A quantitative study of the coexistence of peptides in varicosities within the superficial laminae of the dorsal horn of the rat spinal cord

While several peptides have been shown to coexist in perikarya within dorsal root ganglia of rat, coexistence of peptides has not been confirmed in axons associated with these neurons. In this study, the coexistence of substance P (SP) with somatostatin (SOM), calcitonin gene-related peptide (CGRP), dynorphin A 1-8 (DYN), neurotensin (NT), galanin (GAL), and 5-HT in varicosities was visualized using fluorescence immunohistochemistry. Densities of immunoreactive varicosities within laminae I and II of the dorsal horn of the rat spinal cord were quantified by computer-assisted image analysis. Decreases in densities of immunoreactive varicosities as a result of multiple unilateral dorsal rhizotomies were used to determine proportions of immunoreactive varicosities associated with primary afferent neurons. Three observations were made. (1) Dorsal rhizotomy depleted greater than one-third of the varicosities individually immunoreactive for SP, SOM, GAL, or DYN, confirming the association of these peptides with primary afferent neurons. (2) SP coexisted with CGRP, GAL, and DYN in varicosities within the dorsal horn of normal animals. (3) CGRP-, SP+CGRP-, and SP+GAL-immunoreactive varicosities were nearly depleted following dorsal rhizotomy. The depletion of these peptides, particularly in combination, indicates that they may be used as markers for varicosities of some primary afferent neurons within the superficial laminae of the dorsal horn of the rat spinal cord.     

Adrenalectomy increases the number of substance P and somatostatin immunoreactive nerve cells in the rat lumbar dorsal root ganglia

Using an immunocytochemical technique we have analyzed changes in substance P, somatostatin, calcitonin gene-related peptide, and galanin immunoreactivity pattern in the rat dorsal root ganglia. After 7 days of adrenalectomy, sham operated rats were compared with adrenalectomized animals either receiving a daily intraperitoneal injection of 10 mg/kg b.wt. corticosteronev or vehicle. Three lumbar ganglia from each animal were blocked, serially cut, and immunostained for each neuropeptide by means of the biotin-avidin-peroxidase technique. A systematic sampling of immunoreactive ganglion cells was performed and the sample number of immunoreactive ganglion cells was calculated. After adrenalectomy, the number of substance P and somatostatin immunoreactive ganglion cells markedly increased ((means±S.E.M.): 245 ± 68versus123 ± 12 for sham operated animals, P < 0.01 (substance P) and 42 ± 8 as compared to 22 ± 9 for sham operated animals, P < 0.01 (somatostatin)). No significant changes were found in the number of calcitonin gene-related peptide and galanin immunoreactive cells after adrenalectomu. These results suggest that adrenal steroid hormones may reduce the synthesis of both substance P and somatostatin in the dorsal root ganglion cells. Daily treatment with a high dose of corticosterone, mimicking its serum levels after stress, failed to prevent the increase of peptide contents after adrenalectomy. These observations also indicate that a tonic action of corticosterone on mineralocorticoid receptors may be crucial for peptide regulation in the spinal ganglia. These results may be of relevance to adrenalectomy induced changes in sensory mechanisms, neurogenic inflammation and pain transmission and to a role of substance P and somatostatin in these processes.     

Trigeminal nuclear complex of the ferret: Anatomical and Immunohistochemical studies

