CGRP-like Immunoreactive Fibers Research Articles

Enteric neuropeptides in streptozotocin-diabetic rats; effects of insulin and aldose reductase inhibition

The aim of the present study was to determine whether diabetes-induced changes in the distribution of enteric neuropeptides, could be prevented in 12-week streptozotocin-diabetic rats, by rigorous control of glycaemia, using daily adminstration of insulin, or an aldose reductase inhibitor (ponalrestat). The pattern of distribution of nerve fibres and cell bodies, containing immunoreactive vasoactive intestinal polypeptide (VIP), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P was examined in the myenteric plexus of ileum from control, untreated diabetic, insulin-treated diabetic and aldose reductase inhibitor-treated diabetic rats. The increase in VIP- and GAL-like immunoreactivity, seen in the myenteric plexus of untreated diabetic rat ileum, was not present in the myenteric plexus of ileum from insulin- and aldose reductase inhibitor-treated diabetic rats. With CGRP-like immunoreactive fibres, there was a clear decrease in the ileum of untreated diabetic rats. This was prevented by insulin treatment, but aldose reductase inhibitor treatment had no effect. No alterations in substance P-like immunoreactivity were seen in the myenteric plexus of ileum from any of the groups investigated. Generally, the similarity of effect of ponalrestat and insulin on VIP and galanin expression in this study supports a primary effect of insulin via glycaemic control. The dissimilarity of the effect of the two treatments on CGRP expression may imply a neurotrophic effect of insulin, although there are certainly consequences of hyperglycaemia other than exaggerated flux through the polyol pathway.

Distribution of calcitonin gene-related peptide-like immunoreactivity in the brain of the small-spotted dogfish, Scyliorhinus canicula L.

The distribution of neuropeptides has been useful in comparing neuronal aggregates of elasmobranchs with those in other vertebrates. The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the brain of the dogfish was examined with an antiserum to rat alpha-CGRP. Western blot analysis confirms that our antiserum recognizes a single peptide in the dogfish brain very similar to mammalian CGRP. CGRP-like immunoreactivity was located in discrete neuronal groups. CGRP-like-immunoreactive (CGRP-ir) neurons were found in the motor nuclei III, IV, V, VI, VII, IX, and X of the brainstem motor column and in the octavolateral efferent neurons. In the isthmal region, two groups of CGRP-ir neurons appeared in the parabrachial region and reticular substance. Three other CGRP-ir cell groups were observed in the mesencephalon: in the ventral tegmental area, in the substantia nigra, and one widely scattered but numerous population in superficial layers of the optic tectum. In the diencephalon, CGRP-ir cells were observed in the magnocellular preoptic nucleus and the organon vasculosum hypothalami. A population of CGRP-ir cells was also observed in the entopeduncular nucleus in the impar telencephalon. CGRP-ir fibers of central origin were widely distributed in the brain, but the most conspicuous areas were found in the ventral telencephalon, the hypothalamus, the mesencephalic lateral reticular area, and the dorsolateral isthmal region. The neurointermediate lobe of the hypophysis was also richly innervated by CGRP-ir fibers. CGRP-ir sensory fibers of cranial nerves IX and X and of dorsal spinal roots formed very conspicuous terminal fields in the lobus vagi and Cajal's nucleus commissuralis and in the dorsal region of the substantia gelatinosa, respectively. Comparison of the distribution of fibers and perikarya in dogfish and other vertebrates suggests that this CGRP-ir system has been well conserved during evolution.

Calcitonin gene-related peptide-like immunoreactive (CGRPI) elements in the circadian system of the mouse: an immunohistochemistry combined with retrograde transport study

This study was based on immunohistochemical detection of calcitonin gene-related peptide-like immunoreactive (CGRPI) neurons and fibers in the suprachiasmatic nucleus (SCN) and intergeniculate leaflet (IGL) of the mouse. CGRPI neurons and fibers were found within the ventrolateral part of the SCN, in the whole extent of IGL and sparsely distributed in ventral geniculate body. The presence of CGRPI structures in the SCN and IGL of the mouse further supports the hypothesis of differences in the content of neuroactive substances in the circadian clock between mammalian species. Fluorogold retrograde transport combined with CGRP immunofluorescence demonstrates that CGRPI neurons in the IGL constitute a part of IGL reciprocal connections.

