GAP-43 expression in the developing rat lumbar spinal cord.

The expression of the growth-associated protein GAP-43, detected by immunocytochemistry, has been studied in the developing rat lumbar spinal cord over the period E11 (embryonic day 11), when GAP-43 first appears in the spinal cord, to P29 (postnatal day 29) by which time very little remains. Early GAP-43 expression in the fetal cord (E11-14) is restricted to dorsal root ganglia, motoneurons, dorsal and ventral roots and laterally positioned and contralateral projection neurons and axons. Most of the gray matter is free of stain. The intensity of GAP-43 staining increases markedly as axonal growth increases, allowing clear visualization of the developmental pathways taken by different groups of axons. Later in fetal life (E14-19), as these axons find their targets and new pathways begin to grow, the pattern of GAP-43 expression changes. During the period, GAP-43 staining in dorsal root ganglia, motoneurons, and dorsal and ventral roots decreases, whereas axons within the gray matter begin to express the protein and staining in white matter tracts increases. At E17-P2 there is intense GAP-43 labelling of dorsal horn neurons with axons projecting into the dorsolateral funiculus and GAP-43 is also expressed in axon collaterals growing into the gray matter from lateral and ventral white matter tracts. At E19-P2, GAP-43 is concentrated in axons of substantia gelatinosa. Overall levels decline in the postnatal period, except for late GAP-43 expression in the corticospinal tract, and by P29 only this tract remains stained.     

Up-regulation of GAP-43 and growth of axons in rat spinal cord after compression injury

Summary The growth-associated protein-43 (GAP-43) is an axonal phosphoprotein which is expressed at high levels during development and is reinduced by regeneration in the PNS. Consequently it is believed to be a key molecule in the regulation of axonal growth. However, injury to the CNS does not result in significant regeneration and this has been suggested to correlate with a failure of central neurons to up-regulate GAP-43 after axotomy. We have examined a model of spinal cord injury which is unique in two respects; first dural integrity is maintained by compression of the cord with smooth forceps (thus excluding connective tissue elements) and, secondly, considerable axonal growth has been reported through the resulting lesion. Our previous studies have shown that GAP-43 is extensively distributed in the rat spinal cord (see accompanying paper), but here we have used anti-GAP-43 antiserum at a dilution which did not yield any immunostaining in normal cord. However, supranormal levels of GAP-43 were detected in cell bodies and axons around the lesion within four days of compression injury. Double immunostaining with the RT97 monoclonal antibody indicated that a small subpopulation of neurons local to the site of compression were axotomized and expressed GAP-43 and phosphorylated neurofilament epitopes in their cell bodies. Although damage to long axon tracts was extensive, there was no evidence of regeneration in white matter. On the other hand cavities which formed in grey matter provided an environment for axonal elongation. Immunolabelling with markers for astrocytes and endothelial cells was used to evaluate the interaction of elongating axons with endogenous CNS cell types. Sprouting axons, identified by the presence of elevated levels of GAP-43, did not appear to grow in contact with astrocytes but preliminary evidence suggested that newly formed capillaries provided an appropriate substrate.     

Reinnervation of Pacinian corpuscles by CNS axons after transplantation to the dorsal column: incidence and ultrastructure

