CGRP-IR Axons Research Articles

Distribution and morphology of calcitonin gene-related peptide (CGRP) innervation in flat mounts of whole rat atria and ventricles

Calcitonin gene-related peptide (CGRP) is widely used as a marker for nociceptive afferent axons. However, the distribution of CGRP-IR axons has not been fully determined in the whole rat heart. Immunohistochemically labeled flat-mounts of the right and left atria and ventricles, and the interventricular septum (IVS) in rats for CGRP were assessed with a Zeiss imager to generate complete montages of the entire atria, ventricles, and septum, and a confocal microscope was used to acquire detailed images of selected regions. We found that 1) CGRP-IR axons extensively innervated all regions of the atrial walls including the sinoatrial node region, auricles, atrioventricular node region, superior/inferior vena cava, left pre-caval vein, and pulmonary veins. 2) CGRP-IR axons formed varicose terminals around individual neurons in some cardiac ganglia but passed through other ganglia without making appositions with cardiac neurons. 3) Varicose CGRP-IR axons innervated the walls of blood vessels. 4) CGRP-IR axons extensively innervated the right/left ventricular walls and IVS. Our data shows the rather ubiquitous distribution of CGRP-IR axons in the whole rat heart at single-cell/axon/varicosity resolution for the first time. This study lays the foundation for future studies to quantify the differences in CGRP-IR axon innervation between sexes, disease models, and species.

Organization and morphology of calcitonin gene-related peptide-immunoreactive axons in the whole mouse stomach.

Nociceptive afferent axons innervate the stomach and send signals to the brain and spinal cord. Peripheral nociceptive afferents can be detected with a variety of markers (e.g., substance P [SP] and calcitonin gene-related peptide [CGRP]). We recently examined the topographical organization and morphology of SP-immunoreactive (SP-IR) axons in the whole mouse stomach muscular layer. However, the distribution and morphological structure of CGRP-IR axons remain unclear. We used immunohistochemistry labeling and applied a combination of imaging techniques, including confocal and Zeiss Imager M2 microscopy, Neurolucida 360 tracing, and integration of axon tracing data into a 3D stomach scaffold to characterize CGRP-IR axons and terminals in the whole mouse stomach muscular layers. We found that: (1) CGRP-IR axons formed extensive terminal networks in both ventral and dorsal stomachs. (2) CGRP-IR axons densely innervated the blood vessels. (3) CGRP-IR axons ran in parallel with the longitudinal and circular muscles. Some axons ran at angles through the muscular layers. (4) They also formed varicose terminal contacts with individual myenteric ganglion neurons. (5) CGRP-IR occurred in DiI-labeled gastric-projecting neurons in the dorsal root and vagal nodose ganglia, indicating CGRP-IR axons were visceral afferent axons. (6) CGRP-IR axons did not colocalize with tyrosine hydroxylase or vesicular acetylcholine transporter axons in the stomach, indicating CGRP-IR axons were not visceral efferent axons. (7) CGRP-IR axons were traced and integrated into a 3D stomach scaffold. For the first time, we provided a topographical distribution map of CGRP-IR axon innervation of the whole stomach muscular layers at the cellular/axonal/varicosity scale.

Topographical Distribution and Morphology of CGRP‐IR Axons in the Flat‐Mounts of Whole Mouse and Rat Hearts

To understand and treat cardiac pain, it is important to have an understanding of the topographical distribution of nociceptive axons in the heart. While previous studies have detected nociceptive axons in the sectioned heart, their full distribution has not yet been determined. In this study, we used a nociceptive marker calcitonin gene‐related peptide (CGRP) to determine the distribution of nociceptive afferent axons in the whole heart. We first removed the hearts of male Sprague‐Dawley rats (n=6), and C57BL/6 mice (n=6). The hearts were dissected into left and right atria and ventricles and interventricular septum, and prepared as flat‐mounts. These samples were then processed with immunohistochemical labeling using a primary antibody which binds to CGRP and an Alexa Fluro 488 secondary antibody for fluorescence. The flat‐mounts were imaged with a Zeiss M2 Imager (automatic fluorescence microscope) using a 488 nm excitation wavelength. In both rats and mice, we found that CGRP‐IR nerve bundles entered the heart and innervated the left and right atria and ventricles. For the right/left atria, CGRP‐IR axons entered as large bundles near the superior/inferior vena cava, left pre‐caval vein and the pulmonary veins before bifurcating into small branches, and finally formed single varicose axons and terminals which distributed throughout the tissue, including cardiac ganglia, the SA/AV nodes, auricles, and the blood vessels. In the right/left ventricles and interventricular septum, CGRP‐IR axons entered as large bundles through the base before bifurcating into small branches towards the apex Ultimately, single varicose axons and terminals innervated myocardium and blood vessels. Our data shows the distribution of CGRP‐IR axons in the whole heart at single cell/axon resolution. With this information, we will gain insight into the function of these axons and the pathways that they follow, paving the way for the development of better treatments for cardiac pain and disease. The first two authors contributed equally to this work.

