Calcitonin Gene-related Peptide Fibers Research Articles

Innervation of the equine mature and immature proximal sesamoid bone by calcitonin gene-related peptide and substance P-containing nerves

Abstract Objective To localize and determine relative frequency of occurrence of calcitonin gene-related peptide (CGRP) and substance P (SP) fibers in the proximal sesamoid bones (PSB) and adjacent structures in sound horses. Sample Population 4 foals and 3 adult horses. Procedure Medial PSB and adjacent ligaments of both forelimbs were collected, flushed, and fixed in buffered 4% formalin. The left PSB were cut into 5 longitudinal, sagittally oriented slabs, and the right PSB were cut into 5 transverse slabs. After decalcification in EDTA, slices were transferred to a 30% sucrose solution, deep frozen, sectioned (80 μm), and stored in Tris-buffered saline solution for free-floating immunocytochemical processing, staining of which was performed by use of the peroxidase-antiperoxidase method with polyclonal rabbit antibodies against CGRP and SP. Sections were examined by light microscopy. Results Calcitonin gene-related peptide and SP fibers were present throughout the PSB; in ligaments, they were concentrated at the attachment sites. Presentation of the fibers, the innervation pattern, and intensity in the bones of horses differed from that in foals, in which the fibers were more delicate and frequency of occurrence was lower. In horses, innervation with CGRP and SP fibers was more dense in the periphery of the bone, whereas in foals, local difference in innervation of the bone was not evident. With the exception of the intersesamoidean ligament, ligaments were more richly innervated than was bone. Conclusion Presence of the nociceptive peptides CGRP and SP indicated that the PSB and, more practically, the adjacent ligaments are sensitive structures. (Am J Vet Res 1998;59:1378–1385)

Distribution of Calcitonin Gene-Related Peptide, Atrial Natriuretic Peptide and Neuropeptide Y in the Rat Heart

Calcitonin gene-related peptide (CGRP) is a novel neuropeptide, predicted on the basis of structural analysis of the rat calcitonin gene. It is a neurotransmitter which has been suggested to take part in sensory transmission. In this study, we have examined the distribution of this peptide, α-atrial natriuretic peptide immunoreactivity (irANP) and neuropeptide Y (NPY) within different regions of the rat heart. Attempts were also made to compare the distributions of these peptides in the regions examined, through different methods of immunocytochemistry and further comparing these results with those obtained through radioimmunoassay. The distributions of the peptides in the atria were similar to results obtained with radioimmunoassay, but there were no myocytes containing irANP in the ventricles with immunocytochemistry as opposed to radioimmunoassay. While the staining obtained for irANP in the atrium was more intense in the right, CGRP and NPY nerve fibres were two to three times more abundant in the left atrium. The high local concentration of a vasoconstrictor peptide in the region of coronary vessels may suggest that it is involved in the control of vascular smooth muscle tone. The method of choice with the immunocytochemical studies was that of the susa wax technique for irANP. Caution should therefore be observed when interpreting results based only on a single staining technique.

Hypoxic adaptation of the peptidergic innervation in the rat carotid body

The abundance of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive nerve fibers in the carotid body was compared between normoxic and chronically hypoxic rats (10% O 2 and 3.0–4.0% CO 2 for 3 months). The immunoreactive fibers appeared as thin processes with many varicosities, and were distributed mainly around the vasculatures. In the normoxic control carotid body, NPY fibers were more numerous than VIP, CGRP, and SP fibers. In the chronically hypoxic rats, the carotid body was enlarged several fold, and the mean absolute number of VIP and NPY fibers was 3.88 and 2.22 times higher than in the normoxic carotid body, respectively, although that of SP and CGRP fibers was not changed. When expressed as density per unit area of the parenchyma, the density of SP and CGRP fibers in the chronically hypoxic carotid body decreased significantly to under 50%, the density of VIP fibers increased significantly 1.80 times, and the density of NPY fibers were unchanged. Immunoreactivity for four neuropeptides was not found in the glomus cells of normoxic or chronically hypoxic carotid bodies. These results suggest that altered peptidergic innervation of the chronically hypoxic carotid body is one feature of hypoxic adaptation. Because these neuropeptides are vasoactive in nature, altered carotid body circulation may contribute to modulation of the chemosensory mechanisms by chronic hypoxia.