Summary We have investigated the capacity of injured axons in the spinal dorsal columns of young adult rats to reinnervate grafted Pacinian corpuscles. A branch of the hindlimb interosseous nerve with a group of crural Pacinian corpuscles attached to it was autotransplanted to the surface of the spinal cord and the nerve stump was implanted into the dorsal column. Two to three months later 16 grafts were removed for examination by light and electron microscopy. By 3 months after transplantation almost all Schwann cell columns of the grafted nerve branch were occupied by regenerated myelinated and unmyelinated axons. Of 41 corpuscles examined by electron microscopy 24 were reinnervated by 1–3 myelinated fibres which gave rise to multiple terminals in the inner core. The remaining corpuscles appeared to be denervated. Only two of the reinnervated corpuscles contained regenerated endings which reiterated the distinct ultrastructure of normal presynaptic terminals of CNS axons, characterized by clusters of lucent vesicles and paramembranous densities. All other corpuscles were reinnervated by terminals which resembled peripheral mechanosensory endings as they contained mitochondria and very few vesicles. One such corpuscle was coinnervated by small terminals filled with large dense cored vesicles. We assume that the majority of grafted Pacinian corpuscles have been reinnervated by dorsal column axons and that the regenerated terminals with the ultrastructure of peripheral mechanosensory endings derive from central axons of primary sensory neurons, which are apparently capable of constructing mechanosensory-like terminals in response to signals from the Pacinian corpuscles. The vesicle-filled endings are probably formed by second order sensory neurons, corticospinal neurons and small peptidergic neurons unable to adjust their terminals to the new target.     

Immunocytochemistry of B-50 (GAP-43) in the spinal cord and in dorsal root ganglia of the adult cat

Summary The distribution of the neural-specific growth associated protein B-50 (GAP-43), which persists in the mature spinal cord and dorsal root ganglia, has been studied by light and electron microscopic immunohistochemistry in the cat. Throughout the spinal cord, B-50 immunoreactivity was seen confined to the neuropil, whereas neuronal cell bodies were unreactive. The most conspicuous immunostaining was observed in the dorsal horn, where it gradually decreased from superficial laminae (I–II) toward more ventral laminae (III–V), and in the central portion of the intermediate gray (mainly lamina X). In these regions, the labelling was localized within unmyelinated, small diameter nerve fibres and axon terminals. In the rest of the intermediate zone (laminae VI–VIII), B-50 immunoreactivity was virtually absent. The intermediolateral nucleus in the thoracic and cranial lumbar cord showed a circumscribed intense B-50 immunoreactivity brought about by the labelling of many axon terminals on preganglionic sympathetic neurons. In motor nuclei of the ventral horn (lamina IX), low levels of B-50 immunoreactivity were present in a few axon terminals on dendritic and somal profiles of motoneurons. In dorsal root ganglia, B-50 immunoreactivity was mainly localized in the cell bodies of small and medium-sized sensory neurons. The selective distribution of persisting B-50 immunoreactivity in the mature cat throughout sensory, motor, and autonomie areas of the spinal cord and in dorsal root ganglia suggests that B-50-positive systems retain in adult life the capacity for structural and functional plasticity.     

Identification of dorsal root synaptic terminals on monkey ventral horn cells by electron microscopic autoradiography

Summary The projection of dorsal root fibres to the motor nucleus of the macaque monkey spinal cord has been examined utilizing light and electron microscopic autoradiography. Light microscopy demonstrates a very sparse labelling of primary afferent fibres in the ventral horn. Silver grains overlying radioactive sources are frequently clustered into small groups, often adjacent to dendritic profiles. Under the electron microscope, myelinated axons and a few large synaptic profiles containing rounded synaptic vesicles were overlain by numerous silver grains. These labelled profiles made synaptic contact with dendrites 1–3 m in diameter. The labelled profiles did not contact cell bodies or large proximal dendrites of ventral horn neurons. Frequently, small synaptic profiles containing flattened vesicles were presynaptic to the large labelled terminals and it is suggested that these axoaxonal synapses may mediate presynaptic inhibition of the primary afferent fibres. The relationship of the present findings to previously published physiological and anatomical studies is discussed.     