Quantitative immunohistochemical co-localization of TRPV1 and CGRP in varicose axons of the murine oesophagus, stomach and colorectum

In the gastrointestinal (GI) tract of mammals, endings of spinal afferent neurons with cell bodies in dorsal root ganglia (DRG) detect many stimuli, including those that give rise to pain. Many of these sensory neurons express calcitonin gene-related peptide (CGRP) and TRPV1 in their cell bodies and axons. Indeed, CGRP and TRPV1 have been widely used as immunohistochemical markers of nociceptive spinal afferent axons. Although CGRP and TRPV1 often coexist in the same axons in the GI tract, their degree of coexistence along its length has yet to be quantified. In this study, we used double-labeling immunohistochemistry to quantify the coexistence of CGRP and TRPV1 in varicose axons of the murine oesophagus, stomach and colorectum. The great majority of CGRP-immunoreactive (IR) varicosities in myenteric ganglia of the lower esophagus (97±1%) and stomach (95±1%) were also TRPV1-immunoreactive. Similarly, the majority of TRPV1-IR varicosities in myenteric ganglia of the lower esophagus (95±1%) and stomach (91±1%) were also CGRP-IR. In the colorectum similar observations were made for an intensely immunoreactive population of CGRP-IR axons, of which most (91±1%) were also TRPV1-IR. Of the TRPV1-IR axons in the colorectum, most (96±1%) contained intense CGRP-IR. Another population of axons in myenteric ganglia of the colorectum had low intensity CGRP immunoreactivity; these showed negligible co-existence with TRPV1. Our observations reveal that in the myenteric plexus of murine oesophagus, stomach and colorectum, CGRP and TRPV1 are largely expressed together.

Distribution and morphology of calcitonin gene-related peptide and substance P immunoreactive axons in the whole-mount atria of mice.

The murine model has been used to investigate the role of cardiac sensory axons in various disease states. However, the distribution and morphological structures of cardiac nociceptive axons in normal murine tissues have not yet been well characterized. In this study, whole-mount atria from FVB mice were processed with calcitonin gene-related peptide (CGRP) and substance P (SP) primary antibodies followed by secondary antibodies, and then examined using confocal microscopy. We found: 1) Large CGRP-IR axon bundles entered the atria with the major veins, and these large bundles bifurcated into small bundles and single axons that formed terminal end-nets and free endings in the epicardium. Varicose CGRP-IR axons had close contacts with muscle fibers, and some CGRP-IR axons formed varicosities around principle neurons (PNs) within intrinsic cardiac ganglia (ICGs). 2) SP-IR axons also were found in the same regions of the atria, attached to veins, and within cardiac ganglia. Similar to CGRP-IR axons, these SP-IR axons formed terminal end-nets and free endings in the atrial epicardium and myocardium. Within ICGs, SP-IR axons formed varicose endings around PNs. However, SP-IR nerve fibers were less abundant than CGRP-IR fibers in the atria. 3) None of the PNs were CGRP-IR or SP-IR. 4) CGRP-IR and SP-IR often colocalized in terminal varicosities around PNs. Collectively, our data document the distribution pattern and morphology of CGRP-IR and SP-IR axons and terminals in different regions of the atria. This knowledge provides useful information for CGRP-IR and SP-IR axons that can be referred to in future studies of pathological remodeling.

