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The existence of GABA-like immunoreactivity in the synapses on the primary afferent axons and GABA- and glutamate immunoreactive synapses on the dorsal cell somatic membrane was shown using double postembedding immunogold cytochemistry. These morphological findings suggest that control of the sensory information in the lamprey spinal cord is realized by means of presynaptic inhibition through the synapses on the primary afferent axons as well as directly through the synapses on the somata of the sensory neurons.

Sensory nerves detect an extensive array of somatosensory stimuli, including environmental temperatures. Despite activating only a small cohort of sensory neurons, cold temperatures generate a variety of distinct sensations that range from pleasantly cool to painfully aching, prickling, and burning. Psychophysical and functional data show that cold responses are mediated by both C- and A delta-fibers with separate peripheral receptive zones, each of which likely provides one or more of these distinct cold sensations. With this diversity in the neural basis for cold, it is remarkable that the majority of cold responses in vivo are dependent on the cold and menthol receptor transient receptor potential melastatin 8 (TRPM8). TRPM8-null mice are deficient in temperature discrimination, detection of noxious cold temperatures, injury-evoked hypersensitivity to cold, and nocifensive responses to cooling compounds. To determine how TRPM8 plays such a critical yet diverse role in cold signaling, we generated mice expressing a genetically encoded axonal tracer in TRPM8 neurons. Based on tracer expression, we show that TRPM8 neurons bear the neurochemical hallmarks of both C- and A delta-fibers, and presumptive nociceptors and non-nociceptors. More strikingly, TRPM8 axons diffusely innervate the skin and oral cavity, terminating in peripheral zones that contain nerve endings mediating distinct perceptions of innocuous cool, noxious cold, and first- and second-cold pain. These results further demonstrate that the peripheral neural circuitry of cold sensing is cellularly and anatomically complex, yet suggests that cold fibers, caused by the diverse neuronal context of TRPM8 expression, use a single molecular sensor to convey a wide range of cold sensations.

Most post-crossing commissural axons turn into longitudinal paths to make synaptic connections with their targets. Mechanisms that control their rostrocaudal turning polarity are still poorly understood. We used the hindbrain as a model system to investigate the rostral turning of a laterally located commissural tract, identified as the caudal group of contralateral cerebellar-projecting second-order vestibular neurons (cC-VC). We found that the caudal hindbrain possessed a graded non-permissive/repulsive activity for growing cC-VC axons. This non-permissiveness/repulsion was in part mediated by glycosyl-phosphatidylinositol (GPI)-anchored ephrin A. We further demonstrated that ephrin A2 was distributed in a caudal-high/rostral-low gradient in the caudolateral hindbrain and cC-VC axons expressed EphA receptors. Finally, perturbing ephrin A/EphA signalling both in vitro and in vivo led to rostrocaudal pathfinding errors of post-crossing cC-VC axons. These results suggest that ephrin A/EphA interactions play a key role in regulating the polarity of post-crossing cC-VC axons as they turn into the longitudinal axis.

Background: Neurite growth can be elicited by growth factors and interactions with extracellular matrix molecules like laminin. Among the targets of the signalling pathways activated by these stimuli are cytoskeletal elements, such as actin, tubulin and neurofilaments. The cytoskeleton can also be modulated by other proteins, such as the small heat shock protein Hsp27. Hsp27 interacts with actin and tubulin in non-neuronal cells and while it has been suggested to play a role in the response of some neurons to injury, there have been no direct studies of its contribution to axonal regeneration., Results: We have investigated neurite initiation and process extension using cultures of adult dorsal root ganglion (DRG) sensory neurons and a laminin stimulation paradigm. Employing confocal microscopy and biochemical analyses we have examined localization of Hsp27 at early and later stages of neurite growth. Our results show that Hsp27 is colocalized with actin and tubulin in lamellopodia, filopodia, focal contacts and mature neurites and growth cones. Disruption of the actin cytoskeleton with cytochalasin D results in aberrant neurite initiation and extension, effects which may be attributable to alterations in actin polymerization states. Inhibition of Hsp27 phosphorylation in our cultures results in an atypical growth pattern that may be attributable to an effect of pHsp27 on the stability of the actin cytoskeleton., Conclusion: We observed colocalization of the phosphorylated and non-phosphorylated forms of Hsp27 with actin and tubulin in both very early and later stages of neurite growth from cultured adult DRG neurons. The colocalization of Hsp27 and pHsp27 with actin in lamellopodia and focal contacts at early stages of neurite growth, and in processes, branch points and growth cones at later stages, suggests that Hsp27 may play a role in neuritogenesis and subsequent neurite extension, and potentially in the patterning of this growth. Hsp27 has been reported to play a key role in modulating actin cytoskeletal dynamics as an actin-capping protein in non-neuronal cells. Our results suggest that this may also be the case in neurons and support a role for Hsp27 in neurite outgrowth via its phosphorylation state-dependent interactions with actin.

