CGRP-like Immunoreactive Nerve Research Articles

Influence of liquid diet feeding on calcitonin gene-related peptide-like immunoreactive nerve fibers in rat temporomandibular joints during growth period

Biomechanical factors of masticatory functions are related to the development of the temporomandibular joint (TMJ) components during growth. However, the effects on nerve fibers have not been fully clarified. We investigated the influence of masticatory muscle activity on nerve fibers in the TMJ during growth by changing the consistency of diet. Twenty 3-week-old male Wistar rats were divided into two groups. The experimental group was fed a liquid diet, while the control group had a normal hard diet. Rats were sacrificed at 6-, and 9-week-old. We investigated the expression of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibers, which are most abundant in the TMJ and have a nociceptive function, using immunohistochemistry for CGRP. No significant difference in the number of CGRP-LI nerve fibers was detected in 6-week-old rats, but in 9-week-old ones the number in the experimental group was significantly greater than that in the control. We concluded that masticatory muscle activity in the TMJ closely correlates with peripheral sensory nerve fibers during growth.

Changes in peptidergic nerves in the atrioventricular valves of streptozotocin-induced diabetic rats: a confocal microscopy study.

Several previous studies have described the distribution of neuropeptide Y (NPY)-like and calcitonin gene related peptide (CGRP)-like immunoreactive nerve fibres in the atrioventricular valves of humans and various animals. It has been suggested that peptide-containing nerve fibres might have motor or sensory roles in valvular function. Although there is evidence that diabetic changes occur in the sympathetic (preganglionic and postganglionic), parasympathetic (vagal) and peptidergic nerves of rats, the changes of peptide-containing nerve fibres in the atrioventricular valves of the diabetic rat have not been studied. The distribution, relative density and staining intensity of NPY-like and CGRP-like immunoreactive nerve fibres in the mitral and tricuspid valves were studied in whole mount preparations using confocal microscopy with a computer-assisted image analysis system. Streptozotocin-induced diabetic and control rats were sacrificed at 12 and 24 months. The nerve staining intensity within the tricuspid valve was greater than the mitral valve in both control (P < 0.01) and diabetic (P < 0.001) rats. Nerve density in the anterior leaflet was greater than the posterior leaflet of the mitral valve. However, the anterior leaflet of the mitral and tricuspid valves showed a decreased number of nerve fibres, followed by drastic reduction in the staining intensities for both the peptides studied (P < 0.001) in the long-term diabetic rat. The decrease in the number of nerve fibres that follow the mechanical interruption of nerves raises the possibility that cycles of degeneration may occur. It is suggested that these peptide-containing nerve fibres in the atrioventricular valves may be involved in valvular dysfunction in the diabetic state.

Changes in peptidergic nerves in the atrioventricular valves of streptozotocin-induced diabetic rats: A confocal microscopy study

Several previous studies have described the distribution of neuropeptide Y (NPY)-like and calcitonin gene related peptide (CGRP)-like immunoreactive nerve fibres in the atrioventricular valves of humans and various animals. It has been suggested that peptide-containing nerve fibres might have motor or sensory roles in valvular function. Although there is evidence that diabetic changes occur in the sympathetic (preganglionic and postganglionic), parasympathetic (vagal) and peptidergic nerves of rats, the changes of peptide-containing nerve fibres in the atrioventricular valves of the diabetic rat have not been studied. The distribution, relative density and staining intensity of NPY-like and CGRP-like immunoreactive nerve fibres in the mitral and tricuspid valves were studied in whole mount preparations using confocal microscopy with a computer-assisted image analysis system. Streptozotocin-induced diabetic and control rats were sacrificed at 12 and 24 months. The nerve staining intensity within the tricuspid valve was greater than the mitral valve in both control (P < 0.01) and diabetic (P < 0.001) rats. Nerve density in the anterior leaflet was greater than the posterior leaflet of the mitral valve. However, the anterior leaflet of the mitral and tricuspid valves showed a decreased number of nerve fibres, followed by drastic reduction in the staining intensities for both the peptides studied (P < 0.001) in the long-term diabetic rat. The decrease in the number of nerve fibres that follow the mechanical interruption of nerves raises the possibility that cycles of degeneration may occur. It is suggested that these peptide-containing nerve fibres in the atrioventricular valves may be involved in valvular dysfunction in the diabetic state. Anat Rec 258:277–285, 2000. © 2000 Wiley-Liss, Inc.

