Localization of vasoactive intestinal peptide immunoreactivity in human foetus and newborn infant spinal cord

Using an indirect immunofluorescence method the distribution of vasoactive intestinal peptide (VIP) immunoreactivity was studied in human foetus and newborn infant spinal cord and dorsal root ganglia. Further, for comparison some newborn infant brains were also investigated. Vasoactive intestinal peptide-like immunoreactive fibres were exclusively found in the caudal spinal cord and corresponding dorsal root ganglia. No immunoreactive cell bodies were detected. The first appearance of VIP-like immunoreactive fibres in both spinal cord and dorsal root ganglia was suggested during the fourth month of foetal life. Most immunolabelled fibres, concentrated in the sacral segment, were distributed in the Lissauer tract, along the dorsolateral gray border, in the intermediolateral areas and near the central canal in the dorsolateral commissure. A few VIP-like immunoreactive fibres were also seen in the dorsal funiculus and occasionally in the ventral gray horn and ventral roots. Further, a large population of VIP-like immunoreactive fibres occurs longitudinally in dorsal root, in ganglia and in the spinal nerve exit zone. These findings indicate the early appearance of VIP-like immunoreactive fibres in the human foetus spinal cord and corresponding ganglia. Moreover, they emphasize that in both foetus and newborn infant spinal cord VIP-like immunoreactive fibre distribution is limited to the lumbosacral segment.     

Coexistence of cholecystokinin- and substance P-like peptides in neurons of the dorsal root ganglia of the rat

Using the indirect immunohistochemical technique with antisera to cholecystokinin and to substance P, the spinal dorsal horn and dorsal root ganglia of normal and colchicine-treated rats were studied. In the spinal cord a similar distribution of substance P- and cholecystokinin-positive networks in the superficial layers of the dorsal horn was observed. In the dorsal root ganglia several cholecystokinin and substance P immunoreactive cell bodies were seen in colchicine-treated rats. After elution and restaining for substance P, of sections previously stained for cholecystokinin, it was found that all cholecystokinin-positive cells also contained substance P-like immunoreactivity and vice versa.     

Developmental pattern and distribution of nerve growth factor low-affinity receptor immunoreactivity in human spinal cord and dorsal root ganglia: comparison with synaptophysin, neurofilament and neuropeptide immunoreactivities.

Immunocytochemical expression of the low-affinity nerve growth factor receptor was studied in human fetal and adult tissues using the monoclonal antibody ME20.4. In dorsal root ganglia, a few immunoreactive neurons were first detected in nine-week-old fetuses and many more were found in the following weeks of gestation. However, none was present in adult ganglia. The ME20.4-positive cells were larger than neurons immunostained by substance P, calcitonin gene-related peptide or galanin antibodies. In the spinal cord, fibres immunostained by ME20.4 appeared in a characteristic pattern that differed from the spatial and temporal distributions of synaptophysin- and neurofilament-immunoreactive fibres. Those expressing the low-affinity nerve growth factor receptor were only detected in regions containing collaterals of primary sensory axons: (i) in the dorsal funiculus between seven and 18 weeks of gestation; (ii) in a ventrodorsal bundle reaching the ventral horn from weeks 12-14; (iii) in the medial region of the dorsal horn between weeks 12 and 20; (iv) in the superficial layers and lateral portion of the dorsal horn after the 14th week of gestation and also in adult spinal cord. During the fetal period, ME20.4 immunoreactivity was also found in motoneurons and peripheral nerve fibres in the skin, myotomes and gut. Sheaths of peripheral nerves and the adventitia of blood vessels were stained both in fetal and adult tissues. Thus, the low-affinity nerve growth factor receptor is: (i) strongly expressed in the developing human nervous system; (ii) transiently associated with a subset of large primary sensory neurons and with motoneurons; (iii) transiently and sequentially expressed by various groups of sensory afferents to the spinal cord; (iv) permanently expressed by fibres in the superficial layers of the dorsal horn, Clarke's column, nerve sheaths and the adventitia of blood vessels.     

