Distribution of calcitonin gene-related peptide-like immunoreactivity in the guinea pig heart

Summary The distribution of calcitonin gene-related peptide like-immunoreactivity (CGRP-IR) within the heart and adjacent blood vessels of the guinea pig was investigated anmunohistochemically by use of the peroxidase-antiperoxidase (PAP) technique. Numerous paravascular and perivascular immunoreactive nerve fibers were localized around the aorta, coronary arteries and their branches down to the teminal vasculature. Arterioles in the atria showed greatest density of immunoreactive varicosities of all blood vessels. The epicardium, endocardium and the conductive system also contained numerous CGRP-IR nerve fibers. In the muocardium the number of immunoreactive varicosities was variable. Many were present in both atria, moderate amounts were seen in the right ventricle and parts of the intraventricular septum, and only a few occurred in the left ventricle. CGRP-IR was infrequently found within intracardial ganglionic cells but was abundantly distributed in the surrounding nerve fibers.Supported by the German Research Foundation (DFG, SFB 90)     

Innervation of the dura mater encephali of cat and rat: ultrastructure and calcitonin gene-related peptide-like and substance P-like immunoreactivity

Ultrastructural, immunocytochemical, and immunoelectron microscopical examinations are reported that describe the morphology of putative sensory nerve endings in the dura mater encephali of the rat and the cat. Morphometrical measurements and reconstructions showed that in the cat the mean diameter of axons, the bare area of axolemma, and the content of mitochondria and vesicles are highly variable in dural nerve endings. Nerve fibers with a high volume density of mitochondria are thought to be sensory, while nerve fibers containing many small vesicles are considered autonomic. There is, however, a broad overlap of mitochondria-rich and vesicle-rich nerve fibers in the dura, so that discrimination between sensory and autonomic endings by these characteristics frequently fails. Whole-mount preparations treated cytochemically for detection of substance P- and calcitonin gene-related peptide-like immunoreactivity in the rat and the cat showed a network of immunopositive nerve fibers in the vicinity of dural blood vessels. Most of these peptidergic and probably sensory nerve fibers were found terminating in the dural connective tissue far from vessels. Calcitonin gene-related peptide-positive nerve fibers were much more abundant than substance P-positive fibers. Immunoelectron microscopic preparations revealed that calcitonin gene-related peptide- and substance P-like immunoreactivity is found in a small proportion of generally thin unmyelinated nerve fibers. These proportions were very similar in the rat and the cat. Summarizing the recent literature, the morphological characteristics of putative sensory nerve fibers in the dura mater are discussed in relation to their possible functional significance for neurogenic inflammation and nociception.     

Ontogeny of acetylcholinesterase, substance P and calcitonin gene-related peptide-like immunoreactivity in chick dorsal root ganglia.

The distribution of acetylcholinesterase and of two neuropeptide (substance P and calcitonin gene-related peptide) immunoreactivities has been investigated in sensory neurons of lumbosacral dorsal root ganglia during chick embryo development, combining immunolocalization of neuropeptides with simultaneous histochemical detection of acetylcholinesterase, in order to study co-localization of the two peptides and their relations with acetylcholinesterase. Acetylcholinesterase at E7 of development appears in only a few neurons, usually the larger ones located in the lateroventral region of the ganglia. As development proceeds the number of neurons and intensity of staining increase. Until E12-13 acetylcholinesterase positivity is limited to the region of the ganglion containing larger neurons. At later stages (E20) it spreads progressively, leading to staining of cells over the whole ganglion. Substance P-like immunoreactivity appears at E6 and for calcitonin gene-related peptide at E7. These immunoreactivities progressively increase with development, remaining limited to the small neuron compartment of the dorsomedial region of the ganglion. Immunoreactivity for both neuropeptides reaches a maximum around E10-13 and then declines. Using simultaneous double immunostaining, calcitonin gene-related peptide and substance P-like immunoreactivities are largely co-localized, although their distribution is not completely coincident. Neuropeptide-positive cells are usually devoid of any acetylcholinesterase activity until E15. They become positive for the enzyme at later stages. The significance of acetylcholinesterase expression in sensory neurons and the possible relation of its appearance and neuron size is discussed.     

