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.     

Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury

Radioimmunoassay and immunocytochemistry were used to study the distribution of galanin, a novel 29 amino acid porcine intestinal peptide, in the central nervous system of the rat and pig. The pattern of distribution was similar in the two species, with the highest concentrations of galanin-like immunoreactivity found in the neurohypophysis, hypothalamus and sacral spinal cord. Immunocytochemical studies of these regions localized galanin-like immunoreactivity to cell bodies in the paraventricular and supraoptic nuclei of the hypothalamus, to fibres in the pars nervosa and to numerous cell bodies and fibres in the dorsal horn of the spinal cord. On both gel and high pressure liquid chromatography, galanin-like immunoreactivity in rat and pig nervous tissue eluted as a single peak in a position similar to purified procine intestinal galanin standard. Surgical and pharmacological manipulations in the rat suggest the presence of galanin in afferent fibres. An increase of galanin-like immunoreactivity was observed in the sacral spinal cord of the rat following thoracic spinal cord transection. Thus galanin-like immunoreactivity in the brain is mainly localized in the hypothalamopituitary region. The decrease of galanin-like immunoreactivity in the dorsal horn of the spinal cord, following dorsal rhizotomy and pre-treatment of rats with capsaicin, indicates that many of the fibres, which are of small diameter, may well be derived from spinal sensory neurones.     

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.     

Projections of chemically-specified neurons in the guinea-pig colon.

The arrangement of the enteric nerve plexuses in the colon of the guinea-pig and the distributions and projections of chemically specified neurons in this organ have been studied. Immunoreactivity for neuron specific enolase was used to examine the total population of neurons and individual subpopulations were studied using antibodies raised against calbindin, calcitonin gene-related peptide (CGRP), leu-enkephalin, gastrin releasing peptide (GRP), galanin, gamma aminobutyric acid, neurokinin A, neuropeptide Y (NPY), somatostatin, substance P, tyrosine hydroxylase and vasoactive intestinal peptide (VIP). Neuronal pathways within the colon were lesioned using myotomy and myectomy operations and extrinsic pathways running between the inferior mesenteric ganglia and the colon were also severed. Each of the antibodies revealed nerve cells and nerve fibres or only nerve fibres within the wall of the colon. VIP, galanin and GRP were in anally projecting pathways in the myenteric plexus, as they are in other species. In contrast, there are differences in the projection directions of enkephalin, substance P, NPY and somatostatin nerve fibres between regions and species. Surprisingly, somatostatin and NPY fibres have opposite projections in the small intestine and colon of the guinea-pig. The majority of nerve fibres that innervate the circular muscle, including fibres with immunoreactivity for VIP, enkephalin, substance P, NPY, galanin and GRP come from the myenteric ganglia. The mucosa is innervated by fibres from both the myenteric and submucous ganglia. The present results suggest that the guinea-pig distal colon is a suitable place in which to determine relations between structure, neurochemistry and functions of enteric neural circuits.     

Expression of eight neuropeptides in intraocular spinal cord grafts: organotypical and disturbed patterns as evidenced by immunohistochemistry

The present study examines the distribution of several neuropeptides, as revealed by immunohistochemistry in the isolated cord. Fetal rat spinal cord was grafted to the anterior chamber of the adult Sprague-Dawley albino rats. After intraocular maturation for 2-3 months, the amount and distribution of somatostatin, neuropeptide Y, substance P, enkephalin, vasoactive intestinal peptide, peptide histidine-isoleucine, calcitonin gene-related peptide and cholecystokinin immunoreactive terminals and cell bodies were analysed using indirect fluorescence immunohistochemistry. The visualization of immunoreactive cell bodies in the grafts was enhanced using a novel intraocular colchicine treatment. In the graft a rich network of somatostatin-positive terminals was found with a high density in well-demarcated areas reminiscent of substantia gelatinosa of the dorsal horn of normal spinal cord. A large number of small- to medium-sized somatostatin neurons was found throughout the grafts without colchicine treatment. This is in contrast to normal spinal cord, where positive neurons were difficult to visualize without colchicine and were mainly confined to the dorsal horn. Neuropeptide Y had a distribution in the grafts similar to that of somatostatin and neuropeptide Y cells were found throughout the grafts without colchicine treatment. In normal spinal cord, neuropeptide Y-positive fibers were found mainly in substantia gelatinosa with a sparse network in the ventral horn. Enkephalin-positive fibers were found throughout the grafts. The distribution of fibers resembled that of somatostatin and neuropeptide Y with distinct zones of high fiber density in well-demarcated areas, whereas the density of nerve fibers in the rest of the graft neuropil was moderate to low. The distribution of substance P was similar to that of enkephalin. After colchicine treatment, both enkephalin- and substance P-positive cell bodies were visualized. In the intact spinal cord both peptides were seen in the entire gray matter with the highest concentrations in the superficial laminae of the dorsal horn. Antisera against calcitonin gene related-peptide, revealed a sparse terminal network and many large cells, which might represent motoneurons. A sparse network of varicose cholecystokinin-immunoreactive fibers was found evenly distributed in the grafts. In normal spinal cord a dense cholecystokinin-positive network of primary sensory afferent origin was found in the dorsal horn. In the grafts cholecystokinin cell bodies were seen after colchicine treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Immunocytochemical evidence for calcitonin gene-related peptide-like neurons in the dorsal horn and lateral spinal nucleus of the rat cervical spinal cord