The immunocytochemical distribution of seven peptides in the spinal cord and dorsal root ganglia of horse and pig

Summary The distribution of calcitonin gene-related peptide (CGRP), enkephalin, galanin, neuropeptide Y (NPY), somatostatin, tachykinins and vasoactive intestinal polypeptide (VIP) was compared in cervical, thoracic, lumbar and sacral segmental levels of spinal cord and dorsal root ganglia of horse and pig.In both species, immunoreactivity for the peptides under study was observed at all segmental levels of the spinal cord. Peptide-immunoreactive fibres were generally concentrated in laminae I–III, the region around the central canal, and in the autonomic nuclei. A general increase in the number of immunoreactive nerve fibres was noted in the lumbosacral segments of the spinal cord, which was particularly exaggerated in the case of VIP immunoreactivity. In the horse, some CGRP-, somatostatin- or tachykinin-immunoreactive cell bodies were present in the dorsal horn. In the pig, cells immunoreactive for somatostatin, enkephalin or NPY were noted in a similar location.In the ventral horn most motoneurones were CGRP-immunoreactive in both species. However, in pig many other cell types were CGRP-immunoreactive not only in the ventral horn, but also in laminae V–VI of the dorsal horn.With the exception of enkephalin and NPY immunoreactivity, which was not seen in pig dorsal root ganglia, all peptides studied were localised to neuronal cell bodies and/or fibres in the dorsal root ganglia. In both species, immunolabelled cell bodies were observed in ganglia from cervical, thoracic, lumbar and sacral levels, with the exception of VIP-immunoreactive cells that were detected only in the lumbosacral ganglia. Numerous CGRP- and tachykinin-immunoreactive cell bodies were visualised in both species, while the cells immunolabelled with other peptide antisera were much lower in number.In both species, immunostaining of serial sections revealed that a subset of CGRP-immunoreactive cells co-expressed tachykinin, galanin or somatostatin immunoreactivity. In the horse some enkephalin-immunoreactive cells were also CGRP positive and occasionally combinations of three peptides, e.g. CGRP, tachykinin and galanin or CGRP, tachykinin and enkephalin were identified.The results obtained suggest that the overall pattern of distribution of peptide immunoreactivities is in general agreement with that so far described in other mammals, although some species variations have been observed, particularly regarding the presence of immunoreactive cell bodies in the dorsal horn of the spinal cord.     

Studies on the changes of c-fos protein in spinal cord and neurotransmitter in dorsal root ganglion of the rat with an experimental peripheral neuropathy

Animal models for human chronic pain syndromes have been developed and widely used for pain research. One of these neuropathic pain models by Kim and Chung (1992) has many advantages for operation and pain elicitation. In this neuropathic model we have examined the c-fos protein, substance P, CGRP immunoreactivity in dorsal root ganglia and dorsal horn. 50 Sprague-Dawley rats were used for this study. L5 and L6 spinal nerves were ligated tightly to produce the neuropathic pain model. After 2, 4, 8, 16, and 24 hours and 1 week of surgery, rats were anesthetized and sacrificed by perfusion. After confirmation of the roots transected by the surgery, the L5 and L6 dorsal root ganglions and spinal cord were removed and processed for immunohistochemistry. All tissue sections were immunohistochemically stained for substance P, CGRP and c-fos using the peroxidase-antiperoxidase (PAP) method. The number of immunostained substance P and CGRP dorsal root ganglion cells and c-fos immunoreactive dorsal horn cells were counted and analyzed statistically with Mann-Whitney U test. The results are as follows. The number of c-fos protein immunoreactive neurons in the superficial layer of dorsal horn were increased markedly 2 hours after operation, and gradually decreased to normal level 1 week after operation. The number of c-fos protein immunoreactive neurons in the deep layer of the dorsal horn gradually increased to a peak 24 hours after operation, then decreased to the normal level 1 week after operation. The number of substance P and CGRP immunoreactive L5 and L6 dorsal root ganglion neurons were decreased markedly 1 week after the pain model operation. In conclusion, after neuropathic pain model operation, c-fos proteins were immediately expressed in the superficial layer of spinal dorsal horn, thereafter c-fos proteins in the deep layer of spinal dorsal horn were expressed. CGRP and substance P immunoreactive neurons in DRG were decreased markedly 1 week after neuropathic pain model operation. These decrements do not coincide with the other chronic pain models, which show great increases in these pain transmitting substances. Therefore, the relationship between pain and c-fos, SP and CGRP should be investigated further.     