Distribution of substance P-immunoreactive and calcitonin gene-related peptide-immunoreactive nerves in normal human lungs.

The distribution of substance P (SP)-immunoreactive (IR) and calcitonin gene-related peptide (CGRP)-IR nerves in normal human lungs was immunohistochemically investigated. SP-IR and CGRP-IR nerves were found in lamina propria and adjacent to blood vessels, and also in some of the ganglia and nerve bundles in the submucosal area. Comparison of serial consecutive sections revealed that SP-IR and CGRP-IR nerves represented similar distribution patterns. CGRP-IR nerves were scarce in bronchial smooth muscles, whereas SP-IR nerves were always present therein. Neither SP-IR nor CGRP-IR nerves were found, however, among submucosal glands or in alveolar septa. These localization patterns were closely related to the functions of SP and CGRP in normal lungs.     

Co-localization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs

Calcitonin gene-related peptide (CGRP) has been immunohistochemically co-localized with substance P (SP) in capsaicin-sensitive, varicose axons supplying the skin, viscera and cardiovascular system of the guinea pig. After treatment with colchicine in vitro, 82% of SP neurons in the dorsal root ganglia contained CGRP-like immunoreactivity while 96% of CGRP neurons were immunoreactive for SP. Both CGRP- and SP-like immunoreactive material are transported peripherally and centrally from dorsal root ganglia. Thus, in tissues such as the gut where there are intrinsic nerves containing SP but lacking CGRP, CGRP-like immunoreactivity is a useful means of specifically labelling axons of most sensory neurons containing SP.     

Regional differences in the distribution of nerve fibers showing substance P- and calcitonin gene-related peptide-like immunoreactivity in the rat larynx

Summary The distribution of substance P (SP) — and calcitonin gene-related peptide (CGRP) — containing nerve fibers in the rat larynx was studied using immunohistochemistry. Double-labeling studies revealed a high degree of co-existence of SP- and CGRP-like immunoreactivity (LI) in the nerve fibers in the larynx. There was a considerable regional difference in the number of immunoreactive nerve fibers in the epithelium and lamina propria. Richly supplied sites were the laryngeal side of the epiglottis and the ventral recess, whilst there was no evidence of nerve fibers in the squamous epithelium of the vocal cords. However, where the squamous epithelium of the vocal cords changed into a cuboidal epithelium, a moderate number of nerve fibers was present, and a large number of fibers was seen where the squamous epithelium of the cords was in close contact with cartilage. Nerve fibers showing SP- and CGRP-LI were also observed close to the acini and ducts of the glands, in the blood vessel walls, close to the perichondrium of all the cartilages, and outside the cricothyroid and cricoarytenoid joints. CGRP-LI was detected in epithelial cells facing the lumen of the airway and in cells in the acini and ducts of glands in the subglottic region and trachea. Unilateral sympathectomy did not affect the pattern of SP- and CGRP-innervation in the larynx, whereas after vagotomy, the SP- and CGRP-innervation almost disappeared ipsilaterally in the upper parts of the epiglottis and aryepiglottic folds.     

Reduced levels of calcitonin gene-related peptide-like immunoreactivity in human brain vessels after subarachnoid haemorrhage

Human cerebral vessels were found to contain calcitonin gene-related peptide (CGRP)-like immunoreactivity (-LI) which in the reversed phase HPLC co-eluted with authentic human alpha-CGRP. The level was significantly lower in arteries removed from patients who had died from a subarachnoid haemorrhage (SAH) as compared to patients who died of a coronary infarction. On a molar basis human alpha-CGRP was more potent than human beta-CGRP to dilate human brain vessels and to dilate vasoconstriction elicited by whole blood. It is suggested that the trigemino-cerebrovascular system storing CGRP-LI may be involved in the pathophysiology of SAH in man.     