Postnatal development of corticospinal postsynaptic action

The corticospinal system has a delayed and prolonged postnatal development. In the cat, lesion, inactivation, or stimulation of the system influence motor output minimally when corticospinal (CS) terminals have an immature topographic pattern but produce robust effects immediately after developing the mature pattern by weeks 6-7. In this study, we directly tested if the delay in expression of cortical motor functions is due to the inability of the corticospinal synapse to activate spinal neurons. We stimulated corticospinal axons in the pyramid and recorded evoked field potentials from the surface of the cervical spinal cord and locally from within the gray matter in anesthetized cats during development and in adults. Pyramidal stimulation in animals between week 4 and maturity evoked an initial corticospinal surface volley followed by a postsynaptic field response. Depth recordings from the superficial dorsal horn to the ventral white matter showed that local pre- and postsynaptic field potentials could be recorded over the full extent of the gray matter in 4- to 5-wk animals but were restricted to the intermediate zone in older animals and adults. Dorsoventral refinement of CS field potentials parallels anatomical refinement of individual CS axon terminals shown in our earlier studies. Our present findings indicate that the developing corticospinal system could influence the excitability of virtually the entire contralateral gray matter before cortical motor functions are expressed. Given the importance of activity-dependent axon terminal refinement, this capacity for activating spinal neurons during early postnatal life could play an important role in development of CS circuit connectivity.     

GAP-43 expression in developing cutaneous and muscle nerves in the rat hindlimb.

The expression of the growth associated protein, GAP-43, in developing rat hindlimb peripheral nerves has been studied using immunocytochemistry. GAP-43, is first detected in lumbar spinal nerves at embryonic day (E)12 as the axons grow to the base of the hindlimb. It is expressed along the whole length of the nerves as well as in the growth cones. GAP-43 staining becomes very intense over the next 36 h while the axons remain in the plexus region at the base of the limb bud before forming peripheral nerves at E14. It remains intense along the length of the growing peripheral nerves, the first of which are cutaneous, branching away from the plexus and growing specifically to the skin, their axon tips penetrating the epidermis of the proximal skin at E15 and the toes at E19. GAP-43-containing terminals form a dense plexus throughout the epidermis which subsequently withdraws subepidermally in the postnatal period. GAP-43 staining is also evident along the growing muscle nerves during muscle innervation, which follows behind that of skin. Axons branch over the surface of proximal muscles at E15 but do not form terminals until E17. As target innervation proceeds, GAP-43 staining declines in the proximal part of the nerve but remains intense in the distal portions. Overall GAP-43 expression in the hindlimb decreases in the second postnatal week as axon growth and peripheral terminal formation decline.     

GAP-43 distribution is correlated with development of growth cones and presynaptic terminals

Summary GAP-43 (F1, B-50, pp46) has been associated with neuronal development and regeneration, but precise localization within neurons is not known. Pre-embedding electron microscopic immunocytochemistry using silver-enhanced 1 nm gold particles was used to localize GAP-43 label in cell cultures of cerebellar neurons. In the plasma membranes of early cultures, high levels of GAP-43 were seen in all parts of the neuron. In older cultures, consistent with previous reports, the first loss of GAP-43 label was seen in the soma and then the axon. Growth cones had high levels of GAP-43 label on the plasma membrane, with increased distribution over unattached relative to attached filopodia. The amount of GAP-43 seen over the plasma membrane of forming presynaptic terminals is lower than over growth cones, indicating a possible correlation between the presence of GAP-43 and the stage of presynaptic terminal development. Intracellular GAP-43 in axons and growth cones was highest in membranes of smooth cisternae. The levels of GAP-43 in smooth cisternae in axons fell by seven days in culture while the levels of GAP-43 in smooth cisternae of growth cones fell at 14 days. When mini-explant cerebellar cultures were examined with light microscopic immunocytochemistry, GAP-43 label of plasma membrane was highest at the periphery of the radial axonal outgrowth, suggesting that addition of GAP-43 to the plasma membrane can occur in the distal axon or at the growth cone.     