Alterations in mystacial pad innervation in the aged rat.

It is well established that sensory perception becomes impaired with advancing age and that, in parallel, dystrophy and degeneration of axons occur in sensory pathways. In this study, the impact of aging was examined in the mystacial pad, which receives a large variety of sensory nerve endings organized in a highly predictable pattern. Mystacial pad specimens from aged (30 months old) and young adult (2-3 months old) female Sprague-Dawley rats were processed, in parallel, for immunohistochemical analyses with antibodies against human neuronal cytoplasmic protein (protein gene product 9.5), transmitter enzymes, and several neuropeptides. Several changes in cutaneous innervation including both degenerative and regenerative processes were evident in the aged rat: (1) the Merkel endings and lanceolate endings that emanate from large-caliber afferents in the whisker follicles were reduced and showed signs of degeneration. Furthermore, a reduction of piloneural complexes at the intervibrissal hairs were evident, but only in aged rats that showed more severe behavioral sensorimotor disturbances. In contrast, Ruffini endings as well as mechanoreceptors emanating from medium-caliber axons, i.e., transverse lanceolate and reticular endings, appeared normal. (2) A reduction was evident among two sets of unmyelinated epidermal endings; however, the epidermal innervation affiliated with the intervibrissal hairs appeared normal in the aged rat. (3) A loss of sympathetic neuropeptide tyrosine (NPY) or tyrosine hydroxylase-immunoreactive (IR) and somatosensory Calcitonin gene-related peptide (CGRP)-IR perivascular axons was paralleled by an increase in presumed parasympathetic NPY/CGRP-IR axons. (4) Two "novel" networks of fine-caliber axons were observed in the outer and inner root sheaths of the whisker follicles in the aged rat. (5) NPY was present in a population of small-caliber, somatosensory CGRP-IR axons in the aged rat. This may represent a de novo synthesis, since, normally, NPY-like immunoreactivity is not observed in this set of axons. Our results suggest that the sensory impairments occurring with advancing age are part of a peripheral process instigated by changes in nerve-target interactions and/or incapacitation of the neuronal machinery to sustain the axonal integrity.

Efferent targets of osseous CGRP-immunoreactive nerve fiber before and after bone destruction in adjuvant arthritic rat: an ultramorphological study on their terminal-target relations.

We report the ultramorphological characterization of the terminal-target relation of sensory peptidergic nerve fibers in healthy and diseased osseous tissues. Bone tissue sections were immunoelectronmicroscopically investigated for calcitonin gene-related peptide (CGRP), a neuropeptide widely distributed in sensory peptidergic fibers. Ultramorphological relation of the osseous CGRP-immunoreactive (ir) nerve terminals and their target cells was comparatively analyzed using healthy, arthritic, and postarthritic bone specimens from control and adjuvant-induced arthritic rats. Terminal-like profiles of the osseous CGRP-ir axons were evidenced in direct contact with the metaphyseal osteoblasts and osteoclasts of the control animals. Terminal-like profiles were also noted in the vicinity of the periosteal lining cells. Nonterminal-like profiles did not make intimate spatial relation to the cells/structures surrounding the nerve. Osseous CGRP-ir terminals and axons, which are either uncovered or thinly ensheathed by the supportive tissues, were extensively degenerated in adjuvant-induced infiltration, whereas larger fibers were relatively resistant. Numerous CGRP-ir axons with distinctive features reinnervated the postarthritic, ossifying periosteum. CGRP-ir axons appeared to reinnervate the eroded surface of metaphyseal bone and cartilage as early as the recruited osteoblasts resume osteogenesis in the postarthritic metaphysis. The observed terminal-target relations in the healthy and diseased bone tissues give an ultramorphological basis for the putative trophic, modulatory actions of CGRP innervation of the bone cells.

Innervation of two peptidergic (substance P and calcitonin gene-related peptide) nerves in the cerebral arteries and choroid plexus of the Japanese Newt (Triturus pyrrhogaster).