Calcitonin gene-related peptide and spinal afferents partly mediate postoperative colonic ileus in the rat

Background: Calcitonin gene-related peptide (CGRP) is a widely distributed neuropeptide contained in intrinsic and extrinsic neurons of the gastrointestinal wall that has been shown to be released by noxious stimulation, to be involved in nociception, to inhibit gastrointestinal motility, and to partly mediate postoperative gastric ileus. We hypothesized that abdominal surgery-induced release of CGRP might inhibit postoperative colonic motility and food intake. Methods: Colonic transit, stool pellet number, stool pellet weight, and food intake were measured for 48 hours after induction of postoperative ileus in rats. CGRP was immunoneutralized by preoperative injection of CGRP monoclonal antibody, or visceral afferent nerve fibers containing CGRP were functionally ablated by topical capsaicin treatment of the vagus nerves or of the celiac/superior mesenteric ganglia before abdominal surgery. Results: Abdominal surgery increased colonic transit time and decreased 24-hour cumulative stool pellet number, stool pellet weight, and food intake. CGRP immunoneutralization reversed postoperative inhibition of colonic transit, 24-hour cumulative stool pellet number, stool pellet weight, and food intake by 77%, 82%, 80%, and 52%, respectively. Whereas ablation of vagal afferent nerve fibers had no effect, spinal afferent nerve fiber ablation reversed postoperative inhibition of 24-hour cumulative stool pellet number, stool pellet weight, and food intake by 41%, 38%, and 19%, respectively. Conclusions: CGRP and spinal afferent nerve fibers partly mediate postoperative colonic ileus and inhibition of food intake in the rat. By the magnitude of reversal of postoperative ileus, CGRP seems to be an important mediator of postoperative colonic ileus. Our results for the first time show involvement of a neuropeptide and spinal afferents in the mediation of postoperative colonic ileus and postoperative inhibition of food intake in rats. (Surgery 1998;123:518-27.)

Ontogeny of innervation of rat and ovine fetal adrenals

The formation of adrenocortical zonation occurs in rats during late gestation. Since adult cortical function is modulated by neural mediators, it is possible that the development of differentiated function is dependent on cortical innervation. The goal of this study was to compare the pattern and timing of rodent and ovine adrenal innervation during late organogenesis by staining with antibodies directed against the neuropeptides vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY) and the catecholamine biosynthetic enzyme, tyrosine hydrorylase (TOH). Rat adrenals were collected from fetal days 17–21 (term=21 days) and ovine adrenals from fetal days 101–136 (term=145 days). Adrenals were fixed, cryosectioned at 100μ and immunostained using Cy3-conjugated secondary antibodies. In both species, staining of VIP, CGRP, NPY and TOH fibers was observed in the capsule and subcapsular layers of the cortex during gestation. In late gestation, VIP- and NPY-positive ganglions cells were observed near the medulla extending processes toward the outer cortex; in ovine adrenals, fibers from ganglion cells appeared to surround nests of outer cortical (presumably, zona glomerulosa) cells. These data show that phenotypically distinct neural elements appear at different stages of adrenocortical development. The presence of neural elements in contact with adrenal cortical cells supports the possibility for neural control of adrenocortical development.