Effect of peripheral nerve cut on neuropeptides in dorsal root ganglia and the spinal cord of monkey with special reference to galanin

Summary Using the indirect immunofluorescence method andin situ hybridization, the localization and levels of immunoreactivities and mRNAs for several neuropeptides were studied in lumbar dorsal root ganglia and spinal cord of untreated monkeys (Macaca mulatto) and after unilateral transection of the sciatic nerve. Immunoreactive galanin, calcitonin gene-related peptide, substance P and somatostatin and their mRNAs were found in cell bodies in dorsal root ganglia of untreated monkeys and on the contralateral side of the monkeys with unilateral sciatic nerve lesion. After axotomy there was a marked decrease in the number of calcitonin gene-related peptide-, substance P- and somatostatin-positive neurons in dorsal root ganglia ipsilateral to the lesion, whereas the number of galanin positive cells strongly increased. A few neuropeptide tyrosine-positive cells were seen in after axotomy, whereas no such neurons were found in controls. No vasoactive intestinal polypeptide-, peptide histidine isoleucine-, cholecystokinin-, dynorphin-, enkephalin-, neurotensin-or thyrotrophin releasing hormone-positive cell bodies were seen in dorsal root ganglia of any of the groups studied. In the dorsal horn of the spinal cord all peptide immunoreactivities described above, except thyrotropin releasing hormone, were found in varying numbers of nerve fibres with a similar distribution in untreated monkeys and in the contralateral dorsal horn in monkey with unilateral sciatic nerve lesion. Two cholecystokinin antisera were used directed against the C- and N-terminal portions, respectively, showing a distinctly different distribution pattern in the dorsal horn. Somatostatin- and dynorphin-like immunoreactivities were also observed in small neurons in the dorsal horn. No certain effect of axotomy on these interneurons could be seen. However, marked changes were observed after this type of lesion for some peptide containing fibres in the ipsilateral dorsal horn. Thus, there was a marked increase in galanin-like immunoreactivity, whereas calcitonin gene-related peptide-, substance P-, somatostatin-, peptide histidine isoleucine neurotensin- and cholecystokinin-like immunoreactivities decreased. No changes could be observed in neuropeptide tyrosine or enkephalin-positive fibres. The present results demonstrate marked ganglionic and transganglionic changes in peptide levels after peripheral axotomy. When compared to published results on the effect of axotomy on peptides in dorsal root ganglia and spinal cord of rat, both similarities and differences were encountered. Thus, in contrast to rat there was no marked upregulation of vasoactive intestinal polypeptide/peptide histidine isoleucine or neuropeptide tyrosine after axotomy in the monkey, whereas galanin was increased in both species. Both in monkey and rat, calcitonin gene-related peptide, substance P and somatostatin decreased. The decrease in neurotensin, peptide histidine isoleucine, and genuine cholecystokinin seen in monkey after axotomy has not been reported in the rat. Experimental studies on rat suggest that galanin may be an endogenous analgesic compound, active particularly after peripheral nerve lesions. We have therefore recently proposed that galanin agonists may be used in treatment of chronic pain, and the present demonstration that galanin is regulated in a similar fashion in a primate gives further support to the proposal to test galanin as an analgesic in human.     

Capsazepine, a novel capsaicin antagonist, selectively antagonises the effects of capsaicin in the mouse spinal cord in vitro.