The pattern of cerebrovascular substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive (-IR) innervation was investigated in the newt. SP-IR nerves supplying the cerebral arterial tree and choroid plexus were positive for CGRP, but negative for vasoactive intestinal polypeptide or neuropeptide Y. It is suggested that cerebrovascular SP- and CGRP-IR axons are sensory in nature. The supply of SP- and CGRP-IR nerves to the major cerebral arteries is relatively poor. Nevertheless, numerous SP- and CGRP-IR axons, which are contained in the fiber bundles on the cerebral carotid artery and the basilar artery, spread widely over the microvascular-epithelial regions of the choroid plexuses. It must be considered in relation to the significant role of SP- and CGRP-IR neuronal mechanisms responsible to the microcirculation, cerebrospinal fluid (CSF) production and transport action within the choroid plexus in the nutrition of the newt brain via the CSF.

Calcitonin gene-related peptide- and substance P-immunoreactive axons in the nucleus gracilis of the rat with special reference to axonal dystrophy: light and electron microscopic observations.

Calcitonin gene-related peptide (CGRP) and substance P (SP)-immunoreactive (IR) axons in the nucleus gracilis of normal rats (1-15 months of age) were studied by light and electron microscopy. Besides many CGRP-IR and SP-IR varicosities with normal appearance, we found a few swollen (nearly round or oval) varicosities with either CGRP or SP immunoreactivity. Swollen CGRP-IR varicosities were more frequently seen than SP-IR ones, appearing from 3 months of age and increasing in number and size (up to approximately 25 microns in diameter) with advancing age. At the electron microscopic (EM) level, CGRP-IR and SP-IR swollen varicosities showed dystrophic changes, i.e., many membranous dense bodies, and proliferation of microtubules and neurofilaments. CGRP-IR or SP-IR dystrophic axons also contained many mitochondria and sometimes made synaptic contacts with nonreactive dendrites (occasionally with non-IR axons). These findings suggest that the dystrophic CGRP and SP axonal profiles represent a functionally distinct subpopulation of axonal dystrophy in the nucleus gracilis and use CGRP or SP as a neuroactive substance. Using a double-immunostaining method, many of normal CGRP-IR axons were identified to be SP-IR. However, no single dystrophic varicosity was found to contain both CGRP and SP immunoreactivities. These findings suggest that CGRP and SP afferents are independently affected and progress to dystrophic changes.

The distribution and colocalization of neuropeptides in fish cardiac neurons

Most if not all intracardiac nerve cell bodies in four species of teleost fish and a primitive air breathing fish contained immunoreactivity (IR) to vasoactive intestinal peptide (VIP). Intracardiac nerve cell bodies contained no other neuropeptide although galanin (GAL)-, substance P (SP)- and calcitonin gene-related peptide (CGRP)-IR were detected in cardiac axons. Varicose VIP-IR axons were observed in close association to the cardiac muscle in the sinus venosus and atrium, but not in the ventricle. Slightly less than half the total number of VIP-IR axons also contained colocalised GAL-IR. A smaller number of varicose axons containing colocalised SP- and CGRP-IR were also present in the sinus venosus and atrium. In addition, a subpopulation of CGRP-IR axons present in the sinus venosus and atrium did not contain SP-IR. SP-IR axons lacking CGRP-IR formed boutons around the axon hillock and soma of the majority of VIP-IR nerve cell bodies. Associated with a small number of VIP/- ganglion cells were VIP/- boutons. No neuropeptides were observed in the ventricle of any species of fish studied here. These results suggest that a VIP-like peptide is localised in the cholinergic postganglionic parasympathetic neurons. Associated with some of these neurons are nerve boutons containing either SP alone or VIP alone. In addition, the fish heart is innervated by extrinsic nerve fibres containing: GAL/VIP; CGRP alone; and CGRP/SP.

Bridging a complete transection lesion of adult rat spinal cord with growth factor-treated nitrocellulose implants.