Stimulatory effect of calcitonin gene-related peptide on adrenocorticotropin release from rat anterior pituitary cells.

In the present study, we examined the direct regulatory effect of rat calcitonin gene-related peptide (CGRP) on adrenocorticotropin (ACTH) release from rat cultured anterior pituitary cells. CGRP significantly increased ACTH release at concentrations of 10(-8)-10(-11) M. The ACTH release was gradually increased by CGRP concentrations lower than 10(-10) M, and was decreased at concentrations higher than 10(-9) M, presenting a bell-shaped dose-response curve. As well as having an additive effect on corticotropin-releasing factor-induced ACTH release, CGRP stimulated the accumulation of intracellular cAMP. The CGRP-induced ACTH release was inhibited by a protein kinase A inhibitor, suggesting that its stimulatory effect on the ACTH release was mediated via an adenylate-cyclase-protein kinase system. CGRP-like immunoreactive nerve fibers have been reported to innervate the anterior pituitary, so that the stimulatory effect of CGRP on the ACTH release suggests that this peptide may be involved in neural regulation of hormone secretion in the anterior pituitary.

Galanin-like immunoreactive nerve fibres in the anterior pituitary of the normal and adrenalectomized rat

Our previous studies have shown the presence of substantial amounts of substance P and calcitonin gene-related peptide-like immunoreactive nerve fibres in the anterior pituitary of the monkey, dog and rat. Furthermore, synaptic relationships have been demonstrated between these nerve fibres and the gland cells in the dog and rat. The substance P and calcitonin gene-related nerve fibres increase in number following adrenalectomy and ovariectomy, respectively. The present study was aimed to investigate the galanin-containing nerve fibres in the anterior pituitary of normal and adrenalectomized rats. The results showed only a small amount of galanin-like immunoreactive nerve fibres in the normal anterior pituitary, which were present among the gland cells as well as along the blood vessels. Following adrenalectomy, the number of galanin-like immunoreactive nerve fibres increased and ramification appeared more frequently. The results substantiate our hypothesis of a dual neural-humoral regulation of the mammalian anterior pituitary gland.

Response of substance P-immunoreactive nerve fibres in the anterior pituitary to plasma oestrogen levels in the rat.

The mammalian anterior pituitary has recently been shown to be innervated by substantial amounts of peptidergic nerve fibres. It has also been found that adrenalectomy increases the amount of calcitonin gene-related peptide-like immunoreactive nerve fibres in the anterior pituitary of the rat, and that stimulation of the nerve fibres in vitro can enhance release of ACTH. In the present study, the density of substance P-like immunoreactive nerve fibres in the anterior pituitary of the rat were examined in relation to variations in plasma oestrogen status. The amount of substance P-like immunoreactive nerve fibres increased significantly (P < 0.001) after ovariectomy, and decreased significantly (P < 0.01) under oestrogen replacement, in a dose-dependent manner. The results suggest the possibility of the direct neural involvement of oestrogen secretion in anterior pituitary regulation, which further substantiates the hypothesis of neural-humoral dual regulation of the mammalian anterior pituitary.

Postnatal development of protein gene product 9.5‐ and calcitonin gene‐related peptide‐like immunoreactive nerve fibers in the rat temporomandibular joint