Distribution and Quantity of Neuroendocrine Markers in Allergic Rhinitis

Neuroendocrine components exist in the human nasal mucosa. However, the pathophysiological and neuroimmunological roles of the regulatory peptides in allergic rhinitis (AR) require further investigation. To analyse the functional morphology and quantify the tissue concentration of regulatory peptides in the nasal mucosa of AR subjects, human inferior turbinate mucosa specimens from 25 patients with AR, 20 patients with non-allergic rhinitis and 10 patients without any nasal diseases were investigated. Using immunohistochemistry and radioimmunoassays, we detected the presence, distribution and concentrations of various neuropeptides [vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), substance P (SP) and calcitonin gene-related peptide (CGRP)] and general neuroendocrine markers (neuron-specific enolase and chromogranin A). Quantitative analysis of the stained fibres and cells was performed using a graphic AutoCAD program. The presence and distribution of NPY, CGRP and SP nerve fibres and neuroendocrine cells were similar among the three subject groups. AR subjects had significantly higher tissue concentrations of VIP and SP. AR subjects had increased numbers of VIP fibres which predominantly innervated vessels. Thus, VIP and SP play important neuroimmunological roles in the pathogenesis of AR.

Neuropeptide-containing nerve fibres in the human parotid gland: a semiquantitative analysis using an antibody against protein gene product 9.5.

The occurrence and distribution of neuropeptide-containing fibres in the human parotid gland were examined by the peroxidase-antiperoxidase method with attention to the quality of fixation and the condition of patients. Many fibres immunoreactive for neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) and a moderate number of galanin-positive (GAL) fibres were distributed around the acini. A moderate number of NPY and VIP fibres were distributed around the intercalated ducts. The semiquantitative mean densities (+/- SD) of periacinar NPY, VIP and GAL fibres expressed as a percentage of the total protein gene product (PGP) 9.5 immunoreactive fibres were 75.62 +/- 7.25%, 70.52 +/- 9.33% and 41.76 +/- 5.45%, respectively, whereas those of substance P (SP), calcitonin gene-related peptide (CGRP) and FMRF amide (FMRF) fibres were below 10%. The mean densities of NPY and VIP fibres around the intercalated ducts expressed as the percentage of PGP 9.5 fibres associated with these ducts were 52.37 +/- 6.19% and 59.62 +/- 7.02% respectively. Those of SP, CGRP, GAL, and FMRF fibres were below 10%. The densities of NPY, VIP, SP, CGRP, GAL and FMRF fibres around the striated and excretory ducts were also below 10%. In the vasculature, NPY fibres were the most prominent. Similarly, the mean density of perivascular NPY fibres was 93.76 +/- 2.03%. No somatostatin or leucine or methionine enkephalin immunoreactivity was detected around the acini, duct system or blood vessels. These findings suggest that, in this gland, the periacinar NPY, VIP and GAL fibres may participate in regulating the synthesis of saliva and its secretion and that perivascular peptidergic fibres, especially NPY fibres, may be involved in controlling local blood flow.

Ontogeny of regulatory neuropeptides in the bullfrog taste organ

During metamorphic stages (stages XX–XXV), the first appearance of nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), galanin (GAL), and neuropeptide Y (NPY) in the bullfrog taste organs was different for each substance. CGRP fibers appeared first in association with the immature taste organs at stage XX. Up to stage XXV, the taste organs, epithelial disks, are close to their adult form, and SP, VIP, and GAL fibers appeared within them. Throughout these stages, NPY fibers were absent, and no taste cells had immunoreactivity of the five neuropeptides. The present findings and previous physiological studies suggest that the immature taste organs in metamorphic stages already function as chemical and mechanical receptors and that these sensory mechanisms are under the control of peptidergic innervation.

Ontogeny of regulatory neuropeptides in the bullfrog taste organ

During metamorphic stages (stages XX–XXV), the first appearance of nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), galanin (GAL), and neuropeptide Y (NPY) in the bullfrog taste organs was different for each substance. CGRP fibers appeared first in association with the immature taste organs at stage XX. Up to stage XXV, the taste organs, epithelial disks, are close to their adult form, and SP, VIP, and GAL fibers appeared within them. Throughout these stages, NPY fibers were absent, and no taste cells had immunoreactivity of the five neuropeptides. The present findings and previous physiological studies suggest that the immature taste organs in metamorphic stages already function as chemical and mechanical receptors and that these sensory mechanisms are under the control of peptidergic innervation.