Olfactory receptors are supposed to act not only as molecular sensors for odorants but also as cell recognition molecules guiding the axons of olfactory neurons to their appropriate glomerulus in the olfactory bulb. This concept implies that olfactory receptor proteins are located in sensory cilia and in the axons. To approach this critical issue, antibodies were generated against two peptides, one derived from olfactory receptor mOR256-17, one derived from the "mOR37" subfamily. By means of immunohistochemistry and double-labeling studies using transgenic mouse lines as well as Western blot analyses, it was demonstrated that the newly generated antibodies specifically recognized the receptor proteins. To scrutinize the hypothesis that olfactory receptor proteins may also be present in the axonal processes and the nerve terminals, serial sections through the olfactory bulb were probed with the antibodies. Two glomeruli in each bulb were stained by anti-mOR256-17, one positioned in the medial, one in the lateral hemisphere. Fiber bundles approaching the glomeruli through the outer nerve layer also displayed intense immunofluorescence. A similar picture emerged for the antibody anti-mOR37, a small number of glomeruli in the ventral domain of the bulb was stained. On serial sections through the olfactory bulb of mOR37-transgenic mouse lines, double-labeling experiments demonstrated that distinct immunoreactive glomeruli corresponded to glomeruli that were targeted by neurons expressing a particular member of the mOR37 receptor subfamily. These data indicate that olfactory receptor (OR) proteins are indeed present in the axonal processes and nerve terminals of olfactory sensory neurons, thus supporting the notion that ORs may participate in the molecular processes underlying the fasciculation and targeting of olfactory axons.

The transcription factor Engrailed (En) controls axon pathfinding and synaptic target choice in an identified neuron (6m) of the cockroach cercal sensory system. Knock-out of En using double-stranded RNA interference (dsRNAi) transforms 6m so that it resembles a neighboring neuron that normally does not express the en gene, has a different arbor anatomy, and makes different connections. Like many animals, the cockroach has two En paralogs, Pa-En1 and Pa-En2. In this study we tested the hypothesis that the paralogs have different effects on axon guidance and synaptic target recognition, using RNAi to knock out each one individually. Using dye injections into 6m and intracellular recordings from target interneurons, we obtained evidence that both Pa-En1 and Pa-En2 determine the axonal arborization, but only Pa-En1 controls synaptic connections. However, because immunocytochemical quantification of En protein in 6m after RNAi showed that Pa-En1 represents 65% of the total En activity and Pa-En2 only 35%, our results could be caused by dosage effects. We measured the effects of diluting the mixture of both dsRNAs on the amounts of En protein. From this dose-response curve, we calculated the appropriate dilutions of the dsRNA mixture that would titrate total En protein to levels equivalent to knock-out of either paralog. RNAi using these dilutions showed that Pa-En1 and Pa-En2 both contribute toward the control of axonal guidance and confirmed that Pa-En1 has the paralog-specific function of controlling synaptic target recognition.