Alpha calcium/calmodulin-dependent protein kinase II immunoreactivity in corticospinal neurons: combination of axonal transport method and immunofluorescence

A combination of either retrograde or anterograde fluorescent tracer and immunofluorescence histochemistry using the monoclonal antibody specific for the alpha isoform of calcium/calmodulin-dependent protein kinase II (CaM kinase II) was employed to test whether CaM kinase II is expressed in somata of corticospinal neurons and their axons over their whole course. After the injection of carbocyanine dye DiI into the hindlimb area of the primary motor cortex of the rat, corticospinal axons and their terminal arbors were anterogradely labeled: DiI-labeled corticospinal fibers proceeded caudally in the ipsilateral internal capsule, cerebral peduncle and medullary pyramid, crossed at the pyramidal decussation and descended in the ventralmost area of the contralateral dorsal funiculus of the spinal cord. These DiI-labeled corticospinal axons expressed strong CaM kinase II immunoreactivity along their course. However, their terminal arbors within the gray matter of the lumbar cord were very weakly immunostained. With the injection of Fast Blue into the lumbar enlargement of the rat, somata of corticospinal neurons in layer V of the motor cortex were retrogradely labeled. The subsequent immunofluorescent histochemistry revealed that more than 80% of Fast Blue-labeled corticospinal neurons were immunostained with CaM kinase II antibody. The present immunohistochemical study demonstrated that CaM kinase II is strongly expressed in both somata and axons of a majority of corticospinal neurons, although we could not detect this enzyme in the corticospinal terminals in the spinal target areas.     

Evaluation of cellular organization and axonal regeneration through linear PLA foam implants in acute and chronic spinal cord injury

There are few studies of neural implants in spinal cord injury (SCI) focused on supporting directed axon growth. In this study, we fabricated a macroporous poly (lactic acid) (PLA) foam with oriented inner channels. Amorphous foam without linear channels served as a control in an acute SCI injury model, and the effectiveness of foam with linear channels was further investigated in a chronic SCI model. Implants were placed into a 2 mm hemisection lesion cavity at the T8 spinal cord level in adult rats. Two weeks post-implantation, tissue sections including the implants were examined using antibodies against GFAP, p75, ED-1, laminin, GAP-43, and CGRP. Foam implants were well-integrated with the host spinal cord. In linear foams, numerous DAPI-stained cells were found within the inner channels. Schwann cells but not astrocytes had migrated within the channels. Intense laminin staining was observed throughout the extracellular matrix substrate. GAP-43- and CGRP-positive axons grew through the implants following the linear channels. In the amorphous control foams, DAPI staining distributed evenly through the pores. However, the growth of GAP-43 or CGRP-positive axons was misguided and impeded at the entrance area of the foam. Higher numbers of GAP-43 and CGRP-positive axons grew into linear foam implants after chronic SCI than acute SCI. These results suggest the potential application of linear foam implants in cell and axon guidance for SCI repair, especially for chronic SCI.     

Comparative electron histochemistry of thiamine monophosphatase and substance P in the upper dorsal horn

The genuine marker enzyme of primary nociceptive neurons, thiamine monophosphatase (TMPase) has been localized in the substantia gelatinosa of the rat spinal cord by means of light-and electron microscopic histochemistry; localization of substance P has been studied by light-and and electron microscopic immunohistochemical methods. It has been shown that TMPase and substance P are located in two, regionally and structurally different populations of axon terminals. Substance P is contained both in A delta and in drC axons. In the postero-lateral funiculus of the white matter, substance P-positive axons establish axo-somatic synaptic contacts with large multipolar neurons of Cajal's interstitial nucleus.     