In order to establish the ferret as an animal model for studies of trigeminal pain, we describe the cytoarchitecture and neurochemistry of the trigeminal nuclear complex in the ferret and compare them to those of the cat and rat. The complex was divided as previously described, but the ferret differed in the extent of the nuclear boundaries. The neuroantomical distribution of substance P-, calcitonin gene-related peptide-, galanin-, enkephalin-, serotonin-, somatostatin-, neuropeptide Y-, and neurotensin-immunoreactivity was determined throughout the rostrocaudal extent of the complex. In subnucleus caudalis, substance P-, calcitonin gene-related peptide-, enkephalin-, serotonin-, somatostatin-, neuropeptide Y-, and galanin-immunoreactivity was densest in laminae I and II. In subnucleus interpolaris, immunoreactivity for all the above neurochemicals was most dense along the lateral border and the ventral third of the caudal part of the subnucleus. Enkephalin-immunoreactive cell bodies were present in subnucleus caudalis and interpolaris. In subnucleus oralis, labelling for substance P, calcitonin gene-related peptide, galanin, enkephalin, and serotonin was most prominent in the dorsomedial part of the subnucleus. Somatostatin-immunoreactive cell bodies were distributed throughout the spinal nucleus. Labelling of serotonin, substance P, calcitonin gene-related peptide, galanin, enkephalin, and somatostatin was present in the main sensory nucleus. The motor nucleus contained fibers immunoreactive for substance P, enkephalin, serotonin and neuropeptide Y, and cell bodies immunoreactive for calcitonin gene-related peptide. The majority of neurotensin-immunoreactivity was found at the level of subnucleus caudalis, where it was densest in the trigeminal extension of the lateral cervical nucleus. The distribution of peptides in this species throughout the spinal nucleus is consistent with the notion that all the subnuclei may be involved in the processing of nociceptive inputs. © 1993 Wiley-Liss, Inc.     

Ontogeny of peptide- and amine-containing neurones in motor, sensory, and autonomic regions of rat and human spinal cord, dorsal root ganglia, and rat skin

The developmental patterns of neurofilament triplet proteins, peptide and amine immunoreactivities were compared in motor (ventral spinal cord), sensory (dorsal spinal cord, dorsal root ganglia, epidermis), and autonomic (intermediolateral cell columns, dermis) regions in the rat and human. In the rat, neurofilament triplet proteins first appeared in motoneurones (embryonic day 13). In the youngest human fetuses studied (6 weeks), immunoreactivity was present throughout the spinal cord. Peptides and amines occurred later. Calcitonin gene-related peptide, galanin, somatostatin, neuropeptide Y and its C-flanking peptide (CPON) were the first to appear localized to motoneurones (embryonic days 15-17 rat; fetal weeks 6-14 human). Numbers of immunoreactive motoneurones decreased toward birth, but immunoreactive fibers increased in the ventral horn with enkephalin, thyrotrophin-releasing hormone, and the monoaminergic markers 5-hydroxytryptamine and tyrosine hydroxylase (all presumably of supraspinal origin) the last to appear perinatally. In the dorsal horn, particularly in the rat, a transient expression of substance P-, somatostatin-, and neuropeptide Y/CPON-immunoreactive cells was detected (embryonic days 15-17). A pronounced increase of calcitonin gene-related peptide-, galanin-, somatostatin- and substance P- immunoreactive fibers was found perinatally in both species. This coincided with an increased detection of cells in the dorsal root ganglia containing these peptides and the earliest appearance of calcitonin gene-related peptide-, somatostatin-, and substance P-immunoreactive fibers in the rat epidermis. Few antigens were localized to the intermediolateral cell columns before embryonic day 20 (rat), fetal week 20 (human), with thyrotrophin-releasing hormone-, 5-hydroxytryptamine-, tyrosine hydroxylase-, and vasoactive intestinal polypeptide-immunoreactive nerves appearing perinatally. In the rat dermis, tyrosine hydroxylase-immunoreactive fibers (sympathetic fibers) and fibers immunoreactive for neuropeptide Y/CPON and vasoactive intestinal polypeptide were detected from postnatal day 1. In conclusion, 1) peptide and amine immunoreactivity develops in motor before sensory or autonomic regions, 2) many peptide-containing cells are transient in fetal life, and 3) central terminals of dorsal root ganglion cells express peptides before terminals in the skin.     

Baclofen and the Release of Neuropeptides in the Cat Spinal Cord

The antibody microprobe technique was used to study the effect of baclofen on the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of pentobarbitone-anaesthetized spinalized cats. Both peptides were released in the region of the substantia gelatinosa during ipsilateral noxious cutaneous stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of baclofen suppressed the excitation of lumbar dorsal horn neurons, but did not produce detectable alterations of the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial grey matter dorsal to these neurons. The results suggest that the antinociceptive action of baclofen does not involve a reduction of the intraspinal release of substance P or calcitonin gene-related peptide from the central terminals of nociceptive sensory fibres.     