In three frog species Rana esculenta, Rana temporaria and Xenopus laevis, the contacts established by gamma-aminobutyric acid and glutamate decarboxylase immunoreactive (-ir) terminals upon primary afferent fibers were studied using confocal and electron microscopy. For confocal microscopy, the primary afferent fibers were labeled through the dorsal root with Dextran-Texas Red, whereas gamma-aminobutyric acid and glutamate decarboxylase immunoreactivity were revealed with fluorescein isothiocyanate. Appositions of gamma-aminobutyric acid and glutamate decarboxylase immunoreactive profiles onto primary afferent fibers were observed and were considered as putative axo-axonic contacts of GABAergic terminals upon primary afferents. The latter was confirmed by the ultrastructural finding of axo-axonic synapses from gamma-aminobutyric acid immunopositive boutons upon the HRP-labeled primary afferent fibers in postembedding immunoelectron microscopic study. Such synapses may represent the morphological basis of GABAergic presynaptic inhibition of primary afferent fibers.

It has been found that heterodimers of kappa- and delta-opioid receptors can occur in vitro, but it has been unclear whether they also occur in intact animals. In the present study we examined whether kappa-delta heterodimers might occur in vivo by staining for these receptors with two-color fluorescence immunocytochemistry. Sections of rat spinal cord were double-stained using rabbit anti-kappa opioid receptor combined with rat anti-delta-opioid receptor. It was found that axons in the superficial dorsal horn of the spinal cord were double-labeled. In addition, structures within axonal varicosities were sometimes double-labeled. We conclude that single axons, and single structures within axons, express both kappa- and delta-opioid receptors. These observations are consistent with heterodimers of these receptors existing in vivo.

We investigated the possibility that monoaminergic axons make contacts with spinal interneurons which project to motor nuclei and are monosynaptically activated by group II muscle afferents. Interneurons in midlumbar spinal segments of adult cats were characterized electrophysiologically and intracellularly labelled with tetramethylrhodamine dextran. Serotoninergic and noradrenergic axons were identified with immunofluorescence in sections containing labelled cells. Contacts between monoaminergic axons and interneurons were investigated with three-colour confocal laser scanning microscopy and analysed with a computer reconstruction program. Cell bodies and dendritic trees of five cells were reconstructed and putative contacts were plotted. The average number of contacts formed by serotoninergic axons was 140 and the average number of noradrenergic contacts was 38. The majority (95%) of contacts were formed with dendrites; these were distributed over the entire dendritic tree, even on the most distal branches. These findings provide a morphological basis for the modulatory actions of monoamines on premotor spinal interneurons in pathways from group II muscle afferents.

To help understand how axons interact as they grow into the developing chick hindlimb, we used electron microscopy in conjunction with immunoperoxidase staining for the cell adhesion molecule axonin-1 to label sensory axons. The results showed that sensory axons travel together in bundles, tightly apposed to one another. In contrast, motoneuron axons are more widely spaced, although motoneuron axons situated at the perimeter of sensory axon bundles are found in close contact with neighboring sensory axons. Sensory growth cones and lamellipodia tend to be located centrally within the bundles, with several lamellipodia typically being found stacked together. Strikingly, regions of close axonal apposition are accompanied by axonin-1 expression, suggesting that such contacts are indeed adhesive. Taken together, these observations suggest that groups of sensory axons of a similar age grow together, with some of the older sensory axons fasciculating along motoneuron axons and younger sensory axons later fasciculating along older sensory axons. Axons situated at the periphery of sensory bundles are typically partly labelled, such that axonin-1 is expressed on membranes apposing other labelled axons but not on those facing unlabelled axons or unlabelled Schwann cells. Thus, axonin-1 appears to become redistributed within the membranes of axons growing into the limb, as it does on cultured neurons. In contrast, the neuron-glia cell adhesion molecule (NgCAM), which binds heterophilically to axonin-1, appears uniformly distributed on even those axons that would have an asymmetric distribution of axonin-1. Thus, the localization of axonin-1 strongly suggests that it plays an important role in sensory axon fasciculation, but the relative contributions of its interactions with various potential ligands are unclear. Finally, we found that some sensory growth cones have lamellipodia that are spread over considerable expanses. This suggests that although fasciculation is important in sensory axon guidance, sensory axons may also explore the local environment.