Postnatal development of the corticospinal tract in the rat

Summary Horseradish-peroxidase was used to anterogradely label and thus to trace the growth of corticospinal axons in rats ranging in age from one day to six months. Three to eight HRP-gels were implanted in the left cerebral hemisphere of the cortex. In each spinal cord three levels were studied, the cervical intumescence (C5), the mid-thoracic region (T5) and the lumbar enlargement (L3). The methodology employed for the electron microscopic visualization of HRP has been described previously (Joosten et al. 1987a).The outgrowth of labelled unmyelinated corticospinal tract axons in the rat spinal cord primarily occurs during the first ten postnatal days. The outgrowth of the main weve of these fibres is preceded by a number of pathfinding axons, characterized by dilatations at their distal ends, the growth cones. By contrast, later appearing unmyelinated axons, which presumably grow along the pathfinding axons, do not exhibit such growth cones. The first labelled pioneer axons can be observed in the cervical intumescence at postnatal day one (P1), in the mid-thoracic region at day three (P3) and in the lumbar enlargement at day five (P5).Prior to the entrance of the axons, the prospective corticospinal area or the pre-arrival zone is composed of fascicles consisting of unlabelled, unmyelinated fibres surrounded by lucent amorphous structures. During the outgrowth phase of the corticospinal fibres some myelinated axons could be observed within the outgrowth area even before day 14. These axons, however, were never labelled. These findings strongly suggest that the outgrowth area, which is generally denoted as the pyramidal tract, contains other axons besides the corticospinal fibres (and glial cells). The process of myelination of the labelled corticospinal tract axons in the rat spinal cord starts rostrally (C5) at about day 14 and progresses caudally during the third and fourth postnatal weeks. Although myelination seems to be largely complete at day 28 at all three spinal cord levels, some labelled unmyelinated axons are still present in the adult stage.     

Expression of GAP-43 mRNA in the adult mammalian spinal cord under normal conditions and after different types of lesions,with special reference to motoneurons

Summary In situ hybridization histochemistry was used to detect cell bodies expressing mRNA encoding for the phosphoprotein GAP-43 in the lumbosacral spinal cord of the adult rat, cat and monkey under normal conditions and, in the cat and rat, also after different types of lesions. In the normal spinal cord, a large number of neurons throughout the spinal cord gray matter were found to express GAP-43 mRNA. All neurons, both large and small, in the motor nucleus (Rexed's lamina IX) appeared labeled, indicating that both alpha and gamma motoneurons express GAP-43 mRNA under normal conditions. After axotomy by an incision in the ventral funiculus or a transection of ventral roots or peripheral nerves, GAP-43 mRNA was clearly upregulated in axotomized motoneurons, including both alpha and gamma motoneurons. An increase in GAP-43 mRNA expression was already detectable 24 h postoperatively in lumbar motoneurons both after a transection of the sciatic nerve at knee level and after a transection of ventral roots. At this time, a stronger response was seen in the motoneurons which had been subjected to the distal sciatic nerve transection than was apparent for the more proximal ventral root lesion. An upregulation of GAP-43 mRNA could also be found in intact motoneurons located on the side contralateral to the lesion, but only after a peripheral nerve transection, indicating that the concomitant influence of dorsal root afferents may play a role in GAP-43 mRNA regulation. However, a dorsal root transection alone did not seem to have any detectable influence on the expression of GAP-43 mRNA in spinal motoneurons, while the neurons located in the superficial laminae of the dorsal horn responded with an upregulation of GAP-43 mRNA. The presence of high levels of GAP-43 in neurons has been correlated with periods of axonal growth during both development and regeneration. The role for GAP-43 in neurons under normal conditions is not clear, but it may be linked with events underlying remodelling of synaptic relationships or transmitter release. Our findings provide an anatomical substrate to support such a hypothesis in the normal spinal cord, and indicate a potential role for GAP-43 in axon regeneration of the motoneurons, since GAP-43 mRNA levels was strongly upregulated following both peripheral axotomy and axotomy within the spinal cord. The upregulation of GAP-43 mRNA found in contralateral, presumably uninjured motoneurons after peripheral nerve transection, as well as in dorsal horn neurons after a dorsal root transection, indicates that GAP-43 levels are altered not only as a direct consequence of a lesion, but also after changes in the synaptic input to the neurons.     