Ontogeny of the peptidergic fibers in the male mouse submandibular gland

The first appearance of substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), galanin (GAL), leucine-enkephalin (l-ENK), and methionine-enkephalin (m-ENK) in the male mouse submandibular glands was different for each. VIP immunoreactive fibers first appeared on embryonic day 15 (E15), SP on E16, and CGRP fibers on E18. GAL, l-ENK, and m-ENK fibers appeared in the early postnatal period, and NPY fibers occurred on postnatal day 21 (P21). From P0 to P21, VIP fibers rapidly increased in number, but SP and CGRP fibers increased only slightly. After P21, VIP, SP, and CGRP fibers decreased in number. ENK fibers were found only from P0 to P14. The number of these immunoreactive fibers in the adult phase was low in comparison with that in early postnatal phase. Around the blood vessels, SP, VIP, CGRP, NPY, and GAL fibers appeared by at least P7. These findings suggested that the transient high activity of VIP, CGRP, SP, and GAL and the transient appearance of ENKs in the nerve fibers may be related to the cell proliferation and differentiation of the functionally important structures of the mouse submandibular glands, and that the peptidergic innervation around the vasculature is probably involved in controlling local glandular circulation.

Plasticity of mature sensory cerebrovascular axons following intracranial infusion of nerve growth factor.

Mature perivascular sympathetic axons associated with the intradural segment of the internal carotid artery (ICA) of the adult rat respond by sprouting following a two week infusion of nerve growth factor (NGF) into the lateral ventricle of the brain. Because nonsympathetic axons such as those comprising the sensory and parasympathetic population have been shown to respond to NGF, the present study was carried out to determine whether mature sensory axons respond to in vivo NGF infusion and whether competitive interactions between the innervating populations might affect the responsiveness of these axons to NGF. Standard electron microscopic techniques as well as calcitonin-gene-related peptide (CGRP) immunohistochemistry at the light microscopic level were used to examine the effects of intracerebroventricular NGF infusion on mature perivascular fibers with and without prior sympathetic denervation (i.e., bilateral superior cervical ganglionectomy). Following NGF infusion, CGRP-immunoreactive fibers appeared thicker and more numerous in the longitudinal plane when compared with vehicle controls. However, at the ultrastructural level, a significant increase in the total number of axons was not observed, although there was an increase in the number of large granular vesicles, suggesting that the CGRP fibers responded to exogenous NGF with an increase in neurotransmitter content, but not by sprouting. Sympathetic denervation, on the other hand, resulted in a significant increase in the number of fibers passing in the circumferential plane. The most dramatic change in CGRP immunoreactivity was observed following combined sympathetic denervation and subsequent NGF infusion, where, in addition to the presence of thicker immunoreactive fibers, a significant increase in the perivascular density of immunoreactive fibers associated with the intradural blood vessels was observed. These findings suggest that exogenous NGF has different effects on mature sympathetic and nonsympathetic fibers that innervate intradural blood vessels. The former exhibit robust sprouting, whereas the latter do not sprout in response to NGF but show evidence for increased neuropeptide content. In addition, the heightened response by sensory axons following denervation and subsequent NGF infusion provides support for the idea that sensory and sympathetic axons normally compete for target space and/or target-derived neurotrophic factors.