Confocal microscopy was used to detect GABA-immunoreactive axo-axonic appositions, indicating possible synaptic contacts, on Ib fiber terminals in the lumbosacral spinal cord. A Ib fiber from posterior biceps-semitendinosus muscles was labeled by intra-axonal ejection of tetramethylrhodamine dextran (red), and serial sections of S1-L7 spinal cord segments were processed for GABA immunocytochemistry revealed by fluorescein isothiocynate (green). Appositions between GABA-immunoreactive structures and the labeled fiber appeared as yellow spots because of the presence of both fluorochromes in small volumes (0.3 * 0.3 * 0.5 micrometer(3)) of tissue. These spots were identified as probable axo-axonic contacts when: (1) they were observed in two to four serial confocal planes, indicating that they did not occur by chance; and (2) their sizes, shapes, and locations were similar to those of axo-axonic contacts found on Ia terminals, known to bear presynaptic boutons, and resembled the axo-axonic synapses described in electron microscope studies of Ib boutons in Clarke's column. A total of 59 presumed axo-axonic contacts was observed on two Ib collaterals, representing an estimated 20% of the total complement. In a three-dimensional reconstruction of one collateral, they were mostly located in terminal positions, and some branches bore more contacts than others. Such differential distribution could not result from chance appositions between GABAergic structures and Ib arborization and further supported the identification of axo-axonic contacts. Segmental Ib collaterals bear axo-axonic synapses that might ensure differential funneling of information toward different targets.

The wild white-footed mouse, Peromyscus leucopus, is commonly used for photoperiod studies utilizing physiological, behavioral, and other biological measures indicative of hypothalamic functions. Indoleamines, like melatonin and serotonin, are implicated in regulating these hypothalamic functions. Although neurochemical analyses of hypothalamic serotonin and its receptors have been reported for this species, the relevant neuroanatomy of the serotonin system within mouse hypothalamus has not been studied. A sensitive immunohistochemical method was used to detect serotonin within axons of coronal sections of formaldehyde fixed forebrain from P. leucopus. Large, medium and small diameter serotonin axons were evaluated in most regions, or nuclei, of the hypothalamus rostral to the mammillary region. A fourth type of serotonin axon was observed to have morphology characteristic of terminal arbors. The density of serotonin axons ranged from no staining to very high density similar to other species for which reports exist, i.e., rat, cat, and monkey. The ventromedial hypothalamic nucleus had distinctively lesser density of serotonin axons in this mouse than other species. Evidence of terminal arborization in hypothalamic nuclei and regions was evident. Neuroendocrine, autonomic, and behavioral functions of the hypothalamus are suggested to be regulated by input from serotonin terminals in this wild species of mouse, in correlation with receptor localization as reported by others., (Copyright 1998 Elsevier Science B.V.)

Two distinct populations of Na+ channels (kinetically fast and slow) are present on the cell bodies and axons of cutaneous afferent neurons; the fast current is increased and the slow current reduced in amplitude following nerve injury. The present study was undertaken to determine if similar changes occur on the axons of these neurons following peripheral nerve injury. The compound action potentials from rat sural nerves were recorded in a sucrose gap chamber. Following application of 4-aminopyridine, a prominent and well-characterized depolarization (the delayed depolarization) followed the action potential. This potential, only present on cutaneous afferent axons, has been correlated with activation of a slow Na+ current. The delayed depolarization was reduced after nerve transection. The refractory period of transmission of the action potential was shortened in the transected nerves, but that of the delayed depolarization was prolonged. The changes were largest when the sural nerve was cut and ligated [control: 38.1 +/- 1.7% (n = 5); injury: 24.5 +/- 2.8% (n = 5), P < 0.05], which prevented reconnection to its peripheral target. When the nerve was crushed and allowed to reestablish peripheral target connections, the delayed depolarization was minimally effected. These results indicate that the changes in Na+ channel organization following peripheral target disconnection observed on cutaneous afferent cell bodies also occur on their axons.