Immunohistochemical analysis of the ontogeny of calcitonin gene-related peptide-like immunoreactivity in the rat central nervous system

The ontogeny of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the rat central nervous system was examined using the indirect immunofluorescence technique. Caudally located neurons displayed CGRP-LI relatively early in development. For instance, CGRP-immunoreactive (CGRP-IR) cells were first visualized in the hypoglossal nucleus and dorsal nucleus of the lateral lemniscus at day 19 of gestation, while by day 1 postnatal the facial and ambiguus nuclei possessed their full complement of immunoreactive neurons relative to that observed in the adult. By contrast, CGRP-IR somata in more rostral regions such as the lateral septal nucleus were not visualized until much later in development, i.e. day 12 postnatal. By day 15 postnatal, however, CGRP-LI was at a maximum in almost all areas scrutinized. This pattern of immunostaining persisted up to the oldest age studied (day 28 postnatal), except in the lateral septal and parabrachial nuclei, along the dorsal border of the medial lemniscus, and in the inferior olive, where many of the previously immunopositive cells could no longer be detected. In summary, CGRP-IR somata in the rat central nervous system appear in a principally caudal to rostal direction, beginning in late gestation. In some regions, CGRP is present only ephemerally. The ontogeny of CGRP during maturation of the rat brain is consistent with its suggested neurotrophic and neurotransmitter roles.     

Increased intracellular levels of calcitonin gene-related peptide-like immunoreactivity in pulmonary endocrine cells of hypoxic rats

The mammalian respiratory tract contains innervated groups of endocrine cells which are believed to respond to hypoxia. We have demonstrated the involvement of a specific regulatory peptide produced by the cells, calcitonin gene-related peptide (CGRP), in this response. Cells immunoreactive for CGRP or for protein gene product 9.5 (PGP 9.5), a general marker of nerves and endocrine cells, were quantified in sections of lungs from hypoxic (21 days, 10 per cent O2) and normoxic rats. An immunostaining method employing supra-optimal dilutions of primary antiserum was used. This detects variations in antigen concentration which may be masked if the routine, optimal dilution is used. The number of CGRP-immunoreactive endocrine cells was significantly (P less than 0.001) greater in the lungs of hypoxic rats (76.9 +/- 10.1 cells/cm2, mean +/- SEM) compared with controls (19.7 +/- 2.4). However, the numbers of PGP 9.5-immunoreactive cells were the same in both groups (81.3 +/- 12.2, hypoxic; 79.5 +/- 9.8 control), suggesting that the total number of endocrine cells did not change. It is concluded therefore that the apparent increase in CGRP-immunoreactive endocrine cells in hypoxic rat lungs is due to increased intracellular levels of the peptide. Since CGRP is a vasodilator, this could have important implications in the vasoconstrictor response to hypoxia.     

Cell type and conduction velocity of rat primary sensory neurons with calcitonin gene-related peptide-like immunoreactivity

An immunocytochemical double labelling study of L4 dorsal root ganglia from rats aged seven to 10 weeks was made with an antibody to calcitonin gene-related peptide and with RT97, an anti-neurofilament antibody which specifically labels the light neuron population. Peptide immunoreactivity was found in an average of 46.5% of all neurons. Sixty-two per cent of the small dark (RT97-negative) and 30% of the light (RT97-positive) neuron populations contained the peptide. About one-third (32%) of the cells with peptide immunoreactivity were light cells and about two-thirds (68%) were small dark cells. Intracellular electrophysiological recordings were made in vitro from neurons in lumbar (L4, L5 and L6) dorsal root ganglia from six- to eight-week-old rats, followed by dye-injection and immunocytochemistry. This showed that conduction velocities of neurons with calcitonin gene-related peptide-like immunoreactivity ranged from 0.5 to 28.6 m/s. Seventy-three neurons were successfully processed. Of these, calcitonin gene-related peptide-like immunoreactivity was found in 46% of C-fibre neurons, 33% of A delta-fibre neurons and in 17% of the A alpha/beta-fibre neurons. The peptide-like immunoreactivity was found in approximately 25% of all A-fibre neurons sampled.     