The mouse hemisected spinal cord with attached dorsal roots and spinal ganglia in vitro preparation was used to investigate the effects of the capsaicin antagonist, capsazepine (2-[2-(4-chlorophenyl)ethylamino-thiocarbonyl]-7,8-dihydroxy-2,3,4 ,5- tetrahydro-1H-2-benzazepine). The spinal cord and the ganglia were separated by a perspex gap, allowing application of drugs separately to each compartment. Intracellular recordings were made from 37 cells in laminae II-VI of 12 to 20-day-old mice. Brief applications (30 s) of capsaicin (0.8 microM) excited dorsal horn neurones by activating small diameter primary afferent fibres. The response to capsaicin administered to the spinal cord or to the spinal ganglia was antagonised by the capsaicin antagonist, capsazepine (1.5 microM), administered to the same site. Excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of the dorsal root were not affected by capsazepine. Capsazepine itself (5 microM) did not affect the membrane potential of the dorsal horn cells. Capsazepine did not depress the depolarization evoked by substance P. When capsazepine was applied to the spinal cord and capsaicin to the dorsal root ganglion the capsaicin effect was not antagonised. These data suggest that capsaicin-induced depolarization of spinal dorsal horn neurones was mediated via activation of a specific receptor on primary afferent neurones.     

Enkephalin and capsaicin-resistant substance P-like immunoreactivities in intra-ocular grafts of different fetal spinal cord areas

Fetal spinal cord tissue was grafted to the anterior chamber of the eye of adult rats in order to evaluate survival and distribution of substance P- and enkephalin-immunoreactive neurons. Capsaicin treatment was used to evaluate any possible contribution of host iris-derived substance P fibres to the innervation of the grafts and to check for capsaicin sensitivity of graft substance P-positive systems. Substance P- and enkephalin-immunoreactive nerve fibres were present in grafts of half-transverse segments of the spinal cord and were clearly co-distributed throughout the grafts. Areas with a high density of substance P- and enkephalin-positive fibres resembling substantia gelatinosa were seen. Grafts of the dorsal horn alone had a moderate to high density of substance P- and enkephalin-positive fibres, while ventral horn grafts contained a low amount of such fibres. Capsaicin eliminated the substance P innervation of the host iris and the dorsal root ganglion-derived substance P innervation of the host spinal cord, while sparing the intrinsic substance P innervation of both host spinal cord and spinal cord grafts. These experiments show that intra-ocular grafts of defined spinal cord areas express relatively organotypic amounts of substance P- and enkephalin-positive nerve fibres, and thus emphasize the importance of intrinsic genetic determinants for spinal cord development.     

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.     

Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide and monoamine neurons using antisera to substance P, gastrin/CCK, somatostatin, VIP, enkephalin, neurotensin and 5-hydroxytryptamine

Summary After neonatal treatment of rats with capsaicin, the spinal cord, the spinal trigeminal nucleus and spinal and trigeminal ganglia were analysed with immunohistochemistry using antisera to several peptides and 5-hydroxytryptamine. A marked decrease was observed in substance P-, cholecystokinin-, somatostatin- and VIP-like immunoreactivity present in the central branches of primary sensory neurons in the spinal cord and in substance P- and somatostatin-like immunoreactivity in sensory ganglion cells. No definite depleting effect of capsaicin could be established on 5-hydroxytryptamine and peptides, such as enkephalin and neurotensin, present in centrally originating fibres in the dorsal horn of the spinal cord. The results demonstrate that the effects of capsaicin are not confined to substance P immunoreactive primary sensory neurons. The possibility is discussed that capsaicin effects specifically functioning rather than chemically specific primary sensory neurons.     