Neuropeptides in dissociated cell cultures of mammalian spinal cord and dorsal root ganglion

Immunohistochemical studies of leucine-enkephalin, somatostatin, vasoactive intestinal polypeptide and neurotensin were carried out in dissociated cell co-cultures of embryonic mouse spinal cord and dorsal root ganglion, using the peroxidase-antiperoxidase technique. Leucine-enkephalin immunoreactivity exceeded that of the other peptides in these coculture preparations. Leucine-enkephalin, substance P and somatostatin were also studied in spinal cord cultures (without dorsal root ganglia) and in dorsal root ganglia cultures (without spinal cord). Each of these peptides was present in only a small percentage (<10%) of perikarya and processes in spinal cord cultures. No leucine-enkephalin immunoreactivity was seen in dorsal root ganglion cultures; a considerable proportion of the processes were immunoreactive for substance P or somatostatin. These observations suggest that co-cultures of spinal cord and dorsal root ganglia can provide a simplified in vitro “model” of the nervous system for the study of peptidergic interactions.     

Further studies on galanin-, substance P-, and CGRP-like immunoreactivities in primary sensory neurons and spinal cord: effects of dorsal rhizotomies and sciatic nerve lesions.

The peptides galanin (GAL), substance P (SP), and calcitonin gene-related peptide (CGRP) were analyzed with immunohistochemistry and radioimmunoassay in the spinal cord, dorsal root ganglia, dorsal roots, and sciatic nerve of normal rats and rats subjected to several experimental procedures, including ligation, crush, and/or sectioning of nerves. The results show that peripheral nerve transection induces a dramatic increase in GAL content both in dorsal roots and sciatic nerve, demonstrating that this lesion causes an increased out-transport of the newly synthesized peptide both into the central and peripheral branches of the primary sensory neurons. In contrast evidence was obtained for decreased out-transport of SP and CGRP. The functional significance of these findings remains to be analyzed.     

Immunohistochemical Characteristics and Distribution of Sensory Dorsal Root Ganglia Neurons Supplying the Urinary Bladder in the Male Pig

The study determined the distribution and immunohistochemical coding of the sensory neurons innervating the male pig urinary bladder. Retrograde tracer Fast Blue was injected bilaterally into the bladder trigone, base or dome. The presence of neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP) and substance P (SP) were studied with immunofluorescence. Fast Blue-positive neurons were localized bilaterally in dorsal root ganglia from L1 to L6 and from S3 to S4 with specific differences regarding the injection site. The number of Fast Blue-positive neurons was higher in the right ganglia. Immunohistochemistry revealed that sensory neurons innervating the urinary bladder trigone, base and dome displayed immunoreactivities to CGRP, SP, NOS, GAL and SOM. Differences in the neuropeptide content were observed between the Fast Blue-positive neurons in lumbar and sacral ganglia. Taken together, these data indicate that the lumbar and sacral pathways probably play different roles in sensory transmission from the urinary bladder trigone, base and dome.     

Extrinsic Primary Afferent Neurons Projecting to the Pylorus in the Domestic Pig—Localization and Neurochemical Characteristics

The pig, as an omnivorous animal, seems to be especially valuable species in “gastrointestinal” experiments. The importance of the pylorus in the proper functioning of the digestive tract is widely accepted. Although it is commonly known that sensory innervation plays an important role in the regulation of gastric activity and gastrointestinal tissue resistance, there is complete lack of data on the extrinsic afferents projecting to the swine pylorus. The present experiment has been designed to discover the precise localization and neurochemical properties of the primary sensory neurons projecting to the porcine pylorus. Combined retrograde tracing technique and double immunocytochemistry were applied in five piglets. An additional RT-PCR reaction was used to confirm the presence of all investigated neurotransmitters in the studied ganglia. Traced neurons were localized within the bilateral nodose ganglia of the vagus and bilateral dorsal root ganglia spreading from Th4 to L1. Fast Blue-positive afferents expressed immunoreactivity to substance P, calcitonin gene-related peptide, neuronal isoform of nitric oxide synthase, and galanin. In the vagal and spinal ganglia, the percentages of traced neurons immunoreactive to these substances were 54.8, 10.7, 49.6, 7.4 % and 22.2, 75.5, 95.2 %, respectively, and the solitary perikarya were Gal immunoreactive. The presence of all substances studied in the ganglion tissue was confirmed by RT-PCR technique.     