Development of PGP 9.5- and calcitonin gene-related peptide-like immunoreactivity in organ cultured fetal rat lungs

Knowing that small-granule endocrine cells develop in organ cultured fetal lungs, we investigated whether the cells produce regulatory peptides in vitro, and if sufficient amounts appear to permit using the cultures as an experimental system for physiological study of secretory mechanisms. The paired lungs from 14-day and 15-day fetal rats were organ cultured for 1–8 days and examined daily for development of immunoreactivity against marker proteins and regulatory peptides associated with small-granule endocrine cells and nerves. They proved reactive against protein gene product 9.5 (PGP) and calcitonin-gene related peptide (CGRP) but not against calcitonin or neurofilament protein 200 K, although positive controls were obtained for these substances in lungs from postnatal animals. Initially PGP-like immunoreactivity is associated with cell bodies and processes of neuroblasts which run medial to the bronchial axis on day 14 and are increasingly prevalent on day 15. In 15-day explants PGP becomes detectable after a day in vitro in rare “clear cell” precursors of small-granule cells located in the epithelium lining proximate parts of the lungs, although in 14-day explants comparable reactivity is not seen until the third day (14 + 3 days). In culture PGP-positive neuroblasts increase in number, and nerve processes gradually extend down the airway to encircle the sleeve of smooth muscle that develops as the bronchial tree expands. Concurrently, the initially small clusters of small-granule cells increase in size, and new ones appear in the airway lining. By 15 + 5 days they extend to the boundary between a taller, more proximal epithelium and a glycogen-rich cuboidal layer that lines one or two most-distal generations of branches. Thereafter, the trachea and central, cartilage-bound segments of the primary bronchi mainly contain solitary endocrine cells and the more peripheral lung a mixture of single cells and clusters, much as in near-term lungs in vivo. At this stage PGP-positive nerves extend as far as the entrances of the terminal sacs, and most are distributed to the airway muscle plexus. Exceptionally, they may innervate a small-granule cell cluster, converting it into a neuroepithelial body. CGRP-like immunoreactivity initially appears in small-granule cells of 15 + 2-day cultures but does not develop in ganglion cells or nerves. It localizes to endocrine cells at all conducting airway levels, increasing in staining intensity and accounting for most if not all of the PGP-positive population between 15 + 4 ? 15 + 8 days. Similar localizations with anti-PGP and anti-CGRP were obtained in lungs of 16-day explants grown for 7 days. CGRP is present in sensory nerves of intact rat lungs. Its absence from nerves in vitro confirms that the cultures are deafferented and the PGP-positive nerves are mainly parasympathetic efferents. Small-granule cells and ganglion cells become concentrated in lung organ cultures because elongation of airway branches is severely curtailed, and this affords an advantage for functional studies on these populations. In addition, any experimentally induced fluctuation in release of CGRP to the medium can be ascribed to effects on the endocrine cells and not the sensory nerves. © 1993 Wiley-Liss, Inc.     

Release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from organs of the genitourinary tract in rats

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was detected in the rat urinary bladder, ureter and vas deferens and was depleted by systemic capsaicin desensitization. Exposure to capsaicin in vitro produced a prompt increase in CGRP-LI outflow in superfusates of these tissues, while a second application of the drug was ineffective indicating desensitization. These findings provide further evidence for a transmitter role of CGRP from peripheral endings of sensory nerves and the involvement of CGRP-LI in the specific motor response to capsaicin in the rat genitourinary tract.     

Substance P-, somatostatin- and calcitonin gene-related peptide-like immunoreactivity and fluoride resistant acid phosphatase-activity in relation to retrogradely labeled cutaneous, muscular and visceral primary sensory neurons in the rat

The distribution of several peptides in cutaneous, muscular and visceral primary sensory neurons was investigated in the adult rat. The fluorescent dye Fast blue was applied to the proximal ends of transected saphenous (cutaneous), gastrocnemius (muscular) and greater splanchnic (visceral) nerves. Sections from corresponding dorsal root ganglia were incubated for simultaneous indirect immunocytochemical demonstration of calcitonin gene-related peptide (CGRP)-, substance P (SP)- or somatostatin (SOM)-like immunoreactivity (-LI) and Fast blue. In addition, the presence of fluoride resistant acid phosphatase (FRAP)-enzyme activity (-EA) in retrogradely Fast blue-labeled saphenous and gastrocnemius nerves was investigated by subsequent enzyme cytochemical analysis. The results revealed the presence of CGRP-LI, SP-LI, SOM-LI and FRAP-EA in cell bodies of primary sensory neurons which project to the saphenous and gastrocnemius nerves. CGRP-LI and SP-LI, but not SOM-LI, were found in splanchnic sensory neurons. The vast majority of the visceral sensory neurons were found to contain CGRP-LI.     