Cholecystokinin-like immunoreactivity in the dorsal horn of the spinal cord of the rat: a light and electron microscopic study

Cholecystokinin-like immunoreactivity was investigated with an indirect immunoperoxidase technique in the whole spinal cord with the light microscope and in the dorsal horn with the electron microscope. Intraparenchymal injections of colchicine were performed to allow the detection of cholecystokinin-like immunoreactive cell bodies. Rats treated at birth with capsaicin were also studied at the light microscope. Numerous cholecystokinin-like immunoreactive fibres and varicosities were found in the two superficial layers of the dorsal horn and in the intermedio-medial nucleus; cholecystokinin-like immunoreactive cell bodies were also present in these two regions. After neonatal capsaicin treatment, the number of cholecystokinin-like immunoreactive fibres and varicosities was strongly reduced in the dorsal horn. At the electron microscope level, cholecystokinin-like immunoreactivity was localized in numerous neurites often filled with vesicles (axon terminals and dendrites containing vesicles) and in few cell bodies and dendrites. The immunoreaction was found mainly associated with ribosomes, granular reticulum, neurotubules and vesicles. Large granular vesicles were filled with the reaction product whereas small and medium-sized vesicles showed a varying degree of immunoprecipitate around their membrane. In addition dense "granules" of precipitate were observed in numerous presynaptic neurites. Cholecystokinin-like immunoreactive axons were of small calibre and mostly unmyelinated. Cholecystokinin-like immunoreactive axon terminals made asymmetric synaptic contacts with generally unlabelled dendrites or dendritic spines. A single labelled nerve terminal could contact several different dendrites in structures resembling glomeruli. Few axo-somatic synapses but a relatively high number of axo-axonic contacts were seen. About half of these axo-axonic contacts involved pre- and postsynaptic profiles. Both light and electron microscopic observations led us to the conclusion that some of the cholecystokinin-like immunoreactive fibres of the dorsal horn originate in the spinal ganglia via capsaicin-sensitive C afferents; and some from intrinsic neurons, particularly islet cells. Other fibres may come from supraspinal centres, other local neurons or capsaicin-insensitive afferents from the spinal ganglia. The results are discussed with regard to data in the literature, particularly those concerned with the specificity of the cholecystokinin antibodies; it is hypothesized that several types of cholecystokinin-like immunoreactive peptides may be present in the dorsal horn, depending on their origin (supraspinal, intrinsic or peripheral).(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of monoamine neurotoxins on peptides in the rat spinal cord

The coexistence of two neuronally-localised peptides, substance P and thyrotropin-releasing hormone (TRH), in descending serotoninergic nerve fibres to the spinal cord was investigated using immunocytochemical and biochemical methods. Substance P-like material in the spinal cord was shown to be identical to the undecapeptide substance P by the criteria of gel filtration, high performance liquid chromatography and behaviour in substance P specific radioimmunoassays. Immunocytochemical staining for 5-hydroxytryptamine, substance P, and TRH showed that all three substances had a similar distribution in nerve fibres and terminals in the ventral and lateral grey matter of the spinal cord. After treatment with the serotonin neurotoxin 5,7-dihydroxytryptamine, neuronal elements containing 5-hydroxytryptamine, substance P and TRH degenerated and disappeared from these parts of the spinal cord in parallel with one another.Biochemical measurements of 5-hydroxytryptamine, substance P and TRH in the spinal cord after treatment with 5,7-dihydroxytryptamine confirmed that these three substances were all depleted from the ventral horn and, in addition, showed that there was a small depletion of substance P from the dorsal horn. Two other neuropeptides, somatostatin and methionine-enkephalin were not depleted from the spinal cord by treatment with 5,7-dihydroxytryptamine nor was substance P in other parts of the brain. Substance P in the spinal cord was unaffected by 6-hydroxydopamine, a drug known to destroy catecholamine-containing neurones.These results are consistent with coexistence of substance P and TRH together with 5-hydroxytryptamine in the descending axons and terminals of bulbospinal neurones.     