Amino acid immunoreactivity in corticospinal terminals

Anterogradely labeled corticospinal axons and their terminals were identified after injections of wheat germ-agglutinin conjugated to horseradish peroxidase in the sensorimotor cortex of rats. Thin myelinated axons were labeled in the corticospinal tract. Terminal labeling was densest in the internal basilar nucleus and laminae III–IV of the dorsal horn throughout the spinal cord; electron microscopical observations were mainly from the cervical enlargement. Labeled terminals were most often small and dome-shaped with densely packed, clear round vesicles and sparse mitochondria. These terminals established asymmetric synapses with small dendrites or spines and were never involved in axoaxonic contacts.Postembedding immunocytochemistry was used to study the subcellular distribution of glutamate, aspartate, and gamma-aminobutyric acid (GABA). Corticospinal terminals appeared enriched in glutamate, but not GABA. Some corticospinal terminals appeared enriched in aspartate, though the labeling was less selective than in the case of glutamate. GABA immunolabeling was very dense in about 20% of terminals. These were most often small, rich in mitochondria, and made symmetric synapses; they were not anterogradely labeled from the cortex. Quantitative analysis on double immunolabeled material allowed a direct comparison of particle density for different antigens in the same section. Terminals with a high density of particles coding for glutamate were not enriched with GABA, and terminals immunolabeled for GABA were not enriched with glutamate. There was no significant correlation between glutamate and aspartate immunolabeling in corticospinal terminals; a subpopulation of these terminals may be enriched in aspartate. Aspartate immunolabeling was consistently higher in dendrites postsynaptic (in the plane of section) to corticospinal terminals than in other dendrites. That neither aspartate nor GABA was enriched in dendrites postsynaptic to GABAergic terminals suggests that the phenomenon is not a generic feature of synapses.     

Ultrastructure of primary afferent fibres and terminals expressing alpha-galactose extended oligosaccharides in the spinal cord and brainstem of the rat

Summary The ultrastructural characteristics of primary afferent fibres, which express -galactose extended oligosaccharides recognized by LD2 and LA4 monoclonal antibodies, and the subcellular localization of these oligosaccharides were studied. LD2 and LA4 antibodies both label intensely the plasma membrane of primary afferent fibres, and with LD2 antibody all immunoreactive profiles also possessed strong intracellular staining. In contrast, intracellular staining with LA4 antibody was observed in only a subpopulation of stained profiles. LD2-immunoreactive fibres were detected in trigeminal and Lissauer tracts and in lamina I (LI) and lamina II (LII), and appeared as a mixture of unmyelinated and myelinated fibres. The highest density of LD2-immunoreactive synaptic boutons was found in lamina II outer (LII_o). Many of the terminals were simple dome-shaped terminals, making single asymmetric synapses over small and medium-sized dendritic shafts and dendritic spines. All LA4-immunoreactive fibres were unmyelinated. In addition, some small scalloped central-glomerular terminals contacting two or three dendrites were found. LA4-immunoreactive fibres were found more frequently than terminals and appeared most heavily immunostained in trigeminal and Lissauer tracts. In the neuropil of LI and LII, LA4 profiles were generally very weakly immunostained, although a small sample of immunostained synaptic boutons was detected. All LA4-immunoreactive terminals were found in lamina II inner (LII_i) and made simple asymmetric axodendritic synapses. In addition to axons and terminals, some dendrites exhibited LD2 immunoreactivity and this was most intense in the region of synaptic vesicles. In addition to neurons, some endothelial cells were immunostained with LD2 antibody and astrocytes were immunostained with LA4 antibody.     

Sensory neurons and Schwann cells during pharmacologic stimulation of the nerve regeneration

Using the model of rat sciatic nerve transection and crush injury we studied influence of pyrimidine derivative xymedon on efficacy of regeneration of myelinated axons, number and phenotype of surviving sensory neurons (expressing GAP-43 and Bcl-2) and Schwann cells (S100, GAP-43, PCNA) on the 7th, 15th, 30th, 60th and 90th day after nerve injury. We found out that xymedon counteracts posttraumatic death of sensory neurons, stimulates regeneration of myelinated fibres and proliferation of Schwann cells.     