Changes of calcitonin gene-related peptide-like immunoreactivity in cerebrovascular nerve fibers in the dog after experimentally produced subarachnoid hemorrhage

After producing a model of subarachnoid hemorrhage (SAH) by a single injection of fresh autologous arterial blood into the cisterna magna in the dog, we immunohistochemically examined changes of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in perivascular nerve fibers in the large pial arteries by using whole mount preparations. CGRP-LI in cerebrovascular nerve fibers was suppressed after SAH. The suppression was first detected on the 3rd day after SAH, and was most marked during the 7th to 14th day after SAH. CGRP-LI, however, recovered to a normal level by the 42nd day after SAH.     

Amygdaloid projections to the mesencephalon,pons and medulla oblongata in the cat

Summary Amygdalotegmental projections were studied in 26 cats after injections of horseradish peroxidase (HRP) in the diencephalon, midbrain and lower brain stem and in 6 cats after injection of ³H-leucine in the amygdala. Following HRP injections in the posterior hypothalamus, periaqueductal gray (PAG) and tegmentum many retrogradely labeled neurons were present in the central nucleus (CE) of the amygdala, primarily ipsilaterally. Injections of HRP in the posterior hypothalamus and mesencephalon also resulted in the labeling of neurons in the basal nucleus, pars magnocellularis.Following ³H-leucine injections in CE and adjacent structures autoradiographically labeled fibers were present in the stria terminalis and ventral amygdalofugal pathways. In the mesencephalon heavily labeled fiber bundles were located lateral to the red nucleus. Labeled fibers and terminals were distributed to the mesencephalic reticular formation, substantia nigra, ventral tegmental area and PAG. In the pontine and medullary tegmentum the bulk of passing fibers was located laterally in the reticular formation. Many labeled fibers and terminals were distributed to the parabrachial nuclei, locus coeruleus, nucleus subcoeruleus and lateral tegmental fields. Many terminals were also present in the solitary nucleus and dorsal motor nucleus of the vagus nerve.The location of the cells of origin and the distribution of the terminals of the amygdalotegmental projection suggest that this pathway plays an important role in the integration of somatic and autonomic responses associated with affective defense.Abbreviations A nucleus ambiguus - AL lateral amygdaloid nucleus - AQ cerebral aqueduct - BC brachium conjunctivum - BL basal amygdaloid nucleus, pars magnocellularis - BM basal amygdaloid nucleus, pars parvocellularis - BP brachium pontis - CE central amygdaloid nucleus - CI internal capsule - CN cochlear nucleus - CO cortical amygdaloid nucleus - CP cerebral peduncle - DCN dorsal column nuclei - DMV dorsal motor nucleus of the vagus nerve - E entopeduncular nucleus - F fornix - FLA longitudinal association bundle - GP globus pallidus - H hippocampal formation - 1C inferior colliculus - INJ injection site - LC locus coeruleus - IO inferior olive - LG lateral geniculate nucleus - LRN lateral reticular nucleus - LT lateral tegmental field - M medial amygdaloid nucleus - MB mammilary body - MG medial geniculate nucleus - ML medial lemniscus - MT medial tegmental field - MV motor nucleus of the trigeminus - OC optic chiasm - OT optic tract - P putamen - PAG periaqueductal gray - PB parabrachial nuclei - PC posterior commissure - PH posterior hypothalamus - PT pyramidal tract - PV principal sensory nucleus of the trigeminus - PYR pyriform cortex - R red nucleus - RF reticular formation - S solitary nucleus - SC nucleus subcoeruleus - SN substantia nigra - SO superior olive - SOL solitary nucleus - SPV spinal trigeminal complex - ST stria terminalis - VC vestibular complex - VTA ventral tegmental area - VII facial nucleus - XII hypoglossal nucleus     

Effects of diabetes on tissue content and evoked release of calcitonin gene-related peptide-like immunoreactivity from rat sensory nerves

We measured the evoked release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from sensory nerve terminals in tracheas from control and diabetic rats using an in vitro perfusion system and also the CGRP-LI content of the vagus nerve and trachea. Diabetes caused a 29% (P < 0.05) reduction in CGRP-LI content of the vagus nerve and a decrease in CGRP-LI release from nerve endings in the trachea evoked by either capsaicin (30% decrease, P < 0.05) or electrical field stimulation (50% decrease: P < 0.05). In contrast, there was a 50% increase in the CGRP-LI content of the unstimulated trachea. Thus, diabetes induces an impairment in neuropeptide release from peripheral terminals of sensory nerves that corresponds to decreased levels in the supplying nerve but is not reflected in tissue measurements that incorporate nerve terminals. Impaired neuropeptide release may contribute to peripheral and central sensory dysfunction in diabetic rats.     