Immunocytochemical localization of peptides and other neurochemicals in the rat laterodorsal tegmental nucleus and adjacent area

The laterodorsal tegmental nucleus (ntdl) contains a cluster of cells located just medial to the locus coeruleus in the pontine brainstem. The ntdl has been shown to project both rostrally to the forebrain and diencephalon and caudally to the spinal cord. In an effort to characterize this region neurochemically, the present study was conducted to identify a variety of neurochemicals localized within perikarya and fibers of the ntdl and surrounding nuclei. Rats were perfused with formalin, and brain sections were processed for fluorescence immunocytochemistry and acetylcholinesterase (AChE). Of the neurochemicals screened, atrial natriuretic factor (ANF), choline acetyltransferase (ChAT), cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), dynorphin B (Dyn B), galanin, somatostatin, substance P, neurotensin (NT), neuropeptide Y (NPY), vasopressin, vasoactive intestinal polypeptide (VIP), serotonin (5HT), glutamic acid decarboxylase (GAD), and tyrosine hydroxylase (TH) were studied. AChE and ChAT staining revealed that the ntdl contains mostly cholinergic neurons. In addition, brightly reactive substance P and galanin and paler staining CRF, ANF, CGRP, NT, VIP, and Dyn B cell bodies were found within the ntdl. Varicose fibers in this nucleus also contained these peptides in addition to CCK, GAD, TH, 5HT, and NPY. The dorsal tegmental nucleus, dorsal raphe nucleus, locus coeruleus, and the parabrachial region contained a dense and varied assortment of peptides with distinct positions and patterns. This multiplicity of neurochemicals within this area suggests a possible influence on a variety of functions modulated by the ntdl and other closely associated tegmental nuclei.     

Expression of neuropeptides in experimental diabetes; effects of treatment with nerve growth factor or brain-derived neurotrophic factor

Rats with streptozotocin-induced diabetes of 4 to 6 weeks duration showed a depletion of both substance P (P < 0.01) and calcitonin gene-related peptide (P < 0.01) in the sciatic nerve. Since expression of both peptides is sensitive to nerve growth factor (NGF) in vitro we examined the effect of treatment of diabetic rats with NGF, which significantly increased the levels of both peptides in treated diabetic animals (P < 0.01 for both). Treatment of non-diabetic rats with a similar NGF regime raised the mean peptide levels to a value similar to that seen in treated diabetic rats but the change was not statistically significant. In vehicle-treated diabetic rats the depletions of sciatic nerve neuropeptides were accompanied by a significant (P < 0.05) reduction in the level of CGRP mRNA in the 4th and 5th lumbar dorsal root ganglia, this was accompanied by an analogous reduction in the mRNA for γ-preprotachykinin A (γ-PPT), which did not attain statistical significance. Treatment of diabetic rats with NGF also prevented the deficits in the levels of CGRP and γ-PPT mRNA in the lumbar dorsal root ganglia (P < 0.05). Treatment of other diabetic rats with the related neurotrophin, brain-derived neurotrophic factor (BDNF), had no effect on the levels of substance P and calcitonin gene-related peptide in the sciatic nerve.     