Substance P (SP) is a neuropeptide that is released from axons of sensory neurons and causes signal transduction through the activation of the neurokinin-1 receptor (NK1-R). The present study demonstrates the distribution of SP-like-immunoreactive (SP-LI) axons and the localization of NK1-Rs in rat bone tissue using the avidin-biotin-peroxidase complex method. Axons with SP-LI were commonly found near the trabecular bone in the temporal bone marrow, but they were only sparsely distributed in the mandible, femur, and tibia. Immunoreactivity for NK1-Rs was found on the plasma membrane and in the cytoplasm of the osteoclasts. In the osteoblasts and osteocytes, a small number of weak, punctate immunoreactive products of NK1-Rs were distributed close to the plasma membrane. At the electron-microscopic level, immunoreactivity for NK1-R was distributed mainly in the whole cytoplasm, except for the clear zone of the osteoclasts, and in pit-like structures along the plasma membrane. The NK1-R-immunoreactive structures in the cytoplasm were divided into two types of organelles, consisting of vesicular and vacuolar structures (probably transport vesicles and early endosomes). In the osteoblasts and osteocytes, the number of NK1-R-positive vesicular structures was fewer than in the osteoclasts. These results thus suggest that SP secreted by the sensory axons could directly modulate bone metabolism via NK1-Rs.

The present study examined the organization of afferent and efferent connections of the rat ventrolateral tegmental area (VLTg) by employing the retrograde and anterograde axonal transport of Fluorogold and Phaseolus vulgaris-leucoagglutinin, respectively. Our interest was focused on whether the anatomical connections of the VLTg would provide evidence as to the involvement of this reticular area in audiomotor behavior. Our retrograde experiments revealed that minor inputs to the VLTg arise in various telencephalic structures, including the cerebral cortex. Stronger projections originate in the lateral preoptic area, the zona incerta, the nucleus of the posterior commissure and some other thalamic areas, the lateral substantia nigra, the deep layers of the superior colliculus, the dorsal and lateral central gray, the deep mesencephalic nucleus, the paralemniscal zone, the intercollicular nucleus, the external cortex of the inferior colliculus, the oral and caudal pontine reticular nucleus, the deep cerebellar nuclei, the gigantocellular and lateral paragigantocellular reticular nuclei, the prepositus hypoglossal nucleus, the spinal trigeminal nuclei, and the intermediate layers of the spinal cord. Most importantly, we disclosed strong auditory afferents arising in the dorsal and ventral cochlear nuclei and in the cochlear root nucleus. The efferent projections of the VLTg were found to be less widespread. Telencephalic structures do not receive any input from the VLTg. Moderate projections were seen to diencephalic reticular areas, the zona incerta, the nucleus of the posterior commissure, and to various other thalamic areas. The major VLTg projections terminate in the deep layers of the superior colliculus, the deep mesencephalic nucleus, the intercollicular nucleus and external cortex of the inferior colliculus, the oral and caudal pontine reticular nucleus, the gigantocellular and lateral paragigantocellular reticular nuclei, and in the medial column of the facial nucleus. From our data, we conclude that the VLTg might play a role in sensorimotor behavior.

Serotonin (5-hydroxytryptamine, 5-HT) is a well known inflammatory mediator and algesic substance. It has been hypothesized that 5-HT can have a direct action on peripheral sensory axons, but there has been no anatomical demonstration of 5-HT receptors on peripheral primary afferent processes. The present study shows that 32% of unmyelinated axons at the dermal-epidermal junction are immunohistochemically stained with antibodies directed against the 5-HT2A receptor providing anatomical evidence that 5-HT can have a direct effect on sensory fibers in the skin. Furthermore, encapsulated nerve endings in Pacinian corpuscles also contain reaction product following immunostaining for 5-HT2A receptors, indicating that large myelinated axons can be activated by endogenous serotonin. These data suggest that peripherally acting 5-HT2A antagonists may be effective in reducing pain of peripheral origin.