Distribution of immunoreactive neuropeptides in the pancreas of the bullfrog, Rana catesbeiana, demonstrated by immunofluorescence

Indirect double immunofluorescence labelling for eight neuropeptides in the pancreas of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of certain neuropeptides in the exocrine and endocrine pancreas. Immunoreactivity of substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), FMRFamide (FMRF), and galanin (GAL) was localized in nerve fibers distributed between the acini and around the duct system and vasculature of the exocrine pancreas. In these regions, CGRP-immunoreactive fibers were more numerous than those containing the other five peptides. Almost all SP fibers showed coexistence of SP with CGRP, and about one third of fibers also showed coexistence of SP with VIP, NPY, FMRF, and GAL. In the endocrine pancreas, SP, CGRP, VIP, and GAL were recognized in the nerve fibers around and within the islets of Langerhans, and VIP and GAL fibers were more numerous than SP and CGRP fibers. All CGRP fibers, and about half of the VIP and GAL fibers were immunoreactive for SP. NPY- and FMRF-immunoreactive cells were found at the periphery of the islets. These findings suggest that the exocrine and endocrine pancreatic functions of the bullfrog are under the control of peptidergic innervation.

Regional differences of CGRP-immunoreactive nerve fibers in nasal epithelium of the rat.

The regional distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the epithelium throughout the nasal cavity was determined using semiserial cryostat sections and whole mount preparations. Both respiratory and olfactory epithelia displayed, in their basal portion, an extensive nerve plexus projecting beaded nerve fibers toward the luminal surface. However, the density of the intraepithelial CGRP nerve fibers conspicuously varied according to regions. They were abundant on the septal mucosa, the ventromedial side of the nasoturbinates and the dorsal surface of the maxilloturbinates, while less numerous on the lateral side of the naso- and maxilloturbinates and on the lateral nasal wall. Another difference in the regional distribution of nerves was recognized between the anterior and posterior portions of the respiratory area: the anterior portion received a denser supply of intraepithelial CGRP fibers than the posterior portion. Characteristically, the transepithelial CGRP-immunoreactive nerve fibers were densest on the anteroventral aspect of the nasoturbinates and on the anterodorsal surface of the maxilloturbinates. Some of them appeared to penetrate through the epithelium to come into contact with the lumen of the nasal cavity. These results suggest that the CGRP fibers in the epithelium display a region-specific distribution, apparently disposed more densely over the areas which are more directly exposed to inhaled air, possibly that containing irritants and toxicants.

Localization of immunoreactive neuropeptides in the kidney of the bullfrog, Rana catesbeiana, by immunofluorescence

Indirect double immunofluorescence labelling for demonstrating nine neuropeptides in the kidney of the bullfrog, Rana catesbeiana, revealed for the first time the occurrence, distribution, and coexistence of certain neuropeptides in the kidney of the submammalian vertebrates. Substance P, neuropeptide Y, and calcitonin gene-related peptide were localized in nerve fibers distributed along the afferent arterioles connected with the glomeruli, and along the capillary network between uriniferous tubules. Neuropeptide Y and calcitonin gene-related peptide immunoreactive fibers were more numerous than substance P immunoreactive fibers. In these two regions, about one half of the neuropeptide Y or calcitonin gene-related peptide fibers contained substance P. No immunoreactivity of vasoactive intestinal polypeptide, somatostatin, FMRFamide, or leucine- and methionine-enkephalins was detected in the bullfrog kidney.

Nerve fibers immunoreactive for substance P and calcitonin gene-related peptide in the cervical spinal ventral roots of the mouse.

We demonstrate the existence of nerve fibers possessing substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity in the mouse cervical ventral roots. The distribution of the SP and CGRP fibers was similar, but CGRP fibers were generally more numerous. Both types entered the ventral pia mater or formed hairpin loops, but they did not enter the spinal cord directly through these roots. SP and CGRP fibers in the ventral roots were thin and had many varicosities. We suggest that these SP and CGRP fibers are involved not only in a sensory mechanism, but also in other functions, via the release of SP and CGRP from varicosities in the ventral roots.

Presence of neuropeptides in human nasal polyps.