Acute and Chronic Phases of Unilateral Inflammation in Rat's Ankle are Associated with an Increase in the Proportion of Calcitonin Gene-related Peptide-immunoreactive Dorsal Root Ganglion Cells

Using immunocytochemical methods, the proportion of calcitonin gene-related peptide immunoreactive perikarya was determined in dorsal root ganglia L4–L6 in four control rats and in ten rats with a unilateral inflammation in the ankle region of the left hindlimb. The inflammation was induced by subdermal injection of Freund's complete adjuvant at the ankle. Swelling and cellular infiltration of the ankle region developed within 2 days, and were stable and restricted to the injected ankle for the duration of the 3-week study. In control rats ∼24% of 20 419 perikarya showed calcitonin gene-related peptide (CGRP)-like immunoreactivity. In rats with unilateral inflammation the proportion of CGRP-positive neurons was increased on the inflamed side to ∼32% of 11 454 cells at day 2 ( P < 0.001 with respect to ganglia in normal rats) and ∼29% of 10 739 perikarya at day 20 post inoculation ( P < 0.01). By contrast, no significant changes were found between ganglia in the non-injected side (∼25% at day 2 and ∼24% at day 20). These results demonstrate that peripheral inflammation is associated with an increase in the proportion of neurons in the dorsal root ganglia that synthetize CGRP. This up-regulation is already present at an early stage of inflammation but also at later stages, suggesting that the increased synthesis of CGRP is an important neurobiological reaction associated with the acute and chronic phases of inflammation.     

Immunocytochemical localization of somatostatin receptor sst2A in the rat spinal cord and dorsal root ganglia

Intrathecal administration of octreotide, a stable somatostatin analogue, provides pain relief in patients, and locally applied somatostatin inhibits firing of nociceptive dorsal horn neurons. In the present study, we have raised polyclonal antibodies that specifically detect the somatostatin receptor sst_2A and used these antisera for immunocytochemical localization of the receptor protein in the rat spinal cord and dorsal root ganglia. In the superficial layers of the dorsal horn, sst_2A-like immunoreactivity (Li) formed a dense network consisting of neuronal perikarya and dendrites which were often closely apposed by, but not co-contained within, somatostatin-14-immunoreactive nerve fibres and terminals. sst_2A-Li was resistant to dorsal rhizotomy and did not colocalize with either substance P or calcitonin gene-related peptide suggesting that sst_2A-Li was not located to primary afferents, but rather confined to second-order spinal neurons. The position of sst_2A-Li perikarya and dendrites in the dorsal horn appeared to be similar to those containing μ-opioid receptor-Li; however, double labelling experiments revealed no instances of coexistence of these two receptors. sst_2A-Li was also observed in the dorsal root ganglia predominantly targeted to the somatic plasmalemma of medium size neurons distinct from those expressing somatostatin-14 or δ-opioid receptors. Thus, the present results not only provide a morphological substrate for spinal octreotide analgesia but also show that somatostatin and opioids are poised to modulate nociceptive transmission by distinct anatomical systems.     

Organization of the spinal cord in four species of elasmobranch fish: cytoarchitecture and distribution of serotonin and selected neuropeptides

An analysis of Nissl stained sections of the spinal cord taken from four species of elasmobranch showed that seven distinct cytoarchitectonic laminae are present. These laminae are compared with laminae described previously in the spinal cord of other vertebrates. The distribution of immunoreactivity to serotonin, substance P, somatostatin, calcitonin gene-related peptide, neuropeptide Y, and bombesin was determined in the brown stringray (Dasyatis fluviorum), the eagle ray (Aetobatis narinari), the shovelnose ray (Rhinobatis battilum), and the black-tip shark (Carcharhinus melanopterus). In all species, dense immunoreactivity to most substances tested was found in the outer part of the substantia gelatinosa. Many fibres and varicosities immunoreactive to substance P, calcitonin gene-related peptide, and bombesin were found in this region and smaller numbers of fibres were found in the nucleus proprius. Immunoreactivity to somatostatin consisted of coarse fibre bundles that entered the dorsal horn at the nucleus proprius and radiated dorsally to the substantia gelatinosa. Axons and varicosities immunoreactive to serotonin and neuropeptide Y were found in all regions of the dorsal horn but were concentrated in the outer part of the substantia gelatinosa. The distribution of immunoreactivity to met-enkephalin in the shovelnose ray was concentrated in the lateral third of the substantia gelatinosa and to a lesser extent in the nucleus proprius. The distribution of these substances is compared with that described in other vertebrates. Although the sensory information reaching the elasmobranch spinal cord is limited, compared with that of mammalian species, the distribution of these neuroactive factors in the dorsal horn of the two groups is strikingly similar.     