To determine whether diffusible guidance cues direct retinal axon growth and divergence at the optic chiasm, we cocultured mouse retinal and chiasm explants in collagen gels. The chiasm reduced retinal neurite lengths and numbers, but did not affect commissural or pontine neurite growth. This reduction in growth was equal for all retinal quadrants and occurred without reorienting the direction of neurite extension. The floor plate, another midline guidance locus, also suppressed retinal neurite outgrowth, whereas cortex or cerebellum explants did not. Growth suppression was not mediated by netrin-1, which instead enhanced retinal neurite extension. We propose that chemosuppression may be a general guidance mechanism that acts in intermediate targets to prime growth cones to perceive other, more specific cues.

The present study investigated the cellular distribution of a developmentally regulated phosphorylated form of MAP 1B recognized by monoclonal antibody (mAb) 150 in cultures of dorsal root ganglia. The cell soma and the whole axon, when it first appears, are labelled, but longer axons label with a proximodistal gradient, such that the cell soma and proximal axon become unlabelled, whilst the distal axon and growth cone label strongly. Double-labelling experiments with mAb 150 and a polyclonal antibody (N1-15) that recognizes all forms of MAP 1B demonstrated that MAP 1B is distributed along the entire length of axons with gradients, so the gradient of phosphorylated MAP 1B is not due to a loss or absence of MAP 1B from the proximal axon. The proportion of axons from 20 h cultures that were labelled with a mAb 150 gradient was at least 80% and this proportion was independent of the nerve growth factor concentration of the culture medium. Analysis of axons ranging in length from 100 to 700 microm and labelled with a gradient showed that the unlabelled proximal portions of axons increased in length more slowly than the labelled distal axon. Axons labelled along their entire length accounted for no more than 19% of th axonal population and analysis of these showed them to be frequently < 400 microm long. After simultaneously fixing and detergent-extracting cultures this proportion rose significantly to 93%, suggesting that in the proximal axon the mAb 150 epitope is masked by some factor(s) that is removed by detergent extraction. The possibility that mAb 150 could not access the epitope in the proximal axon was discounted because another IgM, mAb 125, which recognizes a different phosphorylation epitope on MAP 1B, labelled the proximal axon of conventionally fixed cultures. In growth cones of fixed and extracted neurons examined by immunofluorescence, the mAb 150 labelling strongly colocalized to bundled microtubules in the distal axon shaft and the C-domain. In the P-domain, mAb 150 staining was weaker and more widely distributed than the microtubules. Immunogold electron microscopy confirmed that antibody N1-15 and mAb 150 strongly labelled the bundled microtubules in the C-domain and also showed that individual microtubules in the P-domain, some of which lie alongside actin filament bundles of filopodia, were labelled lightly and discontinuously with both antibodies. This suggests that the phosphorylated isoform of MAP 1B recognized by mAb 150 may be microtubules and actin filaments in the P-domain.

A combination of immunocytochemical and electron microscopic methods were employed to assess the organization of the trigeminal (V) spinal tract in adult rats. Immunostaining was employed at the light microscopic level to selectively label large myelinated (by using antibodies against neurofilament protein) and small unmyelinated (by using antibodies against calcitonin gene-related peptide) primary afferents. In addition, the plant lectin Bandeiraea simplicifolia-I was employed to histochemically label small unmyelinated primary afferents. Results from these experiments indicated that larger myelinated axons were distributed throughout the cross-sectional extent of the V spinal tract (TrV), whereas smaller fibers were most numerous just below the pial surface. These results were confirmed with quantitative electron microscopy which demonstrated that the central portion of the V sensory root and TrV were composed primarily of larger myelinated fibers, whereas the periphery of the root and the portion of TrV just below the pial surface contained a higher percentage of smaller myelinated and unmyelinated axons. When considered together with results regarding the birthdates of neurochemically defined classes of V ganglion cells (White et al. [1994] J. Comp. Neurol. 350:397-411), these results suggest that TrV is laid down in a chronotopic fashion with the first axons forming its deeper portion and later arriving axons being added more superficially.