Background The aim of this study was to investigate the little-known postnatal development of innervation in the temporomandibular joint (TMJ). Methods We therefore investigated the protein gene product 9.5-like immunoreactive (PGP-LI) and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerves in the rat TMJ from days 0–24 in wholemount preparations and frozen sections by immunohistochemistry using the avidin-biotin-peroxidase complex method. Results At birth, PGP-LI and CGRP-LI nerves were observed only in the disk attachment but not in the peripheral portion of the disk. From days 3–6, the peptidergic nerves associated with the development of synovial cells were first found in the anterior (3rd day) or posterior (6th day) peripheral portion of the disk in which synovial cells later appear. From days 7–10, incisal occlusion started and simultaneously the distinct networks of the nerves were formed in four peripheral portions of the disk. On the 14th day, the PGP-LI nerves were first more numerous than the CGRP-LI nerves only in the anterior disk attachment. From days 18–24, the occlusion between the first molars started. Simultaneously, the distribution and number of the nerves were comparable to those of adult rats. However, from days 0–24, the central portion of the disk (actual disk) did not contain any nerves. Conclusions These findings suggest that functional stimuli such as sucking and mastication might be associated with the development of nerves in the TMJ during the early and late postnatal days, whereas the actual disk except for both disk attachment and peripheral portion of the disk is not innervated and does not contribute to the afferent sensory information from the TMJ. The presence of peptidergic nerves prior to the appearance of synovial lining cells furthermore suggests that the neuropeptide released from these nerves may affect on the differenciation and maturation of synovial cells. © 1996 Wiley-Liss, Inc.

Postnatal development of protein gene product 9.5- and calcitonin gene-related peptide-like immunoreactive nerve fibers in the rat temporomandibular joint.

The aim of this study was to investigate the little-known postnatal development of innervation in the temporomandibular joint (TMJ). We therefore investigated the protein gene product 9.5-like immunoreactive (PGP-LI) and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerves in the rat TMJ from days 0-24 in wholemount preparations and frozen sections by immunohistochemistry using the avidin-biotin-peroxidase complex method. At birth, PGP-LI and CGRP-LI nerves were observed only in the disk attachment but not in the peripheral portion of the disk. From days 3-6, the peptidergic nerves associated with the development of synovial cells were first found in the anterior (3rd day) or posterior (6th day) peripheral portion of the disk in which synovial cells later appear. From days 7-10, incisal occlusion started and simultaneously the distinct networks of the nerves were formed in four peripheral portions of the disk. On the 14th day, the PGP-LI nerves were first more numerous than the CGRP-LI nerves only in the anterior disk attachment. From days 18-24, the occlusion between the first molars started. Simultaneously, the distribution and number of the nerves were comparable to those of adult rats. However, from days 0-24, the central portion of the disk (actual disk) did not contain any nerves. These findings suggest that functional stimuli such as sucking and mastication might be associated with the development of nerves in the TMJ during the early and late postnatal days, whereas the actual disk except for both disk attachment and peripheral portion of the disk is not innervated and does not contribute to the afferent sensory information from the TMJ. The presence of peptidergic nerves prior to the appearance of synovial lining cells furthermore suggests that the neuropeptide released from these nerves may affect on the differenciation and maturation of synovial cells.

NPY- and CGRP-like immunoreactive nerve fibers in the testis and mesorchium of the toad (Bufo arenarum)

The presence and distribution of peptidergic nerve fibers were studied in the testis and mesorchium of the toad by means of immunohistochemistry. Cryostat sections of the testis and whole-mount preparations of mesorchia were immunostained with antisera to calcitonin gene-related peptide (CGRP) and neuropeptide tyrosine (NPY). After leaving the mesorchium CGRP-immunoreactive (IR) fibers were seen predominantly running in between the seminiferous tubules. In addition, a small population of CGRP-IR nerve fibers formed thin plexuses around blood vessels. Conversely, NPY-like immunoreactivity predominated in nerve fibers that formed dense plexuses around vessels both in the mesorchium and testis. Additionally, some single NPY-IR nerve fibers could be seen in both structures studied. The functional significance of these peptidergic systems in the testis of the toad remains to be analyzed.