Distribution and expression of developmentally regulated phosphorylation epitopes on MAP 1B and neurofilament proteins in the developing rat spinal cord

Summary The distribution and expression of developmentally regulated phosphorylation epitopes on the microtubule-associated protein 1B and on neurofilament proteins recognized by monoclonal antibody (mAb) 150 and mAb SMI-31 was investigated in the developing rat spinal cord. In the embryonic day 11 spinal cord, mAb 150 stained the first axons to appear, whereas mAb SMI-31 staining did not appear until embryonic day 12. At the start of axonogenesis, mAb 150 stained neuronal cell bodies and axons whereas at later times only the distal axon was stained, this is the first demonstrationin vivo of a mAb 150 axonal gradient similar to that seen previouslyin vitro (Mansfield et al., 1991). During the postnatal period, axonal staining by mAb 150 dramatically declined so that by the third postnatal week, only the corticospinal tract, which contains axons that are still growing, was labelled. There was no evidence of dendritic staining except of adult primary motoneurons. In contrast, mAb SMI-31 staining of axons was not present as a gradient. Instead, mAb SMI-31 staining increased progressively throughout this period, persisted into adulthood and was shown by immunoblotting to be related to the increased phosphorylation of the medium and heavy neurofilament proteins. Axonal staining by mAb 150 re-appears in a sub-population of the SMI-31-labelled myelinated axons in the adult spinal cord and PNS and in the perikarya and dendrites of primary motoneurons, where it probably recognizes a phosphorylation epitope on heavy neurofilament proteins. This late appearing epitope has some similarities to that recognized by mAb SMI-31 on neurofilaments, but it is not identical. These cross-reactivities of mAbs that recognize phosphorylation epitopes on otherwise unrelated proteins dictate caution in interpreting immunohistochemical data. It may now be necessary in some cases to re-appraise published studies using these two antibodies.     

Characterisation of axon terminals in the rat dorsal horn that are immunoreactive for serotonin 5-HT3A receptor subunits

Serotonin 5-HT₃ receptors are abundant in the superficial dorsal horn and are likely to have an involvement in processing of nociceptive information. It has been shown previously that 5-HT₃ receptors are present on primary afferent terminals and some dorsal horn cells. The primary aim of the present study was to determine what classes of primary afferent possess 5-HT₃A receptor subunits. We performed a series of double- and triple-labelling immunofluorescence experiments. Subunits were labelled with an anti-peptide antibody and primary afferent axons were identified by the presence of calcitonin gene-related peptide (CGRP) and binding of the lectin IB4. Quantitative confocal microscopic analysis revealed that approximately 10% of axons displaying 5-HT₃A immunoreactivity were also labelled for CGRP but that only 3% of these fibres bind IB4. We also investigated the relationship between immunoreactivity for the subunit and descending serotoninergic systems, axons originating from inhibitory neurons that contain glutamic acid decarboxylase, and axons of a subpopulation of excitatory neurons that contain neurotensin. None of these types of axon was associated with immunoreactivity for receptor subunits. Ultrastructural studies confirmed that punctate immunoreactive structures observed with the light microscope were axon terminals. These terminals invariably formed asymmetric synaptic junctions with dendritic profiles and often contained a mixture of granular and agranular vesicles. Some terminals formed glomerular-like arrangements. Immunoreactive cells were also examined and were found to contain intense patches of reaction product within the cytoplasm. We conclude that the majority (about 87%) of dorsal horn axons that are immunoreactive for 5-HT₃A receptor subunits do not originate from the subtypes of primary afferent fibres that bind IB4 or contain CGRP. It is likely that most of these axons have an excitatory action and they may originate from dorsal horn interneurons and/or fine myelinated primary afferent fibres. Electronic Publication     