The innervation of calcitonin gene-related peptide to the Purkinje cells and granule cells in the developing mouse cerebellum

The present study analyzed the ontogeny of calcitonin gene-related peptide-like immunoreactive (CGRP-IR) structures in the mouse cerebellum. No CGRP-IR neurons were detected at any stage, but three types of CGRP-IR fibers were seen: (1) CGRP-IR dense fiber plexuses which appeared transiently in the developing cerebellum, (2) thin varicose fibers, and (3) mossy fiber-like fibers. The CGRP-IR dense fiber plexuses appeared in the developing Purkinje cell layer at postnatal day 2. From postnatal days 6 to 11, these fibers formed pericellular nests around Purkinje cells. After that stage, these fibers rapidly disappeared and no such plexuses were seen in the adult cerebellum. CGRP-IR fiber plexuses were not evenly distributed, and they had a parasagittal banded pattern in the frontal sections. These plexuses existed in the region of all vermis, crus 1 of the ansiform lobe, simplex lobule, and flocculus, while the other lobules were devoid of such fibers. Under electron microscopy, these CGRP-IR fibers were seen to make synaptic contacts with somatic spines of Purkinje cells, suggesting that CGRP-IR plexuses were closely related to the developing Purkinje cells. Mossy fiber-like CGRP-IR fibers appeared in the granular layer on postnatal day 2, and increased in number to reach a peak on postnatal day 12. Thereafter, they decreased slightly to reach a plateau on postnatal day 30. Under electron microscopy these CGRP-IR fibers were revealed to be the mossy fibers which regulate the granule cells. Thin varicose CGRP-IR fibers were rarely seen at birth, but on postnatal day 8, many fibers appeared in all layers and increased by postnatal day 30. They distributed equally throughout the cerebellar cortex with a slight predominance in density in the molecular and Purkinje cell layer. Immunoelectron microscopic analysis showed that these fibers made synaptic contacts with small dendrites in the molecular layer.

Calcitonin gene-related peptide in the efferent system of the inner ear. A review.

Many calcitonin gene-related peptide-like immunoreactive (CGRP-IR) fibers were found in the vestibular end-organs and the cochlea of rats. CGRP-IR fibers in the vestibular end-organs originated in the bilateral cell groups dorsolateral to the genu of the facial nerve. These fibers formed a fiber plexus at the base of the sensory epithelia in the maculae of the otolith organs and the ampullae of the semicircular canals, and formed synaptic contacts with the nerve chalice on type I vestibular sensory cells. CGRP-IR fibers in the cochlea originated in the ipsilateral lateral superior olivary nucleus. Most of them existed in the inner spiral bundle under the inner hair cells, and formed synaptic contacts with afferent terminals on the inner hair cells. These findings suggest a postsynaptic modulation of CGRP on the vestibular and cochlear information at the level of type I vestibular sensory cells and the inner hair cells.

Effects of topical capsaicin on autonomic nerves in experimentally-induced nasal hypersensitivity. An immunocytochemical study.

Effects of capsaicin on autonomic nerves in the nasal mucosa and olfactory bulb of toluene diisocyanate sensitized guinea pigs were studied using immunocytochemistry. In the nasal mucosa, substance P (SP)- and tyrosine hydroxylase (TH)-like immunoreactive (SPI and THI) fibers seemed to decrease after capsaicin application. Calcitonin gene-related peptide (CGRP)-like immunoreactive (CGRPI) fibers did not show obvious alterations. In the olfactory bulb, SPI and CGRPI fibers were few and the effects of capsaicin on those fibers were difficult to evaluate. THI fibers seemed not to be affected by capsaicin. It is suggested that capsaicin affects not only sensory nerves but that it also impacts on THI sympathetic nerves in the nasal mucosa.

