Two distinct calcitonin gene-related peptide-containing peripheral nervous systems: Distribution and quantitative differences between the iris and cerebral artery with special reference to substance P

The present study first shows that calcitonin gene-related peptide(CGRP)-like immunoreactive (CGRPI) nerve fibers in the cerebral arteries contained substance P (SP) and originated from small- to medium-sized CGRPI cells exhibiting SP immunoreactivity (SPI) in the trigeminal ganglion. The iris contained CGRPI/SPI-costorage nerve fibers and many CGRPI fibers lacking SPI. These fibers originated from large CGRPI cells lacking SPI in the trigeminal ganglion. The heterogenous subpopulations of CGRPI fibers in the iris suggest that CGRP is involved in a variety of functions in this structure.     

Origins and distribution of calcitonin gene-related peptide-containing nerves in the wall of the cerebral arteries of the guinea pig with special reference to the coexistence with substance P

The origins and overall distribution of calcitonin gene-related peptide-like immunoreactivity (CGRPI) in the wall of the cerebral arteries were investigated in the guinea pig by using whole-mounts. Two types of CGRPI fibers were seen; one forming dense fiber bands, located among the periadventitial nerves, and the other forming a meshwork. CGRPI fibers in the periadventitial nerves often leave these nerves to form a meshwork, of a density that varies according to the diameter or location of the blood vessel. The present study showed that CGRPI fibers in the walls of the carotid arterial system originated from the trigeminal ganglion, and those in the vertebrobasilar arterial system from other origins besides the trigeminal ganglion. We also examined the coexistence of this peptide with substance P-like immunoreactive (SPI) structures in a single neuron system. Double staining immunocytochemistry showed that the patterns of the running of CGRPI and SPI fibers in the wall of the cerebral arteries were similar, and this method also demonstrated the presence of neurons containing both CGRPI and SPI structures in single cells of the trigeminal ganglion, which is the major origin of these fibers in the cerebral arteries.     

Distribution and origin of calcitonin gene-related peptide in the rat stomach and duodenum: An immunocytochemical analysis

Summary We studied the three-dimensional distribution of structures with calcitonin gene-related peptide-like immunoreactivity (CGRPI) in the rat stomach and duodenum, including the origins of these structures, using indirect immunofluorescence in both muscle strips and frozen sections. There was a very dense meshwork of CGRPI fibers in the circular and longitudinal muscle layers, and also in the myenteric and submucous plexuses of the stomach and duodenum. No CGRPI neurons were seen in the stomach, even in rats treated with colchicine; in the duodenum, there was a group of CGRPI cells in the myenteric and submucous ganglia. No regional differences were seen in the stomach and duodenum.We found by experimental manipulations that CGRPI fibers in the stomach were exclusively extrinsic in origin; some of such fibers in the duodenum were intrinsic in origin, though most were supplied by CGRPI cells outside the duodenum.     

Occurrence of calcitonin gene-related peptide in the chicken amacrine cells

The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (CGRPI) in the chicken retina was investigated by means of immunohistochemistry. CGRPI was localized in the stratified amacrine cells. The terminal branching pattern of these cells was different between the central and peripheral retinal regions. Flat-mount preparations revealed these CGRPI cells to be evenly distributed in the entire retina with surprisingly rich arborization.     

Calcitonin gene-related peptide-like immunoreactive sensory fibers form synaptic contact with sympathetic neurons in the rat celiac ganglion

The present study demonstrates synaptic contact between calcitonin gene-related peptide (CGRP)-like immunoreactive axon terminals and sympathetic neurons in the rat celiac ganglion. Our observations suggest that sensory ganglion neurons directly regulate the sympathetic activity via synapses, because CGRP immunoreactive (CGRPI) fibers in this ganglion are supplied by the sensory ganglia.     

Preganglionic parasympathetic neurons projecting to the sphenopalatine ganglion contain nitric oxide synthase in the rabbit

We have investigated the possible presence of nitric oxide synthase (NOS) and choline acetyltransferase (ChAT) in brainstem preganglionic parasympathetic neurons projecting to the sphenopalatine ganglion in rabbits, using combined retrograde axonal tracing and immunohistochemistry. Retrogradely labeled neurons were observed in the ipsilateral rostral medulla and caudal pons, in a region laterodorsal to the facial motor nucleus. Double-labeling experiments demonstrated that 75±5% of retrogradely labeled neurons contained NOS immunoreactivity, while all of retrogradely labeled neurons contained ChAT immunoreactivity. These observations suggest that nitric oxide could influence cholinergic transmission from preganglionic endings in the sphenopalatine ganglion.     