NPY- and CGRP-like immunoreactive nerve fibers in the testis and mesorchium of the toad ( Bufo arenarum )

NPY- and CGRP-like immunoreactive nerve fibers in the testis and mesorchium of the toad ( Bufo arenarum )

Innervation of the rat gastrointestinal sphincters: Changes during development and aging

The effect of age on the adrenergic and peptidergic innervation of the lower oesophageal, pyloric and ileocaecal sphincters of the rat was investigated using immunohistochemical techniques. The distribution of nerve fibres containing the neuronal protein, growth associated protein-43, was also studied to determine the integrity of the enteric nervous system during development and aging. The four age groups examined were 2–3 days, 6 weeks, 3 months and 25 months old rats.Using protein gene product 9.5 antibody (a non-specific general neuronal marker), it was revealed that the myenteric ganglia in all sphincter regions were compactly arranged and were smaller in size at neonatal stage getting more spaced out and larger in size with age. There was no obvious change in the structure of the neuronal elements with age. In the lower oesophageal sphincter, calcitonin gene-related peptide- and substance P-like immunoreactive nerve fibres showed notable changes in density and fluorescence intensity with age, decreasing and increasing, respectively, with no obvious change in vasoactive intestinal polypeptide- and growth-associated protein-like immunoreactivity. A slight increase in dopamine-β-hydroxylase-like immunoreactivity was seen in old age. In the pyloric sphincter, there was an increase in calcitonin gene-related peptide- and substance P-like immunoreactivity with a less notable increase in dopamine-β-hydroxylase-like immunoreactivity. A decrease in vasoactive intestinal polypeptide- and growth-associated protein-43-like immunoreactivity in the circular muscle of the sphincter was seen in old age. In the ileocaecal sphincter there was a marked increase in growth associated protein-43-, vasoactive intestinal polypeptide-, dopamine-β-hydroxylase and substance P-like immunoreactivity. There was a decrease in the density of calcitonin gene-related peptide-like immuno-reactive nerve fibres in old age.In summary, two main conclusions can be drawn from the results of the present study. First, there was an age-related differential change in the density of immunoreactive nerve fibres containing various neuroactive substances. This indicates a level of plasticity of the various enteric nerve types and may reflect the degree of importance of the different neurotransmitters in the physiological activities of the specific sphincter. Second, in all three sphincters of aged rats, the density of nerve fibres containing the markers for the excitatory neurotransmitters noradrenaline and substance P (although the precise role of substance P in ileocaecal sphincter is not known) was increased, while the density of nerve fibres containing the inhibitory neurotransmitters calcitonin gene-related peptide in the lower oesophageal and possibly ileocaecal sphincter, and vasoactive intestinal polypeptide in the pylorus were decreased. This is likely to underlie the physiological activity of the sphincters and may be associated with malfunction in old age. Investigations to determine the functional implications of these changes would be of great interest.

NADPH-diaphorase activity as a marker for nitric oxide synthase in neurons of the guinea pig respiratory tract.

Recent studies in physiology have suggested that part of the inhibitory nonadrenergic noncholinergic (iN-ANC) response of airway smooth muscle is mediated by nitric oxide (NO). To examine this point morphologically, the guinea pig respiratory tract was investigated histochemically for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (NADPH-d), a marker for NO synthase (NOS). In addition, coexpression of NOS and vasoactive intestinal peptide (VIP) or calcitonin gene-related peptide (CGRP) was studied using a combination of histochemistry for NADPH-d and immunohistochemistry for VIP or CGRP. Nerve fibers showing NADPH-d activity were abundantly observed in the respiratory tract. They were distributed throughout smooth-muscle bundles, lamina propria, submucosal glands, and around bronchial and pulmonary arteries. NADPH-d-containing nerve-cell bodies were occasionally found within airway ganglia. The colocalization study demonstrated that NADPH-d-containing nerve fibers frequently coincided with VIP-like immunoreactive nerve fibers but not with CGRP-like immunoreactive nerve fibers. Among nonneural tissues, NADPH-d activity was noticed in the endothelium of both bronchial and pulmonary vessels, and in the pleura. These observations indicated that NO may be produced by neurons and vascular endothelium of the guinea pig respiratory tract, and may function as a neuronal mediator as well as endothelium-derived relaxing factor (EDRF). Colocalization of NADPH-d and VIP-like immunoreactivity in nerve fibers suggested that NO and VIP may function as cotransmitters.

Distribution of SP- and CGRP-like immunoreactive nerve fibers in the lower respiratory tract of neonatal foals: evidence for loss during development.