The ability of a substrate bound neurotrophic factor to promote growth of ascending sensory axons across a complete transection lesion of the rat spinal cord was examined in a transplantation model. Aspiration lesions created a 3 mm long cavity in the upper lumbar spinal cord of adult rats. Five weeks after injury two strips of nerve growth factortreated nitrocellulose, were implanted, each in a medio-lateral position, and apposed to the rostral and caudal surfaces of the cavity. Control animals received untreated nitrocellulose implants. Fetal spinal cord tissue was transplanted alongsideand between these strips. Six weeks post transplantation, animals were sacrificed and vibratome sections through the grafts were processed for immunocytochemical demonstration of ingrowing axons expressing calcitonin gene-related peptide (CGRP-IR), Immunolabeled axons were abundant at the caudal interface between host tissue and the NGF-treated nitrocellulose implants, with dense fascicles of fibers abutting the grafts. As the distance from the caudal surface increased some CGRP-IR fibers extended into the fetal tissue although most appeared to remain oriented in a longitudinal course adjacent to the nitrocellulose. Labeled axons were evident along the entire length of the nitrocellulose and appeared to aggregate at the rostral tip of the implant, with many fibers extending into the host spinal cord rostral to the lesion/transplant site. When untreated nitrocellulose was implanted, fewer labeled axons appeared to extend beyond the caudal host-graft interface. Most CGRP-IR axons displayed limited association or contact with the untreated nitrocellulose in this condition. Computer-assisted quantitative analysis indicated that NGF-treated nitrocellulose supported regrowing host axons for nearly three times the length exhibited by axons associated with non-treated nitrocellulose implants. These results indicate that substrate bound nerve growth factor has the capacity to enhance the regrowth of ascending sensory axons across a traumatic spinal cord injury site. The potential to reestablish functional contacts across such a lesion may be heightened by the ability of neurotrophic factors to promote more extensive axonal regrowth.

Analysis of low affinity nerve growth factor receptor during pulpal healing and regeneration of myelinated and unmyelinated axons in replanted teeth.

Nerve regeneration was examined in rat molars that were briefly extracted and then replanted in the socket for 1-90 days. Immunocytochemistry was used to evaluate neural and nonneural immunoreactivity (IR) for low affinity nerve growth factor receptor (p75-NGFR) and for laminin and calcitonin gene-related peptide (CGRP). Three different types of pulpal response to replantation were found. Type I: Some replanted teeth had mild injury and still contained coronal odontoblasts and associated fibroblasts that retained p75-NGFR-IR; they continued regular dentin formation and had excellent reinnervation. Type II: Teeth with intermediate injury lost most or all of the coronal pulp tissue, but they regenerated odontoblast-like cells that formed irregular dentin, they had numerous dispersed p75-NGFR-IR fibroblasts in crown pulp during early regeneration, and they had excellent reinnervation. Type III: Severely injured teeth lost their original pulp; they filled with dense connective tissue and bone and had poor reinnervation. After Type I or II injury the Schwann cells around degenerating myelinated and unmyelinated axons had increased expression of p75-NGFR by 1-3 days. By 7-10 days those Schwann cells had formed hollow tubes (bands of Bungner) along the degenerating axon tracks. They maintained their increased p75-NGFR-IR during and after regeneration of unmyelinated axons, whereas Schwann cells involved in remyelination lost p75-NGFR-IR at that stage. The number of CGRP-IR axons in the regenerating pulp increased from 7 to 90 days. Laminin-IR increased in all replanted teeth at 3-10 days and only returned to normal patterns in teeth with Type I or Type II response at 20-90 days. The special p75-NGFR-IR of pulpal fibroblasts of adult rat molars did not usually persist in regenerated, reinnervated pulp. The extensive depletion of fibroblast p75-NGFR-IR and the continuing enhanced p75-NGFR-IR in unmyelinated nerve fibers at 90 days show that altered growth factor conditions characterize regenerated pulp of replanted teeth.

Peripheral patterns of calcitonin‐gene‐related peptide general somatic sensory innervation: Cutaneous and deep terminations