In the present paper the ultrastructural similarities among the terminal portions of Pacinian corpuscles, the nodes of Ranvier, and the initial segments of primary sensory neurons are pointed out. Our conclusion is based on our observations of cat Pacinian corpuscles and other general knowledge of the node of Ranvier and the initial segment published elsewhere. The morpho-functional similarities of three principal excitable regions of the sensory nerve fibres (the initial segments, the node of Ranvier, and the terminal portions of sensory nerve formations) are illustrated by identical distribution of the enzymes which are associated with ionic transport (alkaline phosphatase, Mg(2+)-ATPase), and non-specific cholinesterase. Furthermore, the polyanionic material revealed by Alcian blue staining in three excitable sites of the sensory axon confirms the supposition that excitable axolemma cannot be considered in the isolation of its surroundings produced by Schwann cells.

Microtubule-associated proteins (MAPs) are essential components necessary for the early growth process of axons and dendrites, and for the structural organization within cells. Both MAP2 and MAP5 are involved in these events, MAP2 occupying a role predominantly in dendrites, and MAP5 being involved in both axonal and dendritic growth. In the chick dorsal root ganglia, pseudo-unipolar sensory neurons have a T-shaped axon and are devoid of any dendrites. Therefore, they offer an ideal model to study the differential expression of MAPs during DRG development, specifically during axonal growth. In this study we have analyzed the expression and localization of MAP2 and MAP5 isoforms during chick dorsal root ganglia development in vivo, and in cell culture. In DRG, both MAPs appeared as early as E5. MAP2 consists of the 3 isoforms MAP2a, b and c. On blots, no MAP2a could be found at any stage. MAP2b increased between E6 and E10 and thereafter diminished slowly in concentration, while MAP2c was found between stages E6 and E10 in DRG. By immunocytochemistry, MAP2 isoforms were mainly located in the neuronal perikarya and in the proximal portion of axons, but could not be localized to distal axonal segments, nor in sciatic nerve at any developmental stage. On blots, MAP5 was present in two isoforms, MAP5a and MAP5b. The concentration of MAP5a was highest at E6 and then decreased to a low level at E18. In contrast, MAP5b increased between E6 and E10, and rapidly decreased after E14. Only MAP5a was present in sciatic nerve up to E14. Immunocytochemistry revealed that MAP5 was localized mainly in axons, although neuronal perikarya exhibited a faint immunostaining. Strong staining of axons was observed between E10 and E14, at a time coincidental to a period of intense axonal outgrowth. After E14 immunolabeling of MAP5 decreased abruptly. In DRG culture, MAP2 was found exclusively in the neuronal perikarya and the most proximal neurite segment. In contrast, MAP5 was detected in the neuronal cell bodies and all along their neurites. In conclusion, MAP2 seems involved in the early establishment of the cytoarchitecture of cell bodies and the proximal axon segment of somatosensory neurons, while MAP5 is clearly related to axonal growth.

The occurrence of sprouting in the spinal cord in response to denervation has been a subject of debate. To test for sprouting of primary afferent fibers after denervation, rats were unilaterally deafferented for 35 days (chronic side) by dorsal rhizotomies performed from T2 to T8 and T10 to L5, thus isolating or sparing the T9 root. The contralateral T9 root was spared by a similar surgery 5 days (acute side) prior to sacrifice. The survival time on the chronic side presumably allows intraspinal sprouting of T9 primary afferents to occur whereas the time on the acute side does not. To test for sprouting of primary afferents, it is necessary to identify these nerve processes. Calcitonin gene-related peptide (CGRP) immunoreactivity has been localized to a subpopulation of primary afferent nerve processes and their terminals within the dorsal horn. Therefore, immunohistochemical methods were used to determine the distribution of CGRP immunoreactivity in laminae I and II on both sides of the spinal cord. Using image analysis, there was an increase of 153 to 704% in the density of CGRP immunoreaction product on the chronic side compared to the acute side in the spared segment. This difference is statistically significant. Furthermore, the increased density on the chronic side extended two segments cranial and two segments caudal to the spared root segment. No difference was found in the laminar distribution between sides. These data support the hypothesis of primary afferent sprouting following spinal cord denervation.