GABA-ergic neurons in the crayfish nervous system: an immunocytochemical census of the segmental ganglia and stomatogastric system

We used an antiserum directed against gamma-aminobutyric acid (GABA) fixed with glutaraldehyde (Hoskins et al., Cell Tissue Res. 244:243-252, '86) to label neurons with GABA-like immunoreactivity (GLI) in wholemounts of the stomatogastric ganglion and each segmental ganglion of crayfish, except the brain. Each abdominal ganglion had an average of 63 labeled neurons, or 10% of all their neurons. Each peripheral nerve of each abdominal ganglion except the last contained labeled axons. Within each segment, the first peripheral nerve, N1, had five axons; the second peripheral nerve, N2, had at most four; and the third peripheral nerve, N3, had two. In the last ganglion, N2 had one labeled axon, N3 had two and N6 had two; the other nerves contained no labeled axons. A tabulation of the identified inhibitory neurons in the abdominal ganglia revealed that 40% of these GABA-ergic neurons have been identified. The subesophageal ganglion had many labeled neurons in clusters that formed a repeating pattern; it also had labeled neurons near its dorsal midline. The thoracic ganglia contained more labeled neurons than did the abdominals, but their patterns of labeling were similar. The commissural ganglia contained three clusters of labeled neurons and sent labeled axons to the esophageal ganglion. The esophageal ganglion contained four labeled neurons and many labeled axons. The stomatogastric ganglion contained labeled axon terminals but not labeled neurons.     

Topography of NPY-, somatostatin-, and VIP-immunoreactive, neuronal subpopulations in the guinea pig celiac-superior mesenteric ganglion and their projection to the pylorus

The topography of the peptidergic neuronal subpopulations in the guinea pig celiac-superior mesenteric ganglion was studied analyzing the distribution of immunoreactivity to neuropeptide Y (NPY), somatostatin (SOM), and vasoactive intestinal polypeptide (VIP)/polypeptide HI (PHI). For comparison, the ganglion was also studied using antisera against the 2 catecholamine-synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). Approximately 65% of the neuronal cell bodies contained NPY-like immunoreactivity (NPY-LI), whereas 25% of the principal ganglion cells contained SOM-like immunoreactivity (SOM-LI). Though occasional cells were found to contain both NPY-LI and SOM-LI, these peptides had a complementary distribution in the ganglion, with NPY cells in the celiac poles and SOM cells in the superior mesenteric pole. The vast majority of both the NPY- and SOM-positive cells also contained TH-like immunoreactivity (TH-LI), confirming their catecholaminergic, presumably noradrenergic, nature. Some noradrenergic neurons seemed to lack NPY- and SOM-LI. Small numbers of VIP/PHI-containing cell bodies were found in areas where the NPY-immunoreactive neurons predominated. Many of the VIP/PHI-positive cells contained NPY-LI and occasionally also TH-LI. The immunohistochemical markers were also observed in fibers. Thus, a comparatively weak NPY-LI was seen in smooth fibers, probably representing axons and axon bundles. SOM-LI was seen in a similar type of fiber but also in more strongly fluorescent fibers with a varicose appearance. The latter fibers were observed only in the SOM-dominated part of the ganglion, often surrounding the ganglion cells. Varicose fibers with a similar distribution containing DBH-like immunoreactivity (DBH-LI) were also seen. In addition, DBH- and TH-LI were seen in smooth axonlike processes. VIP-positive fibers exhibited a very dense fiber network, almost exclusively related to the SOM cell-dominated part of the ganglion. The projection of the postganglionic sympathetic neurons was studied with special reference to the pylorus using a combination of retrograde axonal tracing and indirect immunofluorescence techniques. Seventy-two hours after injection of the fluorescent tracer Fast Blue into the pyloric sphincter, labeled neurons were found in the ganglion. By comparing the Fast Blue-labeled cells with the immunoreactive cell bodies, neurons containing both dye and NPY- or SOM-LI were observed. In elution-restaining experiments, it was established that the majority of these cells were also immunoreactive to TH, indicating that they produce noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)     

Overall distribution of substance P-containing nerves in the wall of the cerebral arteries of the guinea pig and its origins

The overall distribution of substance P-like immunoreactivity (SPI) in the wall of the cerebral arteries and their origins were investigated in the guinea pig by using whole-mounts. Two types of SPI fibers were seen: one forming dense fiber bands and located among the periadventitial nerves, and the other forming a meshwork. The SPI fibers located in the periadventitial nerves often leave these nerves to form a meshwork of SPI fibers of varying density according to the diameter or location of the blood vessels. The present study suggests that: (1) SPI fibers located on the circle of Willis and its branches originate from SPI cells in the trigeminal ganglion; (2) SPI fibers of the rostral one-third of the basilar artery originate partly from trigeminal SPI cells; and (3) SPI fibers in the caudal two-thirds of the basilar artery originate exclusively from other SPI cells, apart from the trigeminal ganglion.     