Tenascin-C expression by neurons and glial cells in the rat spinal cord: Changes during postnatal development and after dorsal root or sciatic nerve injury

Summary We have usedin situ hybridization with a digoxigenin-labelled probe for tenascin-C mRNA and immunocytochemistry with antibodies against tenascin-C, glial fibrillary acidic protein, OX-42 and the 200 kDa neurofilament protein to study the expression, distribution and cellular relationships of tenascin-C mRNA and protein in the developing (postnatal) and adult spinal cord of rat, and the effects thereon of dorsal root, ventral root and sciatic nerve injuries. The most interesting finding was that on postnatal day 7 (P7), P14 and in the adult, but not on P0 or P3, a group of neurons in the lumbar ventral horn expressed the tenascin-C mRNA gene. They represented about 5% of ventral horn neurons in the adult and were among the smaller such neurons. Since 40–60% of such cells were lost at P13 following sciatic nerve crush on P0, some were almost certainly motor neurons. In addition, we found that at P0 and P3, mRNA-containing glial cells were widespread in grey and white matter but sparse in the developing dorsal columns; tenascin-C immunofluorescence showed a similar distribution. By P7 there were fewer mRNA-containing cells in the ventral horns and in the area of the dorsal columns containing the developing corticospinal tract where immunofluorescence was also weak. At P14 there were no glial-like mRNA-containing cells in the grey matter; such cells were confined to the periphery of the lateral and ventral white columns but were present throughout the dorsal columns where tenascin-C immunofluorescence was also strong. No glial-like mRNA-containing cells were present in the adult lumbar spinal cord and tenascin-C immunofluorescence was confined to irregular patches in the ventral horn, especially around immunonegative cell bodies of small neurons, a zone around the central canal, and a thin zone adjacent to the glia limitans. Thus the expression of tenascin-C is differentially developmentally regulated in the grey matter and in different parts of the white matter. Three days after injury of dorsal roots L4–6, many cells containing tenascin-C mRNA, some identified as glial fibrillary acidic protein-positive astrocytes, were present in the ipsilateral dorsal column, but were rare after longer survivals. Immunoreactivity, however, was elevated in the ipsilateral dorsal column at 3 days, remained high for several months and disappeared at 6.5 months. Dorsal root injury had no effect on tenascin-C mRNA or protein in the grey matter. Sciatic nerve or ventral root injury had no effect on these molecules in any part of the spinal cord.     

Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat

The localization of two small peptides, somatostatin and substance P, has been studied with the indirect immunofluorescence technique. Both peptides were present in small neuronal cell bodies in spinal ganglia, in fibers in the dorsal horn of the spinal cord and in fibers in the intestinal wall. By comparing consecutive sections incubated with antisera to somastostatin and to substance P respectively, it was established that somatostatin, or somatostatin-like immunoreactivity and substance P, or substance P-like immunoreactivity are present in different cells. This is possibly indicated also by a somewhat differential distribution of the immunoreactive fibers in the dorsal horn: the highest concentration of somatostatin-positive fibers was observed in lamina II, whereas abundant substance P-positive fibers were present also in lamina I. Furthermore, numerous substance P-, but no somatostatin-positive fibers, were found around the central canal and in the ventral horns. In the intestinal wall more substance P-positive than somatostatin-positive fibers were seen.The present results indicate that two subpopulations of primary sensory neurons exist, one containing somatostatin, or somatostatin-like immunoreactivity, and the other containing substance P, or substance P-like immunoreactivity.     

Distribution of five peptides,three general neuroendocrine markers,and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: An immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I–II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I–III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I–II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

Substance P-, met-enkephalin- and somatostatin-like immunoreactivity distribution in the frog spinal cord

The distribution of substance P-, Met-enkephalin- and somatostatin-like immunoreactivity was studied in the thoracic spinal cord of the frog using immunohistochemical techniques. In fibres, probably nerve terminals, immunoreactivity was greatest in the grey matter (mainly dorsal horn), but it was also present in white matter regions. While substance P- and, perhaps, somatostatin-like immunoreactivity appeared to be contained in primary afferents, the presence of all 3 peptides in neuronal cells of the grey matter indicates the existence of a propriospinal peptidergic system.     

