S.J. Gibson

Ultrastructural studies on calcitonin gene-related peptide-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia: Evidence for the colocalization of different peptides in single secretory granules

SummaryCalcitonin gene-related peptide (CGRP)-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia have been studied by means of single and double immunogold labelling techniques. Peptide-immunoreactive neurones are generally B- or C-type cells of small size, with well developed rough endoplasmic reticulum and scanty neurofilaments. In neurones classifiable as A2-type cells, i.e. larger neurones with a lighter cytoplasm due to the presence of poorly developed Nissl bodies and numerous neurofilaments, only CGRP immunoreactivity was detected. Immunolabelled structures were identified as large (60–100 nm diameter), electron-dense, membranebounded p-type granules. They were observed only in neuronal cell bodies or in the intraganglionic portions of the axons. No granules immunoreactive to the antisera applied in this study were observed in non-neuronal cells. Immunostaining experiments with different combinations of the antisera revealed, in some cells, the presence of double immunolabelled granules; in particular localization of CGRP and tachykinins, CGRP and somatostatin, and tachykinins and somatostatin to single secretory granules was demonstrated. The finding that more than one peptide is localized to the same secretory granule supports the postulate that peptides are co-released upon nerve stimulation providing morphological support for physiological and pharmacological data demonstrating an interaction between different peptides in the modulation of synaptic activity.

Peptide expression is altered when afferent nerves reinnervate inappropriate tissue

Neuropeptides are found in specific subpopulations of primary afferent neurones. Peptide expression can be altered following axotomy or under the influence of nerve growth factor. Here we have examined the consequence of altering the peripheral target of afferent neurones. Many unmyelinated afferents from skin contain substance P-like immunoreactivity (SPLI) whilst those from muscle do not. We have found that fibres will innervate inappropriate tissue types. We have therefore cut and cross-anastomosed a skin and muscle hindlimb nerve in the rat and 10-12 weeks later analysed the regenerated nerves immunocytochemically for SPLI. Muscle afferents inappropriately reinnervating skin were found to contain many SPLI fibres in contrast to control nerves resutured to their own distal stumps. Conversely, skin afferents made to innervate muscle showed reduced levels of peptide staining. These results demonstrate the plasticity of peptide expression and suggest that factors in peripheral tissue or perhaps distal nerve sheaths exert a trophic influence on nervous system function.

Neurogenic extravasation and substance P levels are low in muscle as compared to skin in the rat hindlimb

Activation of cutaneous chemosensitive afferents results in the release of substances which increase the permeability of the microcirculation, producing inflammation. That this inflammation is neurogenic is readily demonstrated by antidromic electrical stimulation of afferent fibres. In the present study we have used the technique of dye extravasation to compare both qualitatively and quantitatively neurogenic extravasation skin and skeletal muscle. Plasma extravasation has been found to occur in skeletal muscle after stimulation but only at less than 10% of the levels seen in skin. We have also found that the levels of the C-fibre markers substance P and fluoride-resistant acid phosphatase are greatly reduced in muscle compared with skin nerves. These results show that there are substantial differences in the population of C-fibres supplying muscle compared with those supplying skin.

Neuropeptide Y in human spinal cord

The distribution of a newly described peptide, neuropeptide Y (NPY) within the human spinal cord has been determined using radioimmunoassay and immunocytochemistry. Higher concentrations were found in the lumbar (49.9 +/- 6.8 pmol/g) and sacral (47.0 +/- 10.6 pmol/g) regions than in the cervical (27.6 +/- 2.7 pmol/g) and thoracic spinal cord (33.8 +/- 5.3 pmol/g). Immunocytochemistry revealed numerous nerve fibers containing NPY in the spinal cord; these were particularly concentrated in the substantia gelatinosa of the dorsal horn. In the ventral spinal cord NPY-containing nerves were sparse becoming more abundant in lumbosacral segments.

A novel substance P pathway linking the dorsal and ventral horn in the upper lumbar segments of the rat spinal cord

We report here the presence of a novel substance P pathway existing at a segmental level in the upper lumbar region (L1/L2) of the rat spinal cord. Substance P-containing fibres were seen directly linking a number of discrete areas of the rat spinal cord, including dorsal and ventral regions. These fibers were found closely associated with a specific group of motoneurones, the mediolateral motor nucleus in the ventral horn. This motoneurone group appeared as a focusing nucleus around which substance P-containing tracts appeared to congregate. An intrinsic spinal cord origin of substance P-containing neurones in these interconnecting pathways is suggested as deafferentation achieved either by section of appropriate dorsal roots, or pharmacologically by neonatal treatment with capsaicin, had no effect on the appearance of the nucleus in the ventral spinal cord or of the associated substance P-containing tracts. Likewise, mid-thoracic lesions of the spinal cord (to determine supraspinal contributions) including hemisection, dorsolateral funiculus lesions and lesions of the region around the central canal, induced no change. The significance of these interconnecting substance P-immunoreactive fibre tracts is not known. However, the intimate association of substance P-containing fibres with a specific group of motoneurons indicates that they may modulate motor function.

