene that encodes SP. In the lumbar spinal cord dorsal horn, EM2 expression was concentrated in presumptive primary afferent terminals in laminae I and outer II. At the ultrastructural level, electron microscopic double-labelling showed co-localization of EM2 and SP in dense core vesicles of lumbar superficial dorsal horn synaptic terminals. Finally, 2 weeks after sciatic nerve axotomy we observed a greater than 50% reduction in EM2 immunoreactivity in the superficial dorsal horn. We suggest that the very strong anatomical relationship between primary afferent nociceptors that express SP and EM2 underlies an EM2 regulation of SP release via mu-opioid autoreceptors.     

Peripheral specification of sensory neurons transplanted to novel locations along the neuraxis

Thoracic dorsal root ganglia in bullfrogs contain sensory neurons that innervate the skin of the trunk and have synaptic connections in the dorsal horn of the spinal cord. The ganglion that innervates the forelimb contains, in addition to cutaneous afferents, many muscle afferents that project more ventrally in the spinal cord and make monosynaptic connections with motoneurons. In the present study, we have transplanted thoracic sensory neurons to the brachial level in tadpoles to discover whether they can innervate forelimb muscles and, if so, whether they form central connections characteristic of forelimb muscle afferents. The ganglion that normally supplies the forelimb was removed from tadpoles and replaced with 2 thoracic ganglia. After the tadpoles completed metamorphosis, the peripheral and central connections of the transplanted thoracic sensory neurons were examined with anatomical and electrophysiological techniques. When the ganglia were transplanted at stage XIV or earlier, transplanted sensory neurons innervated the forelimb and projected into the brachial spinal cord. Electrical stimulation of forelimb muscle nerves evoked impulses in the dorsal root, indicating that some centrally projecting sensory neurons were muscle afferents. Furthermore, muscle afferents were also activated by stretching muscles which suggest that they terminated on spindles. HRP labeling of the central projections revealed that transplanted sensory neurons terminated at sites characteristic of both cutaneous and muscle afferents. The pattern of synaptic connections was assessed by recording intracellularly from motoneurons. Stimulation of muscle afferents produced monosynaptic EPSPs in motoneurons. As in normal frogs, triceps muscle afferents projected more strongly to triceps motoneurons than to subscapularis and pectoralis motoneurons, while subscapularis afferents projected to all 3 types of motoneurons. Thus, the transplanted sensory neurons formed central connections appropriate to their novel peripheral targets. These observations suggest that interactions between sensory neurons and their targets may be important in determining their central connections.     

Expression of lectin binding in the superficial dorsal horn of the rat spinal cord during pre- and postnatal development.

The plant lectin Bandeiraea simplicifolia I-B4 binds to the soma and central terminals of a subpopulation of unmyelinated primary sensory neurones in the adult rat. The binding site of this lectin is thought to be the terminal alpha-D-galactose residue of a membrane associated glycoconjugate which may be involved in the development of specific connections between small diameter primary sensory neurones and second order neurones in the superficial dorsal horn of the spinal cord. To begin to investigate this possibility we have examined the development of lectin binding in the dorsal horn of pre- and postnatal rats. Lectin binding first appeared on axon profiles in the superficial dorsal horn of the spinal cord at embryonic days 18/19. Previous studies in the rat have revealed that the central processes of small diameter primary sensory neurones enter the dorsal horn at embryonic days 18/19. Our findings suggest that the glycoconjugate to which this lectin binds, is expressed by the central processes of small diameter primary sensory neurones as they grow into the spinal cord. It is therefore possible that this glycoconjugate is involved in the development of topographically ordered neural connections within the dorsal horn of the spinal cord.     

Cholecystokinin in Mammalian Primary Sensory Neurons and Spinal Cord: In Situ Hybridization Studies in Rat and Monkey