The pathophysiological effects of non-cholinergic, non-adrenergic neuropeptides are well known in the nasal mucosa, but unclear in the polyps. Since the pathophysiological roles of neuropeptides depend on their presence in the target tissue, specimens of nasal polyps were removed from 20 patients and examined for the presence of vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), dopamine-beta-hydroxylase (DBH), substance P (SP) and calcitonin gene-related peptide (CGRP). To visualize these neuropeptide fibers, immunohistochemical staining by the peroxidase-anti-peroxidase method and color reaction by Nickel enhancement of diaminobenzidine (DAB) were used. Fine varicose neuropeptides immunostained fibers were predominantly distributed in the pedicle of the polyps. No neuropeptides were found in the mucosal epithelium and subepithelium. NPY fibers were predominantly seen around the thick wall vessels, whereas the VIP fibers were mainly to be found in close proximity to the submucosal glands and fairly close to the vessels. SP or CGRP fibers were not found in the polyps. VIP and NPY in the pedicle of the polyps may be present in connection with mucosal inflammation, tissue edema and cystic degeneration of the glands in the early stage of polyp formation. Thus these neuropeptides may contribute to the development and growth of nasal polyps.

Immunohistochemical coexistence of calcitonin gene-related peptide and substance P in the nerve fibers of the internal gills of bullfrog ( Rana catesbeiana) larvae

A double-labelling immunofluorescence study of the internal gills of larval bullfrogs revealed coexistence of calcitonin gene-related peptide (CGRP) and substance P (SP) in the same nerve fibers in the gill tufts. More than about 95% of CGRP fibers showed coexistence of SP, although some fibers contained CGRP without SP. In the branchial muscle, all CGRP fibers demonstrated coexistence of SP. These findings suggest that both CGRP- and SP-containing fibers may use both peptides as neuromodulators of active ion transport and gill movements.

Calcitonin gene-related peptide, substance P and GAP-43/B-50 immunoreactivity in the normal and arthrotic knee joint of the mouse.

The aim of this study was to describe the normal distribution of calcitonin gene-related peptide (CGRP) and substance P (SP) containing fibres in the knee joint of the mouse and to obtain insight into the changes in innervation associated with degenerative processes in the joint. Arthrosis was induced by a single subpatellar intra-articular injection of bacterial collagenase. After decalcification in EDTA solutions, the CGRP and SP fibres were visualized by peroxidase-antiperoxidase pre-embedding immunocytochemistry for light microscopy. Control experiments on the mouse brain as a reference for the effect of EDTA on the immunostaining showed that the decalcification procedure with EDTA had not impaired the immunostaining. A rich innervation of thin varicose CGRP and SP immunoreactive fibres was found in most peri- and intra-articular tissue components. The periosteum, synovial tissues, the joint capsule and the intra-articular fat tissues were richly innervated. Less intense innervations were also found in the subchondral bone plates of the tibio-femoral joint and of the patella. Fibres were also found in the soft tissues between the patellar tendon and the femoral groove. No differences could be found between the location of CGRP and SP fibres with respect to the localization in the joint, but generally more CGRP fibres were found. The collagenase-induced osteoarthrosis was characterized by sclerosis of the subchondral bone, patellar dislocation, osteophyte formation, synovial proliferation and by severe cartilage abrasion, particularly on the medial side of the femoro-tibial joint. The overall distribution of CGRP and SP fibres was the same as in the control joints. However, major differences were found in all studied joints at specific locations around the cruciate ligaments, in the synovium around the patella, in the soft tissues lateral of the patella and in plica tissue between the patella and femoral groove. The CGRP and SP innervation was no longer detectable by immunolabelling with the antibodies. With a polyclonal antibody to the growth associated protein GAP-43/B-50, signs of degenerated axonal profiles were observed in these locations. At other peripheral locations, such as the muscles, the GAP-43/B-50 distribution was normal. In conclusion, the present study provides detailed information on the localization of CGRP and SP fibres, which may be involved in pain perception. Knowledge of the changes that occur during arthrosis may give more insight into the clinical symptoms.

Early Development of Olivocerebellar Projections in the Fetal Rat Using CGRP Immunocytochemistry.