Calcitonin gene-related peptide (CGRP) in the dorsal horn of the rat spinal cord was assumed until now to be principally of primary afferent origin. It is shown here, on the basis of both light and electron microscopic immunocytochemical evidence, that some cell bodies of the dorsal horn and lateral spinal nucleus (LSn) of the rat cervical spinal cord contain a CGRP-like immunoreactivity. At the light microscopic level, immunoreactive cell bodies were observed in animals pretreated with colchicine injected intraventricularly, CGRP-like cell bodies were morphologically heterogeneous and distributed in the three superficial layers of the dorsal horn. They were very rare in lamina I and more numerous in laminae II and III. A group of immunoreactive cell bodies was also observed in the LSn. Using electron microscopic techniques, a few immunoreactive cell bodies were observed even in control animals. In addition, relatively numerous immunoreactive dendrites were observed in lamina II. The specificity of the reaction and the physiological implications of the results are discussed.     

Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin

The temporal course of changes in peptide expression in the dorsal root ganglia L4 and L5 and in the dorsal horn of the spinal cord has been studied in rats subjected to a sciatic nerve transection at a mid-thigh level following different survival times. Galanin-, substance P-, vasoactive intestinal polypeptide-, peptide histidine-isoleucine- and calcitonin gene-related peptide-like immunoreactivities have been studied both by immunohistochemistry and radioimmunoassay. Galanin messenger ribonucleic acid has also been studied by in situ hybridization in the dorsal root ganglia of normal and lesioned animals. In addition, a group of animals with a sciatic nerve crush was studied to compare possible differences in peptide expression after both types of lesions. The results show that the transection induces an increase in the number of cell bodies expressing galanin-like immunoreactivity in the ganglia, and that the galanin levels rise about 120-fold after three and 14 days of survival. This increase reflected increased synthesis of the peptide, since there was a rise in the galanin messenger ribonucleic acid already at 24 h post-lesion, which was maintained for at least 60 days. In the spinal cord there was an increase of staining in the midportion of the outer layers of the dorsal horn that corresponded to fibers thought to arise from cells of the dorsal root ganglia affected by the transection. Also a depletion of substance P-like and an increase in vasoactive intestinal polypeptide- and peptide histidine-isoleucine-like immunoreactivities in the dorsal root ganglia were confirmed. These changes were shown to be rapidly detectable and were paralleled by similar changes in the dorsal horn of the spinal cord. For calcitonin gene-related peptide the immunohistochemistry was inconclusive, and the radioimmunoassay showed no detectable changes. After nerve crush a transient increase in the number of galanin immunoreactive neurons was observed, as well as a decrease in the number of neurons showing substance P-like immunoreactivity. These changes were most noticeable between six and 14 days of survival. After this, peptide expression seemed to return slowly to normal, that is by day 45 post-crush only a few cells showed galanin-like, and many sensory neurons expressed substance P-like immunoreactivity. The results demonstrate that when primary sensory neurons are peripherally lesioned they respond in a complex manner, altering their normal production of peptides by increasing or decreasing their synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

大鼠脊神经后根切断后脊髓和背根神经节CGRP的表达变化

为观察大鼠脊神经后根切断后相应背根神经节(DRG)和脊髓节段CGRP的表达变化,本研究采用25只健康成年SD大鼠随机分为正常对照组、假手术对照组和L4、5后根切断后3d、7d和14d组(n=5),用免疫组织化学方法结合图像分析技术检测各组相应DRG和脊髓节段内CGRP的表达变化。结果如下:后根切断后3d、7d和14d伤侧DRG内CGRP表达较对照组和对侧明显增强;后根切断后3d脊髓后角CGRP免疫阳性纤维减少,7d、14d时进一步减少;后根切断后3d脊髓前角运动神经元内CGRP表达增加,免疫阳性细胞数增多,7d和14d时表达进一步增强。以上结果提示,脊神经后根切断后DRG和脊髓CGRP表达变化呈现一定的时空模式,可能参与了神经损伤后的再生过程。     

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.     