The distribution and colocalization of neuropeptides in perivascular nerves innervating the large arteries and veins of the snake, Elaphe obsoleta.

Single- and dual-labelling immunohistochemistry were used to determine the distribution and coexistence of neuropeptides in perivascular nerves of the large arteries and veins of the snake, Elaphe obsoleta, using antibodies for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, neuropeptide Y, galanin, somatostatin, and leu-enkephalin. Blood vessels were sampled from four regions along the body of the snake: region 1, arteries and veins anterior to the heart; region 2, central vasculature 5 cm anterior and 10 cm posterior to the heart; region 3, arteries and veins in a 30-cm region posterior to the liver; and region 4, dorsal aorta and renal arteries, renal and intestinal veins, 5-30 cm cephalad of the vent. A moderate to dense distribution of vasoactive intestinal polypeptide-like immunoreactive fibres was found in most arteries and veins of regions 1-3, but fibres were absent from the vessels of region 4. The majority of vasoactive intestinal polypeptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were unaffected by either capsaicin or 6-hydroxydopamine (6-OHDA) pretreatment. In the anterior section of the snake, the vagal trunks contained many cell bodies with colocalized vasoactive intestinal polypeptide and substance P-like immunoreactivity. It is suggested that the vasoactive intestinal polypeptide/substance P-like immunoreactive cell bodies and fibres are parasympathetic postganglionic nerves. Neuropeptide Y-like immunoreactive fibres were observed in all arteries and veins, being most dense in regions 3 and 4. The majority of these fibres also contained colocalized galanin-like immunoreactivity, and were absent in tissues from 6-OHDA pretreated snakes, suggesting that neuropeptide Y and galanin are colocalized in adrenergic nerves. A small number of neuropeptide Y-like immunoreactive fibres contained vasoactive intestinal polypeptide but not galanin, and were unaffected by 6-OHDA treatment. All calcitonin gene-related peptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were observed in all vessels, being particularly dense in the carotid artery and jugular veins. All calcitonin gene-related peptide/substance P-like immunoreactive fibres appeared damaged after capsaicin treatment suggesting they represent fibres from afferent sensory neurons. A sparse plexus of somatostatin-like immunoreactive fibres was observed in the vessels only from region 4. No enkephalin-like immunoreactive fibres were found in any blood vessels from any region. This study provides morphological evidence to suggest that there is considerable functional specialization within the components of the rat snake peripheral autonomic system controlling the circulation, in particular the regulation of venous capacitance.

Quantitative autoradiographic distribution of calcitonin gene-related peptide (hCGRP alpha) binding sites in the rat and monkey spinal cord.

Calcitonin gene-related peptide (CGRP) has been implicated in various spinal functions on the basis of its presence in the substantia gelatinosa and motoneurons and the biological effects induced by intrathecal CGRP injections. We investigated here the comparative distribution of [125I]hCGRP alpha binding sites in various segments of the rat and monkey spinal cord. The immunocytochemical localization of CGRP-like material in rat spinal cord was also evaluated for comparison. In the rat spinal cord, high densities of [125I]hCGRP alpha binding sites were observed in lamina I, in a U-shaped band that included lamina X and the medial parts of laminae III-IV and in the intermediolateral and intermediomedial nuclei. The substantia gelatinosa (lamina II) contained relatively lower, but still significant, densities of [125I]hCGRP alpha binding sites, while the ventral horn showed low amounts of specific labeling. CGRP-like immunoreactive fibers, on the other hand, were heavily concentrated in laminae I-II and in the reticulated portion of lamina V of the dorsal horn. Immunoreactivity to CGRP antiserum was also noted in fibers around the central canal and in a number of motoneurons of the ventral horn. In the monkey spinal cord, [125I]hCGRP alpha binding sites were present in lamina I in a U-shaped band that included lamina X and the medial portions of laminae V-VI. Relatively low levels of [125I]hCGRP alpha binding were detected in laminae II to IV of the dorsal horn, while the ventral horn was more enriched with specific [125I]hCGRP alpha binding sites. Thus, it appears that the autoradiographic distribution of [125I]hCGRP alpha sites is species dependent in the spinal cord. Additionally, some differences are observed between the localization of [125I]hCGRP alpha binding sites and immunoreactive material in the rat spinal cord. These differences may be relevant to the purported roles of CGRP-like peptides in spinal functions such as nociception, control of sympathetic output, and motor control.