Catecholaminergic properties of cholinergic neurons and synapses in adult rat ciliary ganglion

Parasympathetic neurons of the ciliary ganglion are innervated by preganglionic cholinergic neurons whose cell bodies lie in the brain stem; the ganglion cells in turn provide cholinergic innervation to the intrinsic muscles of the eye. Noradrenergic innervation of the iris is supplied by sympathetic neurons of the superior cervical ganglion. Using immunocytochemical and histochemical techniques, we have examined the ciliary ganglion of adult rats for the expression of cholinergic and noradrenergic properties. As expected, the postganglionic ciliary neurons possessed detectable levels of choline acetyltransferase immunoreactivity (ChAT-IR). Unexpectedly, many ciliary neurons also exhibited immunoreactivity for tyrosine hydroxylase (TH-IR). Some had dopamine beta-hydroxylase-like (DBH-IR) immunoreactivity, but none contained detectable catecholamines, even after treatment with nialamide and L-DOPA. A sparse plexus of fibers exhibiting faint TH-IR was present in the irises of acutely sympathectomized rats. The terminals of preganglionic axons in the ciliary ganglion exhibited not only immunoreactivity for ChAT, but also for TH and contained stores of endogenous catecholamine. Neither ciliary neurons nor their preganglionic innervation accumulated detectable stores of exogenous catecholamines. Rats sympathectomized as neonates by treatment with 6-hydroxydopamine subsequently had a greater proportion of neurons possessing detectable TH-IR in the ciliary ganglion; both the TH-IR perikarya and their axons in the iris were more intensely immunofluorescent. TH-IR was present in the ciliary neuron cell bodies of mouse, guinea pig, and ferret. These species, however, lacked detectable TH-IR or catecholamine stores in preganglionic terminals. These observations indicate that mature, functionally cholinergic neurons from 2 different embryonic origins, postganglionic ciliary neurons derived from the neural crest and preganglionic neurons derived from the neural tube, display several catecholaminergic properties.     

Effect of ciliary neuronotrophic factor on somatostatin expression in chick ciliary ganglion neurons

The normal development of somatostatin (SOM) expression in neurons of the chick ciliary ganglion and the effects of ciliary neuronotrophic factor (CNTF) on SOM induction in cultured ciliary ganglion neurons, were studied by immunocytochemical techniques. SOM immunoreactivity was first detectable in some neurons of the ganglion at embryonic day (E)8 and between E14 to hatch, 44–46% of the neuronal population contained the peptide. It was inferred that essentially all choroid neurons, which constitute 50% of the neuronal population, contain SOM. Culture studies indicated that CNTF supported both the SOM positive choroid neurons and the SOM negative ciliary neurons. Although CNTF was necessary for the survival and maturation of cultured ciliary ganglion neurons, it did not influence either the induction or maintenance of SOM expression in these neurons. CNTF may instead act as a permissive factor, allowing the induction of SOM in neurons of the ciliary ganglion by other, more specific, factors.     

Double labeling of the paravertebral sympathetic C system in the bullfrog with antisera to LHRH and NPY

The specificity of synaptic contacts between pre- and postganglionic cells in the sympathetic C system has been examined by immunocytochemical localization of two neuropeptides. Sections of bullfrog paravertebral sympathetic ganglia were stained with antibodies to luteinizing hormone releasing hormone (LHRH) and neuropeptide Y (NPY). Preganglionic synaptic boutons containing LHRH immunoreactivity were found to make contact with a subpopulation of postganglionic cell bodies and with some clusters of small intensely fluorescent (SIF) cells. In ganglia 9 and 10, 95.8% of the neurons contacted by LHRH-containing boutons were also positive for NPY-like immunoreactivity and conversely, 99.3% of the neurons that contained NPY-like immunoreactivity were contacted by LHRH-containing boutons. Qualitatively similar results were found in most other paravertebral ganglia. These observations support the conclusions that preganglionic C axons selectively innervate C-type ganglion cells and that virtually all C-type ganglion cells and some SIF cells receive a direct LHRH input. Moreover, they suggest that a pattern of specific connections between two sets of peptidergic neurons is expressed throughout most of the paravertebral sympathetic chain of the bullfrog.     