Distribution and localization of neuromedin B-like immunoreactivity in pig, cat and rat spinal cord

The distribution and localization of neuromedin B, a novel bombesin-like decapeptide isolated from porcine spinal cord, was investigated by newly established radioimmunoassay and immunocytochemistry in the pig, cat and rat spinal cord. Neuromedin B-like immunoreactivity was found to be concentrated particularly in the dorsal part of lumbosacral segments in all species studied and the highest concentration of immunoreactivity was 25.7 +/- 3.4 pmol/g wet wt in the dorsal part of sacral region of pig spinal cord. The nature of the immunoreactivity was studied by gel permeation and high pressure liquid chromatography. Chromatography of spinal cord extracts from three species revealed two major peaks of neuromedin B-like immunoreactivity and the prevalent molecular from co-eluted with synthetic porcine neuromedin B. Immunocytochemistry localized neuromedin B immunoreactivity to fibres and terminals throughout the entire length of the spinal cord of pig, cat and rat. Fibres were most abundant in laminae I and II of the dorsal horn, the area around the central canal (lamina X) and intermediolateral cell columns of thoracic and sacral segments. In lumbosacral segments neuromedin B-immunoreactive fibres were slightly more numerous, in both dorsal and ventral spinal cord, than in cervical and thoracic regions.

PHI-like immunoreactivity co-locates with the VIP-containing system in human lumbosacral spinal cord

Using a specific radioimmunoassay and immunocytochemistry, the quantitative regional distribution and localization of PHI-like immunoreactivity was studied in normal postmortem human spinal cord. The levels of PHI-like immunoreactivity were low in the cervical and thoracic region whereas the lumbar and especially sacral regions showed higher levels, with dorsal sacral concentrations exceeding ventral concentrations. Chromatographic analysis by high pressure liquid chromatography revealed that the PHI-like immunoreactivity in human spinal cord elutes slightly earlier than pure porcine PHI, and may correspond to PHM-27, a PHI-27-like peptide found in human preprovasoactive intestinal polypeptide. Immunocytochemical studies show a distinctive distribution of PHI-like immunoreactive fibres and terminals at the lumbosacral segments. The distribution of PHI-like immunoreactivity is thus similar to that of VIP, and unlike a number of other neuropeptides; with VIP, it may mark a system which has a role in the spinal control of urogenital function in man.

Increase of substance P-like immunoreactivity in the peripheral nerve of the axolotl after injury

That substance P may be a neurotrophic factor in urodele limb regeneration was investigated in the axolotl (Ambystoma mexicanum). Two weeks after section of the peripheral nerves to the forelimb, there was a marked increase in substance P-like immunoreactivity (IR) content of the nerves proximal to the lesion. A smaller increase occurred as early as 3 days after section of nerves innervating a regrowing limb bud (blastema), and substance P-IR fibres were observed by immunocytochemistry to innervate blastema tissue. As substance P-IR by applying capsaicin to peripheral nerve--capsaicin had no effect on substance P-IR in either intact or injured axolotl nerves. Substance P fulfills a number of criteria of a trophic substance in axolotl limb regrowth.

Transient expression of neuropeptide Y and its C-flanking peptide immunoreactivities in the spinal cord and ganglia of human embryos and fetuses

An immunohistochemical study of spinal cord, dorsal root and sympathetic ganglia of human embryos and fetuses demonstrated that neuropeptide Y and its C-flanking peptide could be detected in seven-week-old embryos but were absent or difficult to demonstrate after the 17th week of gestation. The peptides were found in several structures of the spinal cord, e.g. fibres in the dorsal portion of the lateral funiculus, cell bodies and fibres in the dorsal horn, and motoneurons, and also in numerous primary sensory neurons of dorsal root ganglia. They were also present in sympathetic neurons and since these are the only structures expressing neuropeptide Y and its C-flanking peptide in the adult, it must be concluded that their presence in other neurons is a transient developmental feature. To assist in understanding the relationship of these transient structures with other spinal and sensory neurons, a comparison was made with other neuronal structures showing immunoreactivity for two general neuronal markers, neurofilaments and protein gene product 9.5, and two neuropeptides present in primary sensory afferents, somatostatin and substance P. In the dorsal root ganglia, numerous neuropeptide Y- and C-flanking peptide-immunoreactive neurons were observed before substance P- or somatostatin-immunoreactive cells could be detected. Therefore, neuropeptide Y and its C-flanking peptide could represent a primitive peptidergic system appearing before primary sensory neurons express their characteristic adult phenotype. The fibres of the lateral funiculus showing immunoreactivity for neuropeptide Y and its C-flanking peptide were longitudinally orientated and could be detected at all cephalocaudal levels of the spinal cord. Comparison with the other immunohistochemical markers indicated that they were not primary sensory afferents. At least some of them probably originated from neuropeptide Y- and C-flanking peptide-immunoreactive neurons of the dorsal horn, that may be considered to be a subset of early-appearing interneurons.

Studies of vasoactive intestinal polypeptide expression in injured peripheral neurons using capsaicin, sympathectomy and mf mutant rats

The increased expression of vasoactive intestinal polypeptide (VIP) in injured peripheral neurons was studied. In contrast to substance P, there was a marked increase, and maintained fast axonal transport, of VIP in rat sciatic nerve after peripheral axotomy. Local capsaicin application to the nerve trunk failed to inhibit the injury-induced VIP increase, and capsaicin even increased VIP levels when applied locally to uninjured nerves. Pharmacological sympathectomy showed that some of the peripheral VIP increase may occur in post-ganglionic sympathetic fibres. The VIP increase after injury appeared unaffected in the mf mutant rat, in spite of its loss of lumbar dorsal root ganglion cells. VIP-staining fibres in the epi- and peri-neurium and perivascular plexuses of sciatic nerve showed an increase in number in parallel with the changes of the nerve VIP content. These findings suggest that sensory and sympathetic nerve fibres expressing VIP after injury play a role in the regulation of blood flow to nerves, and in the pathophysiological processes in nerve and dorsal spinal cord which follow peripheral nerve injury.