Reciprocal age-related changes in GAP-43/B-50, substance P and calcitonin gene-related peptide (CGRP) expression in rat primary sensory neurones and their terminals in the dorsal horn of the spinal cord and subintima of the knee synovium

Age-related changes in the expression of the growth associated protein GAP-43/B-50, and the neuropeptides substance P and calcitonin gene-related peptide (CGRP) were investigated in the sensory neurones of rat dorsal root ganglia, dorsal horns of the spinal cord and subintimal knee synovium. The two time-points studied were 2 months (young adults) and 14-month (aged)-old Sprague Dawley rats. Dorsal root ganglia: In young adults, 40 and 35% of the L4-L5 dorsal root ganglion neurones were positive for GAP-43/B-50 with a 1.5 fold increase in frequency in aged rats at the L5 ganglion. GAP-43/B-50 was strongly expressed by the non-neuronal satellite cells of some medium and many large sized neurones in aged rats. There were marked reciprocal shifts between small and medium sized sensory neurones in respect to their substance P and CGRP expression profiles. Dorsal horn of the spinal cord: there was a 1.3 fold decrease of substance P at L5 level and a 1.3 and 1.5 fold decrease of CGRP at L4-L5 levels in aged rats, respectively. Synovial membrane: There was a 2.3 fold increase in GAP-43/B-50 and a 2.5 fold decrease of CGRP with no changes in substance P expression. These results indicate that (i) primary sensory neurones undergo age-related changes already in early stages of aging, (ii) aging may result in a reduction of substance P and CGRP axonal transport, and (iii) reduced numbers of CGRP containing synovial perivascular fibres may imply a deficient regulation of the synovial microvasculature and therefore metabolic homeostasis of the joint in aged subjects.     

Longitudinal extent of dorsal root fibres in the spinal cord and brain stem of the frog

The longitudinal arrangement of dorsal root fibres was investigated with a modified cobalt labelling technique in the spinal cord and brain stem of frogs. The topographical order of dorsal root fibres in the dorsal white column closely resembles the well-known scheme of the mammalian spinal cord. A significant difference between frogs and mammals is the extension of fibres up to the cerebellar plate. The ascending fibres of different origin are organized in concentric rings in the medulla. An oval-shaped area and a triangular area in the dorsal horn, and the motor horn, receive fibre collaterals in the spinal cord. Thoracic dorsal root fibres terminate exclusively in the oval-shaped area. Fibre terminations clearly outline the dorsal column nuclei which begin in the obex region and end at the level of the glossopharyngeal nucleus. The spinal nucleus of the trigeminus is richly supplied by both thin and thick calibre dorsal root fibres in its entire rostrocaudal extension. Two parts of the reticular formation receive dorsal root fibres; the first is in the dorsal gray matter ventral and lateral to the solitary fascicle in the medulla, the second is the lateral reticular zone. In the vestibular region, the medial, lateral and superior vestibular nuclei are innervated by dorsal root fibres. The granular layer of the cerebellum receives a significant contingent of dorsal root fibres. Fibres terminating in the vestibular region and in the cerebellum arise from limb-innervating spinal ganglia.The results indicate a close similarity in the longitudinal arrangement of dorsal root fibres in frogs and in higher vertebrates. The several collaterals that terminate in the hindbrain may modulate the function of the receiving structures. On the basis of present and previous findings the aggregation of primary sensory fibres and the convergence of their terminations are surveyed in the hindbrain.     

Fluoride-resistant acid phosphatase-containing neurones in dorsal root ganglia are separate from those containing substance P or somatostatin

The distribution of fluoride-resistant acid phosphatase, substance P and somatostatin were investigated in the dorsal horn of the spinal cord and in dorsal root ganglia. In the dorsal horn, the distribution of fluoride-resistant acid phosphatase closely paralleled that of somatostatin and only partly overlapped with that of substance P. In sensory ganglia, none of the fluoride-resistant acid phosphatase-containing neurones contained either substance P or somatostatin. The results suggest the existence of a population of fluoride-resistant phosphatase-positive sensory neurones which is distinct from neurones containing either of these peptides.