The lungs of neonatal foals contain many nerves immunoreactive for substance P and calcitonin gene-related peptide. These nerves are closely associated with the epithelium, bronchial and pulmonary vessels and the airway smooth muscle of all intrathoracic airways, including non-cartilaginous bronchioles. Activation of sensory nerves in the respiratory epithelium could thus potentially affect, via local axon reflexes, vascular and respiratory smooth muscle in neonatal equine airways. Nerves immunoreactive for these peptides are much more widely distributed within the lung than in adult horses; they may thus play a trophic role before birth, or contribute to the post-natal adaptation to breathing.

Neurodegeneration in sweat glands and skin of aged rats

In order to compare age-associated neurodegenerative changes in peripheral nerves of laboratory mammals and humans, we have investigated the density and pattern of different nerve populations innervating sweat glands of ageing rats and compared our results with a previous study of the innervation of human sweat glands. We have also studied age-changes in subepidermal afferent nerves that may be involved in reflex activation of sweat glands. Total nerve density, measured by immunohistochemical staining for the general neuronal marker, protein gene product (PGP9.5) and image analysis, showed a significant decline around secretory coils of sweat glands of old compared to young rats. Marked reductions of acetylcholinesterase (AChE) histochemical staining and of vasoactive intestinal polypeptide (VIP)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity were observed in nerves around sweat glands. In the sub-epidermis, PGP- and CGRP-like immunoreactive nerves were significantly reduced in old rats. The age-related changes in sweat gland innervation of old rats were comparable to those reported in elderly human subjects suggesting that these tissues may provide a suitable model for experimental studies of neuronal ageing.

Response of calcitonin-gene-related peptide-like immunoreactive nerve fibers of the anterior pituitary to adrenalectomy in the rat.

The mammalian anterior pituitary is known to be regulated by humoral factors only, no direct neural influence having ever been discovered. Our recent studies have shown in several species that there are substantial amounts of substance P and calcitonin-gene-related peptide-like immunoreactive (CGRP-LI) nerve fibers in the anterior pituitary and that they can make synaptic contacts with secretory cells, among which corticotropes. In the present study, we investigated changes in CGRP-LI nerve fibers of the anterior pituitary after bilateral adrenalectomy in the rat. The results show that the number of CGRP-LI nerve fibers increases significantly. In parallel, they cover a wider area of the gland after the operation, suggesting a direct neural influence.

Distribution of substance P and calcitonin gene-related peptide-like immunoreactive nerve fibers in the rat temporomandibular joint.

The density and distribution of substance P-like immunoreactive (SP-LI) and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibers in rat temporomandibular joint (TMJ) were investigated in whole-mount preparations and frozen sections by immunohistochemistry with the avidin-biotin-peroxidase complex method. Both types of immunoreactive nerves were observed primarily in the joint capsule, the peripheral articular disc, the synovial membrane, and the periosteum. The distribution of CGRP-LI nerves was similar to that of SP-LI nerves. The anterior portion of the joint capsule and disc was most densely innervated, followed by the posterior, lateral, and medial portions. In addition, CGRP-LI nerves were more numerous and more dense in immuno-intensity than SP-LI nerves. In the synovial membrane, many SP- and CGRP-LI nerves terminated in the subsynovial layer, but some branches extended into the superficial synovial lining layer close to the joint cavity. Immunolabeled nerves were prominently located in the disc attachment and peripheral portion of the disc, and occasional nerves were located in the dense collagenous disc band as an actual disc. However, no fibers were detected in the central disc band. Thus, most of the disc was not innervated by any nerves. The present study provides a morphological basis for the possible roles of neuropeptides in endocytosis by synoviocytes, regulation of blood flow in the synovial membrane, nociception mechanisms of the TMJ, and modulation of the inflammatory response in the TMJ.