The peptide cholecystokinin (CCK) has been suggested to be involved in nociception, but its exact localization at the level of the spinal cord and in spinal ganglia has been a controversial issue. Therefore the distribution of messenger RNA (mRNA) for CCK was studied by in situ hybridization using oligonucleotide probes on sections of adult rat lumbar dorsal root ganglia following unilateral section of the sciatic nerve and on sections of untreated monkey trigeminal ganglia, spinal cord and spinal ganglia from all levels. For comparison, calcitonin gene-related peptide (CGRP) mRNA was also studied in the monkey tissue using the same techniques. Peripheral sectioning of the sciatic nerve in the rat resulted in the appearance of detectable CCK mRNA in up to 30% of remaining ipsilateral L4 and L5 dorsal root ganglion neurons 3 weeks after surgery, with a distinct but more limited appearance also in the contralateral ganglia. No cells, or only single cells, could be seen in normal control rat ganglia. In contrast, in the normal monkey, ∼20% of dorsal root ganglion neurons, regardless of spinal level, and 10% of trigeminal ganglia neurons expressed mRNA for CCK. CGRP mRNA was expressed at detectable levels in ∼80% of these monkey dorsal root ganglion neurons. In the monkey spinal cord, CCK mRNA was detected in the dorsal horn and in motoneurons, whereas CGRP mRNA was only seen in motoneurons. The present results suggest that CCK peptides can be involved in sensory processing in the dorsal horn of the spinal cord in normal monkeys and in rats after peripheral nerve injury, adding one more possible excitatory peptide to the group of mediators in the dorsal horn.     

Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord

The infusion of BDNF and NT-3 into Schwann cell (SC) grafts promotes regeneration of brainstem neurones into the grafts placed in adult rat spinal cord transected at T8 ( 1 ). Here, we compared normal SCs with SCs genetically modified to secrete human BDNF, grafted as trails 5 mm long in the cord distal to a transection site and also deposited in the transection site, for their ability to stimulate supraspinal axonal regeneration beyond the injury. SCs were infected with the replication-deficient retroviral vector pL(hBDNF)RNL encoding the human preproBDNF cDNA. The amounts of BDNF secreted (as detected by ELISA) were 23 and 5 ng/24 h per 10⁶ cells for infected and normal SCs, respectively. Biological activity of the secreted BDNF was confirmed by retinal ganglion cell bioassay. The adult rat spinal cord was transected at T8. The use of Hoechst prelabelled SCs demonstrated that trails were maintained for a month. In controls, no SCs were grafted. One month after grafting, axons were present in SC trails. More 5-HT-positive and some DβH-positive fibres were observed in the infected vs. normal SC trails. When Fast Blue was injected 5 mm below the transection site (at the end of the trail), as many as 135 retrogradely labelled neurones could be found in the brainstem, mostly in the reticular and raphe nuclei (normal SCs, up to 22, mostly in vestibular nuclei). Numerous neurones were labelled in the ventral hypothalamus (normal SCs, 0). Also, following Fast Blue injection, a mean of 138 labelled cells was present in dorsal root ganglia (normal SCs, 46) and spinal cord (39 vs. 32) rostral to the transection. No labelled spinal neurones rostral to the transection were seen when SCs were not transplanted. Thus, the transplantation of SCs secreting increased amounts of BDNF improved the regenerative response across a transection site in the thoracic cord. Moreover, the enhanced regeneration observed with infected SCs may be specific as the largest response was from neurones known to express trkB.     

Central projections of primary sensory afferents to the spinal dorsal horn in the long-tailed stingray, Himantura fai

The central projections of primary sensory afferents innervating the caudal region of the pectoral fin of the long-tailed stingray (Himantura fai) were labeled by applying the lipophilic carbocyanine dye DiI to the dorsal roots in fixed tissue. These observations were complemented by examination of hemotoxylin and eosin-stained paraffin sections of the dorsal root entry zone, and transmission electron microscopy of the dorsal horn. Transverse sections of the sensory nerve and dorsal root revealed two distinct myelinated axon sizes in the sensory nerve. Although the thick and thin axons do not appear to group together in the sensory nerves and dorsal root, they segregate into a dorsally directed bundle of thin fibers and a more horizontally directed bundle of thick fibers soon after entering the spinal cord. In DiI-labeled horizontal sections, fibers were observed to enter the spinal cord and diverge into rostrally and caudally directed trajectories. Branching varicose axons could be traced in the dorsal horn gray matter in the segment of entry and about half of the adjacent rostral and caudal segments. In transverse and sagittal sections, DiI-labeled afferents were seen to innervate the superficial and, to a lesser extent, deeper laminae of the dorsal horn, but not the ventral horn. Electron microscopy of unlabeled dorsal horn sections revealed a variety of synaptic morphologies including large presynaptic elements (some containing dense-core vesicles) making synaptic contacts with multiple processes in a glomerular arrangement; in this respect, the synaptic ultrastructure is broadly similar to that seen in the dorsal horn of rodents and other mammals.