Developmental alterations in nociceptive threshold, immunoreactive calcitonin gene‐related peptide and substance P, and fluoride‐resistant acid phosphatase in neonatally capsaicin‐treated rats

This study examined the effect of neonatal administration of capsaicin on nociceptive threshold and the distribution of calcitonin gene-related peptide (CGRP), substance P (SP), and fluoride-resistant acid phosphatase (FRAP) in the dorsal horn of the spinal cord during the course of development (10 days to 12 weeks of age) in the rat. As early as 10 days of age, CGRP-like immunoreactivity was reduced in laminae I, II, and V, as well as in the bundles of fibers situated dorsal and ventral to the central canal. However, beginning on or about 6 weeks of age, the density of CGRP-like immunoreactivity in the superficial laminae and in the bundles dorsal and ventral to the central canal increased. Moreover, thick, nonvaricose CGRP-like immunoreactive fibers appeared in laminae III and IV. These recurring fibers were of primary afferent origin as demonstrated by their disappearance after multiple, unilateral rhizotomies. A similar age-dependent alteration in the density of FRAP activity was also observed. Although virtually absent at 10 days of age after neonatal administration of capsaicin, the density of FRAP activity increased in lamina II by 8 weeks of age. This activity disappeared after multiple, unilateral rhizotomies, indicating that the FRAP activity that reappeared was of primary afferent origin. Neonatal administration of capsaicin also reduced the density of SP-like immunoreactivity in the dorsal horn as early as 10 days of age, although the density of SP-like immunoreactivity showed some recovery after 6 weeks of age. However, unlike CGRP-like immunoreactivity or FRAP activity, the density of SP-like immunoreactivity in capsaicin-treated rats was not detectably altered by multiple, unilateral rhizotomies, indicating that it originated principally from intrinsic dorsal horn neurons. Age-dependent alterations in both thermal and mechanical, but not chemical, nociceptive thresholds were also observed in these same animals. Thus, tail flick latency, hot plate latency, and paw withdrawal threshold were maximally increased at 6 weeks of age, after which time thresholds declined to vehicle-treated values. In contrast, capsaicin-treated animals were uniformly insensitive to ophthalmic administration of capsaicin. The correspondence between developmental alterations in CGRP-like immunoreactivity or FRAP activity and in thermal and mechanical nociceptive thresholds is suggestive of a role of CGRP- or FRAP-containing primary afferents in thermal and mechanical nociception.

Striatal calcitonin gene-related peptide-like immunoreactive afferents from the regions ventral and medial to the medial geniculate nucleus of rats

The sources of fibers with immunoreactivity to calcitonin gene-related peptide in the caudal portion of the caudate-putamen were investigated in animals treated by a knife cut, a tracer injection, or an electrolytic lesion of regions ventral and medial to the medial geniculate nucleus. Hemitransection of the brain just caudal to the caudate-putamen decreased ipsilaterally calcitonin gene-related peptide-like immunoreactivity in the caudal portion of the caudate-putamen, suggesting that calcitonin gene-related peptide-like immunoreactive fibers in the caudate-putamen originated from neurons in the area caudal to the caudate-putamen. Some calcitonin gene-related peptide-like immunoreactive neurons in the regions ventral and medial to the medial geniculate nucleus, including the peripeduncular, posterior intralaminar and suprageniculate nuclei, were labeled ipsilaterally with a retrograde tracer after an injection into the caudal portion of the caudate-putamen. In addition, an electrolytic lesion of calcitonin gene-related peptide-like immunoreactive neurons in the regions ventral and medial to the medial geniculate nucleus decreased the number of calcitonin gene-related peptide-like immunoreactive fibers in the ipsilateral caudal portion of the caudate-putamen. These results suggest that some cells containing calcitonin gene-related peptide in several posterior thalamic nuclei project to the caudal caudate-putamen.