Calretinin-immunoreactivity in vagal and glossopharyngeal sensory neurons of the rat: distribution and coexistence with putative transmitter agents.

Immunoreactivity for the calcium binding protein, calretinin (calretinin-ir), was demonstrated in cell bodies of vagal and glossopharyngeal sensory ganglia (jugular, petrosal, and nodose ganglia) and in associated nerve fibers. In the jugular and petrosal ganglia, many calretinin-ir neurons were also immunoreactive for calcitonin gene-related peptide and substance P. In the nodose ganglion, most of the calretinin-ir neurons lacked these peptides. None of the calretinin-ir neurons in these ganglia were also immunoreactive for tyrosine hydroxylase.     

Increased calcitonin gene-related peptide (CGRP), substance P,and enkephalin immunoreactivities in dorsal spinal cord and loss of CGRP-immunoreactive motoneurons in arthritic rats depend on intact peripheral nerve supply

The distribution of peptides thought to be involved in pain modulation—substance P, calcitonin gene-related peptide (CGRP), and enkephalin—were studied in the spinal cord and dorsal root ganglia of polyarthritic rats and in rats with one sciatic nerve sectioned prior to induction of arthritis. In arthritic rats there was a bilateral increase of CGRP- and substance P-immunoreactive fibers and appearance of enkephalin-immunoreactive cell bodies in the dorsal horn of the lumbar (L₄) spinal cord when compared to controls. In the corresponding dorsal root ganglia there were significant increases of CGRP- (P<0.02) and substance P- (P<0.001) immunoreactive cell bodies compared to controls. In the ventral horn of the control rats CGRP-immunoreactive motoneurons were abundant but were significantly (P<0.001) reduced in the arthritic spinal cord. Less pronounced changes were seen in the contralateral L₄ spinal cord of arthritic rats with unilateral sciatic nerve section. In the ipsilateral dorsal horn, however, CGRP- and substance P-immunoreactive fibers were markedly depleted, and no enkephalin cell bodies were present. Furthermore, a number of CGRP-immunoreactive motoneurons were observed. In the ipsilateral L₄ ganglia CGRP- (P<0.02) and substance P- (P<0.02) immunoreactive cells were significantly decreased compared to the contralateral side. The data suggest that pain perception is linked to complex interactions between CGRP, substance P, and enkephalin in sensory pathways and an intact peripheral input. The loss of CGRP-immunoreactive motoneurons may reflect muscular dysfunction associated with the arthritic condition.     

Distribution of substance P and calcitonin gene-related peptide immunoreactive nerve fibers in the trachea of chronically hypoxic rats

The distribution of substance P and calcitonin gene-related peptide immunoreactive nerve fibers in the trachea was compared between normoxic and chronically hypoxic rats (at 380 mm Hg for 10 weeks). In the normoxic trachea, the immunoreactivity to either peptide was seen in the nerve fibers in four principal locations: a) within and b) under the ciliated epithelium, c) within the smooth muscle bundles in the posterior wall, and d) in the connective tissue and around blood vessels in the lamina propria and submucosa. These immunoreactive fibers within the epithelium and smooth muscle bundles, in the connective tissue, and around blood vessels were thin and displayed some varicosities, and those under the epithelium appeared as thick nerve bundles. When the distribution and density of immunoreactive fibers were compared between normoxic and chronically hypoxic tracheas, there was a difference in number of substance P and calcitonin gene-related peptide immunoreactive fibers penetrating into the epithelium, although there was no difference in the other three locations. The mean number of substance P and calcitonin gene-related peptide immunoreactive intraepithelial fibers per section of the chronically hypoxic trachea was significantly increased. Because substance P and calcitonin gene-related peptide are predominant signal peptides of primary sensory neurons, the increase of substance P and calcitonin gene-related peptide immunoreactive fibers suggests that altered airway reflexes may be a feature of hypoxic adaptation.     