生长抑素样免疫反应物在山羊脊髓和背根节的分布─ABC法研究

用免疫细胞化学ＡＢＣ法研究了生长抑素样免疫反应物在山羊脊髓和背根节的分布。在整个脊髓背角，出现浓密的生长抑素样纤维和终末网，在板层Ⅱ和Ⅲ形成不规则的波形带。在带的深面，大量的生长抑素阳性小细胞（直径小于１５μｍ）形成细胞带，这些细胞呈卵圆形或梭形，大多是双极细胞。在背角最表层仅有稀疏的生长抑素阳性纤维。这种分布型式显著不同于以前报道的其它动物。在胸髓中间外侧核有为数不多的生长抑素阳性细胞，在脊髓的其它部位如中间带区和腹角有少量散在纤维和终末。还发现纤细的生长抑素阳性纤维和终末网出现于骶髓中央管的背外侧区域。此外，在所有背根节只见到极少数生长抑素阳性神经元；切断背根时，相应节段的脊髓背角的生长抑素样免疫反应物无明显变化。本研究结果表明，山羊脊髓背角的生长抑素主要来源于脊髓背角的固有神经元，而不是背根节细胞。出现于脊髓板层Ⅱ和Ⅲ的生长抑素阳性神经元可能属于岛细胞。本研究还提示，生长抑素可能在脊髓的感觉和内脏运动系统中起作用，还表明生长抑素免疫反应物的分布存在着种属差异。     

MK-801-induced sprouting by CGRP immunoreactive primary afferent fibers in the dorsal spinal cord of the rat

In the present study, rats received daily injections of the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, over 30 consecutive days. The effects of MK-801 on the distribution of calcitonin gene-related peptide (CGRP)-immunoreactive fibers in the dorsal spinal cord of the rat were subsequently examined. In addition to the normal immunostaining pattern in laminae I, II and lateral V, a dense network of CGRP-immunoreactive fibers was observed along the medial border of the dorsal horn and within the dorsal grey commissure. This marked increase in immunoreactivity was virtually eliminated following dorsal rhizotomy. These observations suggest that MK-801 induces intraspinal sprouting by CGRP immunoreactive primary afferent fibers in vivo.     

In situ hybridization studies on mRNAs for cholecystokinin, calcitonin gene-related peptide and choline acetyltransferase in the lower brain stem, spinal cord and dorsal root ganglia of rat and guinea pig with special reference to motoneurons

In situ hybridization techniques were used to analyse the distribution of cholecystokinin (CCK) mRNA in the lower brain stem, spinal cord and dorsal root ganglia of the rat and guinea pig, in comparison with that of mRNAs for calcitonin gene-related peptide (CGRP) and choline acetyltransferase. In the rat, CCK mRNA was found in numerous motoneurons in the spinal cord as well as in the motor trigeminal, facial and hypoglossal nuclei. Coexistence of CCK mRNA and CGRP mRNA could be established in spinal and brain stem motoneurons. Conversely, in the guinea pig CCK mRNA could only be detected in few motoneurons in the spinal cord. In both species, CCK mRNA was present in the spinal trigeminal nucleus and in the dorsal horn of the spinal cord, in numerous small cells located in the outer laminae (mainly II-IV), and in the rat was also found in large cells in laminae IV and V. Few small cells in laminae VI-VIII and X of the spinal cord and cells in several brain stem nuclei, such as the solitary tract, gracile and cuneate nuclei, also showed CCK mRNA in the rat. In the guinea pig brain stem CCK mRNA was found, among others, in the solitary tract nucleus, pontine reticular formation and pontine periventricular grey. In dorsal root ganglia CCK mRNA was abundant in the guinea pig, but almost absent in the rat, where only single cells were found that expressed low levels of this mRNA.     

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.     

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.     

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.     