Calcitonin gene-related peptide (CGRP) immunoreactivity in the afferents to the caudate-putamen and perirhinal cortex of rats

The posterior caudate-putamen and perirhinal cortex are innervated by fibers immunoreactive to calcitonin gene-related peptide (CGRP). We investigated the origins of these fibers by using immunohistochemistry combined with lesion experiments and fluorescent dye tracers. Lesions of the posterior thalamus surrounding the medial geniculate nucleus, in which groups of CGRP-like immunoreactive (CGRP-LI) cells exist, decreased the number of ipsilaterally CGRP-LI fibers in the posterior caudate-putamen and partly in the perirhinal cortex. Some of CGRP-LI neurons in several posterior thalamic nuclei surrounding the medial geniculate nucleus were labeled with both Fast blue injected into the posterior caudate-putamen and fluoro-gold administered into the anterior perirhinal cortex. Our results indicate that CGRP-LI cells in the posterior thalamus, such as posterior intralaminar, lateral subparafascicular and subparafascicular nuclei, project either separately or simultaneously to innervate the posterior caudate-putamen and perirhinal cortex.

Age-related decrease of calcitonin gene-related peptide-containing vasodilator innervation in the mesenteric resistance vessel of the spontaneously hypertensive rat.

We previously demonstrated that the mesenteric resistance blood vessels have nonadrenergic, noncholinergic vasodilator innervation in which calcitonin gene-related peptide (CGRP) is a possible neurotransmitter. The role of CGRP-containing vasodilator nerves in hypertension was investigated in perfused mesenteric vascular beds isolated from spontaneously hypertensive rats (SHR). The adrenergic vasoconstrictor responses to perivascular nerve stimulation in both SHR (8-, 15-, and 30-week-old) and age-matched Wistar-Kyoto (WKY) rat preparations increased with aging, but the response was greater in SHR than in WKY rats at all ages. The preparation isolated from SHR and WKY rats was precontracted by continuous perfusion of Krebs' solution containing 7 x 10(-6) M methoxamine plus 5 x 10(-6) M guanethidine. In both SHR and WKY rats, perivascular nerve stimulation (1-8 Hz) produced frequency-dependent vasodilation, which was blocked by 1 x 10(-7) M tetrodotoxin, pretreatment with 5 x 10(-7) M capsaicin, and denervation by cold storage (4 degrees C for 72 hours). The vasodilation induced by perivascular nerve stimulation in SHR greatly decreased with age, whereas a slight decrease in the response with age was found in WKY rats. The neurogenic vasodilation in the young SHR preparation was similar in magnitude to the vasodilation in age-matched WKY rats, whereas the vasodilation in 15- and 30-week-old SHR was significantly smaller than that in age-matched WKY rats. In both SHR and WKY rats, perfusion of rat CGRP (1 x 10(-10) to 3 x 10(-8) M) produced marked vasodilation in a concentration-dependent manner. The CGRP-induced vasodilation in SHR increased with age, whereas an age-related decrease in vasodilation was found in WKY rats. Perivascular nerve stimulation (4 and 8 Hz) of the perfused mesenteric vascular bed evoked an increased release of CGRP-like immunoreactive substance in the perfusate, which was significantly less in 15-week-old SHR than in age-matched WKY rats. Immunohistochemical studies showed an age-related decrease in CGRP-like immunoreactive fibers in SHR but not in WKY rats. These results suggest that CGRP-containing vasodilator innervation is greatly decreased when SHR develop and maintain hypertension. It is also suggested that the decreased vasodilator mechanism by CGRP-containing nerves contributes to the development and maintenance of hypertension.