Substance P and calcitonin gene-related peptide expression in dorsal root ganglia in sciatic nerve injury rats

The neuropeptides, substance P and calcitonin gene-related peptide, have been shown to be involved in pain transmission and repair of sciatic nerve injury. A model of sciatic nerve defect was prepared by dissecting the sciatic nerve at the middle, left femur in female Sprague Dawley rats. The two ends of the nerve were encased in a silica gel tube. L5 dorsal root ganglia were harvested 7, 14 and 28 days post sciatic nerve injury for immunohistochemical staining. Results showed that substance P and cal- citonin gene-related peptide expression increased significantly in dorsal root ganglion of rats with sci- atic nerve injury. This increase peaked at 7 days, declined at 14 days, and reduced to normal levels by 28 days post injury. The findings indicate that the neuropeptides, substance P and calcitonin gene- related peptide, mainly increased in the early stages after sciatic nerve injury.     

Comparison of SLN-evoked swallows during rest and chewing in the freely behaving rabbit

Calcitonin-gene related peptide is among a group of peptides whose expressions are down-regulated following peripheral nerve damage. It is known that this is probably due to deprivation of some target-derived neurotrophic factors, mainly of nerve growth factor though positive effect of other factors, for example that of leukemia inhibitory factor on galanin has also been demonstrated. In this study, the effects of leukemia inhibitory factor and nerve growth factor on calcitonin gene related peptide expression in cultured dorsal root ganglion explants and in their outgrowing axons were examined. Lumbar dorsal root ganglia with short pieces of peripheral nerves were removed from adult mice and explanted into collagen gels. They were covered with RPMI 1640 culture medium and left in an incubator for 2 days after which they were fixed. These whole mount preparations with outgrowing axons were stained with an antibody against calcitonin gene related peptide. Following microscopic examination and imaging, sections were cut from the cultured ganglia as well as from some freshly taken normal ones and they were also stained to determine calcitonin gene related peptide immunoreactivity in the primary sensory neurons. The results demonstrated that besides the positive effect of nerve growth factor on the expression of this peptide in outgrowing axons, leukemia inhibitory factor also supported the expression of calcitonin gene related peptide in the primary sensory neurons of adult mouse lumbar dorsal root ganglia and in their outgrowing axons in vitro. When the time course of changes in calcitonin gene related peptide expression in dorsal root ganglia and the up-regulation of leukemia inhibitory factor at the site of a peripheral nerve injury in vivo are considered together, this novel finding may lead to new explanations for the changes in neuropeptide expression following axotomy.     

Transmitter diversity in ganglion cells of the guinea pig gallbladder: an immunohistochemical study.

Several neurotransmitters have been reported to exist in the ganglionated plexus of the guinea pig gallbladder. These include substance P, neuropeptide Y (NPY), calcitonin gene-related peptide, vasoactive intestinal peptide (VIP), acetylcholine, norepinephrine, serotonin, and dopamine. To determine which neuropeptides are intrinsic to gallbladder ganglia, we performed immunohistochemistry on colchicine-treated preparations. In separate, single-labeled preparations, a majority of neurons contained substance P-, NPY-, or somatostatin-like immunoreactivity. In double-labeled preparations, a large majority of the neurons that contained substance P-like immunoreactivity also contained NPY-like immunoreactivity and somatostatin-like immunoreactivity. Immunoreactivity for VIP was present in a small percentage of the gallbladder neurons which did not contain substance P-like immunoreactivity. Additional experiments were done to test for the presence of other compounds, known to exist in the neurons of the gut. Although immunoreactivity was found in control preparations of small intestine, the ganglionated plexus of the gallbladder lacked immunoreactivity for galanin, dynorphin, enkephalin, gastrin-releasing peptide, or gamma-aminobutyric acid. We conclude that ganglia of the guinea pig gallbladder contain at least two populations of neurons, based on transmitter phenotype. One of these populations appears to contain substance P, NPY, and somatostatin. Another population, which represents a small contingent of the total population of neurons, contains VIP.