The immediate early genes c-fos, c-jun and NGFI-A are rapidly and transiently expressed in neurons of the superficial dorsal horn following noxious sensory stimulation. However, using either in situ hybridisation to map mRNA or specific antibodies to detect the protein products we were unable to detect any change in expression of those genes in stimulated dorsal root ganglion cells or motor neurons. In contrast levels of c-jun mRNA and protein-like immunoreactivity (but not c-fos or NGFI-A) are massively increased within dorsal root ganglion cells and motor neurons following sciatic nerve section or crush. However, these changes are neither rapid nor transient. Increased gene product is seen at 24 h but not 2 h after nerve damage and these levels are maintained up to seven days later. These results suggest that there are multiple routes for the control of c-jun gene expression within the nervous system and that c-jun may play a key role in the neuronal response to injury.

Following peripheral axotomy, fluoride resistant acid phosphatase (FRAP) and most neuropeptides are depleted in the central terminals of axotomised nerves and reduced in their corresponding cell bodies (DRG) but vasoactive intestinal polypeptide (VIP) increases. The increase in VIP probably results from a change in gene expression in other ganglion cells which do not normally express VIP. A quantitative study was performed to investigate the proportion of DRG cells immunoreactive for different peptides at increasing times after sciatic nerve section. Retrograde fluorescent neuronal labelling of sciatic nerve cell bodies by injection of fast blue into the proximal stump was combined with unlabelled antibody immunohistochemistry for CGRP and VIP. The proportion of cells immunoreactive for these peptides was quantified between two and fourteen days post-axotomy. The number of VIP immunoreactive profiles increased significantly in the first 4 days post-axotomy, followed by a slight decrease before rising again. In contrast, the number of and CGRP-immunoreactive cell profiles declined to zero by 14 days post-axotomy. 4 days post-axotomy 50% of VIP positive cells were also immunoreactive for CGRP. There was neither colocalisation between VIP and FRAP nor between CGRP and FRAP. It is concluded that many peptidergic DRG cell bodies switch their expression of peptide to VIP after injury, whereas non-peptide-containing subpopulations do not.

The highest molecular weight neurofilament protein (NF-H) is multiply phosphorylated at epitopes which can be distinguished by specific monoclonal antibodies on Western blots. Eight characterized antibodies were used in immunocytochemistry to examine the tissue distributions of phosphorylated variants of NF-H in axons of the adult rat spinal cord. The most striking difference in staining was found between axons in the cuneate tract and those in the neighboring dorsal corticospinal tract. Axons in the cuneate tract reacted intensely with antibodies to phosphorylated epitopes of NF-H and poorly with antibodies to dephosphorylated epitopes of NF-H, whereas the reverse was the case for the axons of the dorsal corticospinal tract. These differences showed that systematic variations in the phosphorylation of NF-H in long-tract axons in the central nervous system occur as a function of cell type. When the cytoskeletons of these axons were compared by electron microscopy, the neurofilaments of the cuneate fibers were seen to be more abundant and formed a latticework, more compactly organized than the neurofilaments of the dorsal corticospinal axons. By comparison, the dorsal corticospinal axons were relatively richer in microtubules than the cuneate axons. Although the cuneate fiber tract contained many more large (greater than 2.0 microns 2 in cross section) axons than did the dorsal corticospinal tract, these differences in cytoskeletal organization were apparent even when myelinated axons of similar sizes (0.4 micron 2 to 2.0 microns 2) were compared. In addition, the number of neurofilaments in cuneate axons in the 0.4 to 2.0 microns 2 size range was significantly better correlated with axon size than was the case for this size range of dorsal corticospinal axons. Thus, the differences seen in the organization of the neurofilament latticework and the phosphorylation of NF-H between axons found in these two tracts both appeared to be correlated with cell type, and were independent of length or caliber of the axons.