Electron microscopic observation of calcitonin gene-related peptide-like immunoreactivity in the organ of Corti of the rat

Calcitonin gene-related peptide (CGRP)-like immunoreactive (CGRP-IR) nerve terminals in the organ of Corti of rats were studied by light and electron microscopy. Surface preparation of the organ of Corti were immunostained using anti-CGRP antiserum for avidin-biotin immunohistochemistry. Dense CGRP-IR fiber bundles were observed by light microscopy in the inner spiral bundles, tunnel spiral bundles and outer spiral bundles. Electron microscopic analysis indicated that CGRP-IR fibers belong to efferent nerves. In the inner spiral bundles, the CGRP-IR fibers showed a direct contact mainly with non-immunoreactive afferent fibers. Some CGRP-IR nerve endings in the inner spiral bundles formed contacts directly with inner hair cells. In the outer spiral bundles, CGRP-IR fibers formed synaptic contacts exclusively with the outer hair cells. It should be noted that the number of synapses of CGRP nerve endings with outer hair cells varied depending upon the sub-row: a falling gradient in number occurred along the inner-outer axis. Our results suggest that CGRP acts as an efferent neuromodulator in the organ of Corti.

Calcitonin gene-related peptide in the rat uterus: Presence in nerves and effects on uterine contraction

The influence of calcitonin gene-related peptide (CGRP) on rat uterine activity was examined in concert with the anatomical distribution of CGRP-like immunoreactivity in the uterus. CGRP-like immunoreactivity was localized in nerve fibers; these peptide-containing nerves were abundant throughout the mesometrium of the uterine horn and appeared to innervate mesometrial smooth muscle and vascular smooth muscle. In the uterine wall, CGRP-like immunoreactive fibers were prevalent in the myometrium, endometrium and the endocervix. Fibers in the endometrium and endocervix appeared to form a plexus subjacent to the epithelium and some fibers penetrated the epithelium as an intraepithelial plexus. The action of CGRP (10 −9 to 10 −6 M) on acetylcholine (10 −6 or 10 −5 M)-stimulated uterine activity was examined in vitro. Exogenously applied CGRP induced a dose-dependent relaxation of acetylcholine-stimulated uterine contractions. CGRP had no effect on basal uterine tension. The localization of CGRP-like immunoreactivity in nerves and the relaxing effect of CGRP suggests a role for CGRP-containing nerve fibers in the regulation of uterine activity.

ラット前庭神経核におけるＣＧＲＰ含有細胞ならびに線維の局在について 免疫組織化学的研究

The distribution and fine structure of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) cells and fibers in the vestibular nuclei of the rat were investigated by light and electron microscopic immunocytochemistry. In addition to the previous report that CGRP-LI cells were found in the lateral vestibular nucleus, the present study clarified that they are found also in the inferior vestibular nucleus, medial vestibular nucleus and nucleus X. The lateral vestibular nucleus contains a high density of CGRP-LI cells. They are medium in size and multipolar in shape. CGRP-LI cells in the inferior vestibular nucleus are small to medium in size and triangular or pea shaped. CGRP-LI cells in the medial vestibular nucleus and nucleus X are both few in number and small in size. Possible colocalization of CGRP with acetylcholine, gamma-aminobutyric acid or substance P in the single neuron of the vestibular nuclei might be suggested. CGRP-LI fibers are more extensively distributed in various areas throughout the vestibular nuclei, though previous studies reported that they were found in the lateral vestibular nucleus and inferior vestibular nucleus. A number of CGRP-LI fibers are clearly observed in the inferior vestibular nucleus. Under electron microscopic analysis, CGRP-LI endoproducts are diffusely localized throughout the cytoplasm and some of CGRP-LI dendrites are identified to receive synaptic inputs form non-immunoreactive axon terminals with small spherical vesicles. It seems likely that CGRP is participated both in the intrinsic neurons in the vestibular nuclei or in the reciprocal innervations between the vestibular nuclei and the reticular formation or the cerebellum.