An immunohistochemical study of neuronal populations containing neuropeptides or gamma-aminobutyrate within the superficial layers of the rat dorsal horn

Immunohistochemical techniques have been used to characterise a number of interneuronal types found within the superficial layers of the rat dorsal horn. Substance P, methionine-enkephalin, neurotensin, avian pancreatic polypeptide, somatostatin and glutamic acid decarboxylase-like immunoreactivities were found within discrete populations of cell bodies within layers I to III of the spinal cord after colchicine pretreatment. Each immunoreactive cell type was distinct in terms of the distribution of terminals, perikaryal size and position within the dorsal horn. Cell bodies containing substance P were predominantly within layers I, II and III, while surgical isolation of the dorsal horn to remove terminals of extrinsic origin indicated that the terminals of the local substance P-containing interneurones were present throughout layers I and II₀. Enkephalin-immunoreactive neurones were most numerous within layer II, with less frequently observed larger enkephalin immunoreactive neurones in layer I. Neurotensin-containing neurones were found in a fairly narrow layer along the II/III border, with dendritic arborisations extending into both layers II and III. Neurotensin-immunoreactive terminals occurred in two bands, a dense band within layer II, and a less dense one within layers I and II₀. Somatostatin-like immunoreactivity was found in occasional large neurones within layer II_i. Avian pancreatic polypeptide immunoreactivity was localized to neurones within layers I, II and III, as was terminal immunoreactivity. Glutamic acid decarboxylase-containing neurones were large and were found predominantly within layers I, II ₀ and on the II/III border, where terminal immunoreactivity was also concentrated. Hemisection combined with dorsal root section indicated that the majority of these immunohistochemically identifiable neurones were interneurones.The interconnections and relations of these neurones to dorsal horn projection neurones are considered in the light of the reported analgesic properties of a number of these substances when applied directly to the spinal cord.     

The effects of dorsal rhizotomy and spinal cord isolation on calcitonin gene-related peptide-labeled terminals in the rat lumbar dorsal horn

In the present study, the origin of calcitonin gene-related peptide (CGRP) to the dorsal horn in the rat lumbar spinal cord is investigated. CGRP immunoreactivity is examined following multiple unilateral and bilateral dorsal rhizotomies and isolated cord preparations (spinal cords are isolated by transecting the cord in two places and cutting all dorsal roots between the transections). Seven to 11 days after surgery, unilateral multiple dorsal rhizotomies result in a drastic decrease in CGRP-stained terminals on the operated side; following bilateral dorsal rhizotomies and isolated cord preparations, one or two CGRP varicosities remain in the dorsal horn in each section. The numbers of CGRP-immunostained varicosities observed in the latter two preparations are not significantly different, suggesting that few if any axons descending from the brain contribute to the CGRP terminal population in the spinal cord dorsal horn. Based on these data, we hypothesize that dorsal root ganglion cells are the only source of CGRP to the rat lumbar dorsal horn.     

Distribution of some peptides (substance P, [Leu]enkephalin, [Met]enkephalin) in the brain stem and spinal cord of a lizard, Varanus exanthematicus

The distribution of substance P-like and [Leu]- and [Met]enkephalin-immunoreactive cell bodies, fibers and terminal structures in the brain stem and spinal cord of a lizard, Varanus exanthematicus, was studied with the indirect immunofluorescence technique, using antibodies to these peptides. Substance P-like immunoreactive cell bodies were found in the hypothalamus, in a periventricular cell group in the rostral mesencephalon, in the interpeduncular nucleus, in and ventral to the descending nucleus of the trigeminal nerve, in and directly ventral to the nucleus of the solitary tract, scattered in the brain stem reticular formation and in the trigeminal and spinal ganglia. A rather widespread distribution of substance P-like immunoreactivity was found in the brain stem and spinal cord, mainly concentrated in striatotegmental projections related to visceral and/or taste information (nucleus of the solitary tract, parabrachial region), in the descending nucleus of the trigeminal nerve and in the dorsal horn of the spinal cord (areas I and II). In the spinal cord also around the central canal (area X and adjacent parts of area V-VI) a distinct substance P innervation was found. The ventral horn receives only a very sparse substance P innervation. The distribution of [Leu]- and [Met]enkephalin in the brain stem and spinal cord of Varanus exanthematicus is less impressive than that of substance P. Enkephalinergic cell bodies were found particularly in the caudal hypothalamus. Small populations of enkephalinergic cell bodies were found in the vestibular nuclear complex, in the nucleus of the solitary tract, in and around the descending nucleus of the trigeminal nerve and throughout the rhombencephalic reticular formation. Enkephalins are likely to be present in efferent projections of the striatum, in projections related to taste and/or visceral information (nucleus of the solitary tract, parabrachial region) and in descending pathways to the spinal cord. Enkephalinergic fibers are present in the lateral funiculus and enkephalin-immunoreactive cell bodies are found in the reticular formation, particularly the inferior reticular nucleus which is known to project to the spinal cord. In the spinal cord enkephalinergic terminal structures were found especially in the superficial layer of the dorsal horn (areas I and II) and around the central canal. The ventral horn including the motoneuron area receives only a relatively sparse enkephalinergic innervation.