The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury

When adult dorsal root ganglion cells are dissociated and maintained in vitro, both the small dark and the large light neurons show increases in the growth-associated protein GAP-43, a membrane phosphoprotein associated with neuronal development and plasticity. Immunoreactivity for GAP-43 appears in the cytoplasm of the cell bodies as early as 3.5 h post axotomy and is present in neurites and growth cones as soon as they develop. At early stages of culture (4 h to eight days) satellite/Schwann cells are also immunoreactive for GAP-43. Neurons in isolated whole dorsal root ganglion maintained in vitro become GAP-43-immunoreactive between 2 and 3 h after axotomy. It takes three days however, after cutting or crushing the sciatic nerve in adult rats in vivo, for GAP-43 immunoreactivity to appear in the axotomized dorsal root ganglion cells. GAP-43 immunoreactivity can be detected in the central terminals of primary afferent neurons in the superficial laminae of the dorsal horn of the lumbar enlargement four days after sciatic cut or crush. The intensity of the GAP-43 staining reaches a peak at 21 days and becomes undetectable nine weeks following crush injury and 36 weeks following sciatic nerve cut. The pattern of GAP-43 staining is identical to the distribution of sciatic small-calibre afferent terminals. Little or no staining is present in the deep dorsal horn, but GAP-43 does appear in the ipsilateral gracile nucleus 22 days after sciatic injury. In investigating the mechanism of GAP-43 regulation, blockade of axon transport in the sciatic nerve with vinblastine (10(-5) M-10(-4) M) or capsaicin (1.5%) was found to produce a pattern of GAP-43 immunoreactivity in the dorsal horn identical to that found with crush, while electrical stimulation of the sciatic nerve had no effect. Axotomy of primary sensory neurons or the interruption of axon transport in the periphery therefore acts to trigger GAP-43 production in the cell body. The GAP-43 is transported to both the peripheral and the central terminals of the afferents. In the CNS the elevated GAP-43 levels may contribute to an inappropriate synaptic reorganization of afferent terminals that could play a role in the sensory disorders that follow nerve injury.     

GAP-43 and p75NGFR immunoreactivity in presynaptic cells following neuromuscular blockade by botulinum toxin in rat

Summary Peripheral nerve lesion results in changes in protein expression by neurons and denervated Schwann cells. In the present study we have addressed the question whether similar changes take place following functional denervation. Using immunohistochemistry and immunoelectron microscopy we examined changes in growth-associated protein (GAP-43) and low-affinity nerve growth factor receptor (p75^NGFR) in rat gastrocnemius muscle following botulinum toxin-induced paralysis. GAP-43 and p75^NGFR were selected because they are not expressed by mature intact motor neurons or Schwann cells, but are expressed following nerve lesion in both motor neurons and denervated Schwann cells. In control muscle, GAP-43 and p75^NGFR immunoreactivity was seen only in nerve fibres near blood vessels. Two weeks after toxin injection, GAP-43 immunoreactivity could be seen at the motor endplates and in axons. Intensity of staining increased with longer survival and reached a peak between 4 and 8 weeks post-injection. Ultrastructurally, GAP-43 immunoreactivity was confined to nerve terminals and axons, whereas Schwann cells remained negative. Immunostaining for p75^NGFR also increased following toxin injection and was detected in some terminal Schwann cells and in perineurial cells of small nerve fascicles near the paralyzed target cells, but not in axons. These results show that changes in expression of GAP-43 in motor neurons following functional denervation closely resemble the changes following anatomical interruption of nerve-muscle contact. GAP-43 was not expressed in Schwann cells, indicating that its upregulation in these cells is induced by loss of axonal contact or nerve degeneration products. There is no support for a role of p75^NGFR in incorporation of neurotrophins in axons. The restriction of p75^NGFR expression to terminal Schwann cells and perineurial cells in close proximity to the paralyzed target suggests a role for a target-derived signal or, alternatively, macrophages in eliciting this expression.