Calcitonin gene-related peptide immunoreactivity in the visceral sensory cortex, thalamus, and related pathways in the rat.

It has been proposed that calcitonin gene-related peptide (CGRP) may serve as a major neuromodulator in visceral sensory pathways, but its exact role in the visceral sensory thalamus and cortex has not been determined. We therefore examined the distribution of CGRP-like immunoreactive (CGRPir) innervation of the insular cortex and the parvicellular division of the ventroposterior nucleus of the thalamus (VPpc) in the rat by using immunohistochemistry for CGRP combined with retrograde transport of the fluorescent dye fluoro-gold. Modest numbers of CGRPir fibers were distributed in the dysgranular and agranular insular cortex, but few were observed in the granular insular cortex. The density of CGRPir innervation increased caudally along the rhinal fissue and was considerably greater in the perirhinal cortex. When fluoro-gold was injected into the insular cortex numerous retrogradely labeled neurons were seen in the VPpc, but few of these were CGRPir. Retrogradely labeled CGRPir neurons were, however, seen in the ventral lateral and medial parabrachial (PB) subnuclei. Injection of fluoro-gold into the perirhinal cortex (which is just caudal to the insular cortex along the rhinal fissure) resulted in many retrogradely labeled CGRPir neurons in the posterior thalamic region, including the subparafascicular, the lateral subparafascicular, and the posterior intralaminar nuclei. The VPpc was heavily innervated by CGRPir fibers but contained few CGRPir cell bodies. Injection of fluoro-gold into the VPpc resulted in many retrogradely labeled CGRPir neurons in the external medial PB subnucleus bilaterally, but with a contralateral predominance. Smaller numbers of retrogradely labeled CGRPir neurons were also observed in the ventrolateral PB subnucleus, bilaterally with an ipsilateral predominance. These results suggest that CGRP may be a neuromodulator in the ascending visceral sensory pathways from the PB to the VPpc and the insular cortex, but not between the latter two structures.

Localization, origin and fine structure of calcitonin gene-related peptide-containing fibers in the vestibular end-organs of the rat

The localization, origin and fine structure of nerve terminals immunoreactive for calcitonin gene-related peptide (CGRP) were examined in the vestibular end-organs of rats using immunocytochemistry. Many CGRP-like immunoreactive (CGRP-IR) fibers were observed in the vestibular sensory epithelial layer. By electronmicroscopy, CGRP-IR terminals were found to make synaptic contacts with the chalyces of the vestibular nerves terminating on type I cells. The origin of these vestibular CGRP-IR fibers was examined by a combination of a retrograde fiber tracing technique (using Fast blue) and immunocytochemistry. Injections f the tracer into the vestibular cistern, resulted in fast blue-labeled neurons bilaterally in the area dorsolateral to the genu of the facial nerve; these labeled neurons also contained CGRP. These findings indicate that CGRP-IR fibers originate bilaterally from the area dorsolateral to the genu of the facial nerve and that CGRP plays a modulatory role in the transmission of vestibular information from type I cells.

Calcitonin gene-related peptide immunoreactivity in the cat lumbosacral spinal cord and the effects of multiple dorsal rhizotomies.