The expression of calcitonin gene-related peptide (CGRP) immunoreactivity in certain inferior olivary neurons is transient and developmentally regulated. Labelled neurons begin to appear at embryonic day 16 (E16), and reach their maximal extent by postnatal day 2 (P2). The extinction of the labelling occurs between P13 and P16. Expression of CGRP immunoreactivity is also observed in a few cerebellar fibres from E17, when axons in the restiform bundle begin to enter medially the cerebellar parenchyma. Their maximal extent is reached by P6, and thereafter they slowly disappear following a precise pattern, although fibre extinction is not complete. The spatio-temporal changes in the olivary distribution of the labelled neurons and the changes in the cerebellar labelled fibres follow the known pattern of topographic arrangement of the olivocerebellar system in adult rats. Moreover, the developmental phases of the CGRP-labelled fibres in postnatal rats correspond to those known for climbing fibre phenotypic acquisition. Thus, CGRP immunocytochemistry identifies in the fetal rat a subset of inferior olivary neurons and their corresponding cerebellar climbing fibres. Using this approach, we have analysed some of the initial events leading to the formation of the olivocerebellar projection, and obtained the following information: (i) Olivocerebellar axons are not randomly distributed in the restiform bundle before they enter the cerebellum. (ii) In the presence of a large spectrum of choices at the surface of the rostral half of the cerebellar plate the labelled olivary axons begin to enter the cerebellum at a precise medial point to abut a region composed solely of migrating Purkinje cells, and establish contacts with their targets before these neurons reach their final cortical location. (iii) From E18 to E19, the bundle of labelled fibres loses its superficial location, being bypassed by migrating Purkinje cells, to occupy a region corresponding to the prospective white matter. This translocation is coincident with the occurrence of a second axonal entry point, somewhat more lateral than the previous one, and with the appearance of a new lateral stripe of labelled fibres. (iv) Both the early and the late appearing labelled stripes remain confined from the time of their formation in precise cerebellar territories, indicating that only some clusters of Purkinje cells are contacted by the CGRP fibres. The results obtained imply that there is neither a waiting period nor an initial phase of randomness in the formation of the olivocerebellar projection map. This absence of chaotic cerebellar invasion, and the high selectivity of the entry points, suggest that the orientation of CGRP-positive olivocerebellar fibres towards their targets is regulated by positional information shared between subsets of olivary neurons and clusters of Purkinje cells. The result of this process would be the formation of a precocious coarse topography that would need further refinement.

Calcitonin gene-related peptide in afferents to the cat's cerebellar cortex: Distribution and origin

In the present study, the distribution and origin of calcitonin gene-related peptide (CGRP) were analyzed in the cat's cerebellum. Following incubation in an antibody generated against rat CGRP and processing with the peroxidase anti-peroxidase (PAP) technique, CGRP immunoreactivity (IR) is found in profiles that have morphological characteristics of both simple and complex mossy fibers. However, all mossy fibers are not CGRP-positive. Further, CGRP-IR mossy fibers have a heterogeneous distribution in the cerebellum. In the vermis, the majority of immunoreactive profiles are in lobules VII, VIII, and the dorsal folia of IX. In anterior vermal lobules, only scattered terminals, located primarily at the apex and along the shoulder of the folia, are present. Laterally, CGRP-IR mossy fibers are located in the paramedian lobule, paraflocculus, and crus II. No CGRP fibers or varicosities are observed in any of the cerebellar nuclei. However, CGRP-positive cell bodies are scattered throughout the nuclear neuropil. A double label technique revealed that CGRP-IR mossy fibers arise from neurons located in the lateral reticular nucleus, external cuneate nucleus, inferior vestibular nucleus, and basilar pons. The present findings, taken together with previous data, indicate that cerebellar afferents are chemically heterogeneous. The findings of the present study suggest that precerebellar nuclei that give rise to the mossy fibers that contain CGRP have the potential for playing a complex role in modulating circuitry in the cerebellar cortex of the cat.