The distribution of calcitonin-gene-related peptide (CGRP) immunoreactivity (IR) was studied in peripheral tissues of rats. The ganglionic origin, somatosensory nature, and anatomic relations of this thin-axon population were evaluated with particular emphasis on possible nociceptive roles. In animals untreated with colchicine, CGRP-IR is found in a vast proportion of small- and medium-diameter sensory ganglion cells that give rise to numerous thinly myelinated and unmyelinated axons that display CGRP-IR throughout the body. The integumentary innervation consists, in part, of an extensive subpapillary network largely traced to dermal blood vessels, sweat glands, and "free" nerve endings, some of which are found within regions containing only mast cells, fibroblasts, and collagen. Dermal papillae contain CGRP-IR axons surrounding each vascular loop; other papillary axons end freely or occasionally surround Meissner corpuscles. Intraepithelial axons enter glabrous epidermal pegs, branching and exhibiting terminals throughout the stratum spinosum. A similar pattern is found in hairy skin with additional innervation entering the base and surrounding the lower third of each hair follicle, but apparently not supplying sebaceous glands and arrector pili muscle. Axons innervating nonkeratinized oral epithelium are similar or greater in number and distribution compared to epidermis, often with more extensive branching. The high density of intraepithelial CGRP-IR innervation does not appear to correlate with the sensitive mechanoreceptor-based increase in spatial sensory discriminative capacities in the distal portions of the limb. In deep somatic tissues, CGRP-IR is principally related to vasculature and motor end plates of striated muscle, but there is an extensive network of thin axons within bone, principally in the periosteum, and focally in joint capsules, but not in relation to muscle spindles or tendon organs. These findings, together with the distribution in cranial tissues described in an accompanying paper (Silverman and Kruger: J. Comp. Neurol. 280:303-330, '89), are considered in the context of a "noceffector" concept incorporating the efferent role of these sensory axons in various tissues. It is suggested that involvement in tissue maintenance and renewal during normal function, as well as following injury, may predominate over the relatively infrequent nociceptive role of this peptidergic sensory system.

Distribution of calcitonin gene-related peptide immunoreactivity in relation to the rat central somatosensory projection.

The distribution of the neuropeptide calcitonin gene-related peptide (CGRP) was studied in relation to the known subcortical somatosensory pathways and contiguous systems in the central nervous system (CNS) of rats by using peroxidase histochemical methods in order to relate zones of immunoreactivity (IR) to cytoarchitecture. CGRP is the most ubiquitous peptide found to date in sensory ganglion cells: principally small and medium-size neurons emitting thin axons inferred to be largely nociceptive in function on the basis of the peripheral distribution of their terminals. Its apparent absence in sympathetic axons provides an especially useful sensory marker. The distribution of CGRP-IR axons displays remarkable selectivity at each level of the CNS. The trigeminal root distributes axons primarily to the pericornual layers (laminae I and II) of spinal V nucleus caudalis and to subnucleus oralis, evading the subnucleus interpolaris and contributing only few axons to principal V. Although there are only a few CGRP-IR somata at each level, heavily labeled axon trajectories can be traced to the nuclei of the solitary tract, the parabrachial nuclei, several sectors of the caudal medial thalamus, and the central nucleus of the amygdala. A sector of labeled neuron somata lies contiguous to each of these axon terminal zones, the largest of which is a thalamic nucleus containing cells of distinctive dendritic architecture extending from the periaqueductal gray across the posterior group nuclei to the peripeduncular nucleus, forming a linear array at the mesodiencephalic junction. The relation of CGRP-IR axonal distribution to spinothalamic, visceral, and gustatory systems is discussed in the context of a specialized "chemosensory" component of the thin-fiber somatosensory system.

Two immunohistochemically identified populations of calcitonin gene-related peptide (CGRP)-immunoreactive axons in human skin

Double labelling immunofluorescence has shown two populations of unmyelinated sensory axons in human skin that contain immunoreactivity (IR) to calcitonin gene-related peptide (CGRP). One population also contains IR to substance P (SP), whilst the other also contains IR to somatostatin (SOM). Axons containing both CGRP-IR and SOM-IR comprised more than 75% of CGRP-IR axons associated with the epidermis; the rest of the CGRP-IR axons contained SP-IR. No axons contained both SP-IR and SOM-IR. Some dermal blood vessels were surrounded by axons containing both CGRP-IR and SP-IR, but most CGRP-IR perivascular axons contained SOM-IR without SP-IR. Sweat glands were well supplied with sensory axons containing CGRP-IR and weak SOM-IR but not SP-IR. Therefore, CGRP is a histochemical marker for a larger number of unmyelinated cutaneous afferents in human skin than is SP. CGRP itself may have a role in the mediation of responses to stimulation of at least two populations of sensory axons.