This study determined the extent of the rostral projection of calcitonin gene-related peptide-like immunoreactive (CGRP-IR) primary afferents in the cat lumbosacral spinal cord. To do this we examined the distribution of CGRP-like immunoreactivity (CGRP-LI) contralateral and ipsilateral to multiple dorsal rhizotomies. In the contralateral dorsal spinal cord, CGRP-IR fibers were mostly observed in Lissauer's tract, the dorsal columns, and laminae I, II, and V. Fewer CGRP-IR fibers were observed in laminae III, IV, and VI and the area around the central canal. The location of the CGRP-LI suggests that the afferents arose from nociceptors. Unilateral dorsal rhizotomies of five consecutive segments in the lumbar enlargement caused a substantial although incomplete loss of CGRP-LI in the rhizotomized dorsal spinal cord ipsilateral to the lesions. The majority of the remaining CGRP-IR fibers were located in Lissauer's tract, the dorsal columns, and the lateral part of laminae I and V. Ventral rhizotomies or an ipsilateral hemisection in the most rostral rhizotomized segment, in addition to the dorsal rhizotomies, had no noticeable effect upon the density or location of the remaining CGRP-LI. These results suggest that the majority of the CGRP-LI within the rhizotomized region of spinal cord was contained within branches of small-diameter primary afferents that entered the spinal cord through intact dorsal roots located caudal to the rhizotomized segments of spinal cord. It is concluded that CGRP-IR small-diameter primary afferents are capable of projecting at least five segments beyond their segment of entry and supplying collaterals to the superficial and deeper layers of the dorsal horn involved in the processing of nociceptive information.

Distribution and fine structure of calcitonin gene-related peptide-like immunoreactive nerve fibers in the rat skin

Distribution of calcitonin gene-related peptide-like immunoreactive (CGRPI) nerve fibers and their fine structure were examined in the skin of rat foot pads using immunocytochemistry. The CGRPI fibers formed bundles in the dermis and subcutaneous tissue. Two types of single-stranded CGRPI fibers were seen to leave the fiber bundles: one was located along the blood vessels or around the eccrine sweat glands, while the other entered the epidermis directly or through the Meissner's corpuscles in the dermal papillae. CGRPI fibers in the epidermis were distributed widely and were occasionally associated with Merkel cells. Immunoelectron microscopic study revealed that CGRPI fibers located around blood vessels, sweat glands, epidermal keratinocytes and Merkel cells, or in the Meissner's corpuscles did not form typical synaptic contacts with underlying cells, despite being varicose and filled with vesicles resembling synaptic ones. These findings suggested that the CGRP is released non-synaptically from these terminals to influence diffusely the organs surrounding the terminals. These cutaneous fibers seemed to originate from CGRPI neurons (both small type B cells and large type A cells) in the dorsal root ganglia (DRG), because injection of fast blue dye into the cutaneous nerve resulted in labeling of these CGRPI cells in the DRG and excision of the L 3-L 6 DRG resulted in the non-detection of cutaneous CGRPI fibers in the foot pads. Analysis of the composition of CGRPI fibers found in the rat skin has revealed that these are mostly unmyelinated, C-type fibers with some of them being thin myelinated fibers. This was true even of CGRPI fibers at the proximal end of peripheral neurites of the DRG. Such results lead us to conclude that CGRPI neurons may constitute a distinct subpopulation of DRG cells, whose axons are exclusively unmyelinated or thinly myelinated.

A light and electron microscopic study of calcitonin gene-related peptide in the rat caudate putamen

Calcitonin gene-related peptide (CGRP)-like immunoreactive (CGRP-IR) structures have been studied in the rat caudate putamen using avidin-biotin peroxidase immunohistochemistry. Immunoreactivity was found in the axons of this nucleus but not in the perikarya. CGRP-IR fibers were most densely concentrated along the ventral border and in the caudal portion of the rat caudate putamen. CGRP-IR fibers were sparsely distributed throughout the rest of this nucleus. Almost all immunoreactive boutons which contained small clear vesicles had formed asymmetrical synapses. Postsynaptic targets included dendritic spines and shafts. Asymmetrical synapses in the caudate putamen are supposed to be extrinsic in origin. These observations, together with the results of other investigations, suggest that CGRP-IR boutons form synapses with spiny striatal neurons, which, most likely, are medium-sized spiny projecting neurons. Moreover, evidence indicates that these boutons are of extrinsic origin.