Quantitative immunocytochemistry shows calcitonin gene-related peptide-like immunoreactivity in lung neuroendocrine cells is increased by chronic hypoxia in the rat

We have previously shown that the vasodilator calcitonin gene-related peptide (CGRP) is increased in pulmonary neuroendocrine cells in response to hypoxia. To quantify the change, we have now examined lung of adult male Wistar rats exposed to hypoxia (FIO2 = 0.1) for 1 wk and littermate controls. Sections of lung were immunostained simultaneously using rabbit antiserum to rat alpha-CGRP with the peroxidase antiperoxidase technique. The area and integrated optical density of each group of endocrine cells were measured using an image analyzer. For each animal, the summed integrated optical density of endocrine cells divided by the sum of their areas was used as a measure of CGRP-like immunoreactivity. The intensity of immunostaining of endocrine cells in the respiratory portion of the lung was 43% greater than that of endocrine cells along the conducting airways (P less than 0.001). The intensity of staining was increased by approximately 12% (P less than 0.04) after 7 d of hypoxia with no apparent difference in the response of central and peripheral endocrine cells. Measurements of staining intensity of CGRP-coupled agarose beads indicated that a 12% change in staining intensity corresponded to a 15 to 20% change in the concentration of CGRP or CGRP-like immunoreactive material. The supra-optimal dilution technique (measurement of the increase in the number of immunoreactive cells upon sequential immunostaining with a supra-optimal and then an optimal dilution of primary antiserum) detected the increase in CGRP-like immunoreactivity after 7 d of hypoxia with a high degree of statistical significance (P less than 0.005) using the same number of sections.     

Metastin-like immunoreactivity in the rat medulla oblongata and spinal cord

Metastin, the product of metastasis suppressor gene KiSS-1, is proposed to be the natural ligand for the G-protein-coupled receptor GPR54, known also as AXOR12. This immunohistochemical study, using a rabbit polyclonal antiserum against the human metastin fragment (45-54)-NH(2), showed that in rats metastin-like immunoreactivity (MTS-LI) was present in neurons of the nucleus of the solitary tract and caudoventrolateral reticular nucleus, and in cell processes of the spinal trigeminal tract and lateral reticular nucleus. MTS-LI was confined mainly to neurons and fibers at or caudal to the area postrema. In the spinal cord, MTS-LI cell processes formed a dense plexus in superficial layers I and II of the dorsal horn. The pattern of distribution of MTS-LI in the medulla and spinal cord suggests that this novel peptide may participate in autonomic and sensory neural signaling.     

Quantitative examination of calcitonin gene-related peptide immunoreactive nerve fibres in the cat knee joint capsule

 The knee joint of the cat has been used extensively to study the morphology and function of primary afferents in a deep somatic tissue. A proportion of these neurones synthesizes various neuropeptides, with calcitonin gene-related peptide being the most prominent. In the present study we examined the distribution and density of nerve fibres immunoreactive for calcitonin gene-related peptide within the medial articular capsule. The fibres were predominantly located in the superficial layer of the capsule. They formed a dense innervation pattern, mainly accompanying blood vessels. Electron microscopy showed that most fibres were in close proximity to small arteries. The highest innervation density was found in parts of the capsule that were located over the epicondyle of the femur with 21±12 fibres per mm² (mean±SD). In the tissue over the joint cleft this density was lower, with 11±6 fibres per mm². In conclusion, the high innervation density of the knee joint capsule by nerve fibres containing calcitonin gene-related peptide supports the hypothesis of an important regulatory function of this peptide in normal tissue. Accepted: 24 January 1997