Neuropeptides in the intestine of two teleost species (Oreochromis mossambicus, Carassius auratus): Localization and electrophysiological effects on the epithelium

The presence of bioactive peptides in the gut and their possible electrophysiological effects on the intestinal epithelium were studied in two teleost species, the tilapia (Oreochromis mossambicus) and the goldfish (Carassius auratus). Vasoactive intestinal polypeptide-like immunoreactive nerve fibres were found beneath the intestinal epithelium of both species. Galanin-, metenkephalin-and calcitonin gene-related peptide-like immunoreactive nerve fibres were found exclusively in the mucosa of the tilapia. Both species had vasoactive intestinal polypeptide-, enkephalin- or neuropeptide Y-like immunoreactive endocrine cells; calcitonin gene-related peptide-like immunoreactive endocrine cells were additionally found in the tilapia. Somatostatin- and dopamine-β-hydroxylase-like immunoreactivities were not observed. Nerve cell bodies in the myenteric plexus of both species showed immunoreactivity for calcitonin gene-related peptide-, vasoactive intestinal polypeptide-, and galanin-like peptide. Enkephalin-like immunoreactive nerve cell bodies were present in the tilapia only. None of the peptides had a pronounced electrogenic effect. However, calcitonin gene-related peptide added to stripped intestinal epithelium of the tilapia, reduced the ion selectivity, and addition of galanin increased the ion selectivity. In goldfish intestine, both galanin and calcitonin gene-related peptide were without effect. Enkephalin counteracted the serotonin-induced reduction of the ion selectivity of the goldfish intestinal epithelium, but had no effect on the tilapia epithelium. In both species, vasoactive intestinal polypeptide reduced the ion selectivity of the intestinal epithelium, and neuropeptide Y induced an increase of the ion selectivity. Somatostatin showed no effect on the epithelial ion selectivity of either species. Tetrodotoxin did not inhibit the effects of the peptides studied. The changes in ion selectivity suggest that the enterocytes may be under the regulatory control of these peptides.

An Electron Microscopic Study on CGRP-like Immunoreactive Nerve Fibers in the Trigeminal and Superior Cervical Ganglia of Rats

An Electron Microscopic Study on CGRP-like Immunoreactive Nerve Fibers in the Trigeminal and Superior Cervical Ganglia of Rats

Calcitonin gene‐related peptide and corneal innervation: A developmental study in the rat

The development of calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerves was studied in neonatal and adult rat corneas stained immunohistochemically according to an avidin biotin peroxidase procedure. At birth, rat corneas already contained dense plexuses of CGRP-LI nerve fibers. Most of the nerves entered the cornea in 12-15 prominent stromal nerve bundles located at regular intervals around the circumference of the cornea. Fibers in these bundles entered the epithelium approximately midway between the limbus and the center of the cornea and supplied extensive central and pericentral areas of the tissue. In addition, smaller numbers of axons entered the cornea individually and in small fascicles located in between the larger bundles and supplied mainly peripheral territory. In the epithelium, the CGRP-LI nerves formed a complex, highly anastomotic meshwork that ramified uniformly throughout central and peripheral areas of the tissues. Fibers in the plexus gave origin to numerous short, stout terminal axons that extended into the adjacent epithelium in all directions with no preferred orientation. During the first week of neonatal life, several changes in CGRP-LI innervation occurred: 1) the innervation density of the central and pericentral cornea increased relative to the peripheral cornea; 2) intraepithelial axons became progressively longer, increased in branching complexity, and oriented preferentially towards the center of the cornea; and 3) a dense innervation of the corneoscleral limbus and, in particular, the branches of the marginal artery, developed. Midway through the second week of life, immature versions of corneal epithelial "leashes," the dominant feature of the adult corneal innervation, were first observed. Over the next 10 days, the leash formations in the central and pericentral cornea gradually became more complex and gave rise to greater numbers of terminal axons, compared to developing leashes in the peripheral cornea. The mature pattern of corneal CGRP-LI innervation was reached on day 21 and remained constant (except for compensatory growth-related elongation of axons) for at least the first 6 months of life. Transection of the ophthalmomaxillary nerve or neonatal administration of the sensory neurotoxin capsaicin resulted in the total loss of CGRP-LI staining from the cornea. In contrast, removal of the superior cervical ganglion had no effect on corneal CGRP-LI staining. The extraordinary density and complexity of the CGRP-LI innervation of the rat cornea demonstrated at all stages of development in this study suggests that these nerves may play important roles in corneal sensory, reflex, and trophic functions.

Calcitonin gene-related peptide-like immunoreactive nerve endings in the rat knee joint

Calcitonin gene-related peptide-like immunoreactive nerve endings in the rat knee joint