Calcitonin gene-related peptide containing autonomic efferent pathways to the pelvic ganglia of the rat

Indirect immunofluorescence method was employed to investigate the involvement of calcitonin gene-related peptide (CGRP) in the autonomic efferent innervations of the pelvic visceral organs of the rat. Cells labeled with Fast blue (FB) injected into the pelvic ganglia were observed in the sacral parasympathetic nucleus; about 30% of these neurons showed CGRP-like immunoreactivity. These CGRP-like immunoreactive neurons were located in the dorsomedial part of the sacral parasympathetic nucleus, extending their dendrites mediolaterally. FB-labeled cells were also found in the upper lumbar level (L1, L2) of the spinal cord. Some of these neurons also showed CGRP-like immunoreactivity. CGRP-like immunoreactive varicose fibers were seen in the pelvic ganglia surrounding individual ganglion cells. Considerable amount of these fibers were not affected by sensory deafferentation, so they probably originated from autonomic efferent neurons.     

Substance P immunoreactivity in the superior cervical ganglia of normotensive and genetically hypertensive rats

Substance P-like immunoreactivity (SPI) was investigated in the superior cervical ganglion of normotensive and genetically hypertensive (GH) Otago Wistar rats aged 1, 2, 8-10 and 50-60 weeks, by used of an indirect immunoperoxidase method. SPI was not seen in neuronal cell bodies but a subpopulation of ganglion cells was supplied by SP-positive terminals which closely invested the cell surface. This subpopulation showed no particular topographical distribution. The number of SP-positive terminal varicosities per unit area was several times higher in GH rats than in normotensive rats at all ages over 2-60 weeks. The proportion of neurons supplied by SP-positive terminals (sampled in 8-10 week-old rats) was also greater in GH than in normotensive rats. Decentralization of the ganglion or chronic capsaicin treatment removed all SP-immunoreactive terminals around the cell bodies, indicating that the SP-positive terminals are collaterals of thoracic sensory afferents. As SP has been reported to have an excitatory effect in sympathetic ganglia, intraganglionic release of SP might contribute to the development of hypertension in the GH strain.     

Calcitonin gene-related peptide- and substance P-immunoreactive nerve fibers in Meissner's corpuscles of rats: an immunohistochemical analysis

The present study demonstrates that about 15% of the Meissner's corpuscles (MCs) of the rat glabrous skin of the feet contain one to two fibers immunoreactive for calcitonin gene-related peptide (CGRP), and that these fibers originate from the dorsal root ganglion. Double immunofluorescent cytochemical investigation has revealed that almost all the CGRP-immunoreactive (CGRPI) fibers in the MCs were immunoreactive for substance P. Subsequent immunoelectron microscopic analysis has demonstrated that the CGRPI fibers in the MCs are unmyelinated. In the course of passage through the MCs, no synaptic contact between the CGRPI fibers and underlying cells or non-CGRPI fibers was found.     

Vasoactive intestinal peptide and neuropeptide Y coexist in non-noradrenergic sympathetic neurons to guinea pig trachea.

Vasoactive intestinal peptide (VIP) has been suggested to be a mediator of vagal inhibition of airway tone and it has been assumed that VIP-containing nerve fibres in the airway arise from intrinsic ganglia. We have used a combination of double- and triple-labelling immunohistochemistry, retrograde axonal tracing, organotypic culture and nerve lesion studies, to identify the origin and distribution of neurons containing immunoreactivity (IR) to VIP in guinea pig airway smooth muscle. We also investigated whether immunoreactivity to other neuropeptides coexisted with VIP-IR within these neurons. We found that all VIP-IR nerve fibres in guinea pig tracheal smooth muscle also contained IR to neuropeptide Y (NPY) but not to tyrosine hydroxylase (TH), a marker for noradrenergic neurons. Both VIP-IR and NPY-IR were absent from nerve cell bodies in the tracheal plexus. After maintenance of isolated trachea in organotypic culture for 4 days, to allow degeneration of extrinsic nerve fibres, nerve fibres containing VIP-IR or NPY-IR were almost completely absent from tracheal smooth muscle. Of ganglia known to supply the trachea, coexistence of VIP-IR and NPY-IR was found only in cell bodies of the stellate ganglion. Retrograde tracing studies using the fluorescent tracer, DiI, confirmed that the stellate ganglion was the site of origin of neurons containing VIP-IR and NPY-IR supplying the airways. These neurons projected to the airways from the stellate ganglion both directly through the mediastinum, and via the cervical sympathetic trunk and vagus nerves. These results suggest that nerve fibres containing both VIP-IR and NPY-IR in the tracheal smooth muscle of the guinea pig are derived from non-noradrenergic cell bodies in the stellate ganglion. The absence of VIP-IR from vagal post-ganglionic neurons suggests that VIP cannot be a mediator of vagal inhibitory transmission in tracheal smooth muscle of this species.     

Vasomotor, pilomotor and secretomotor neurons distinguished by size and neuropeptide content in superior cervical ganglia of mice.

Populations of postganglionic sympathetic neurons projecting to cranial targets from the superior cervical ganglia of mice were identified by retrograde axonal tracing with Fast blue combined with double-labelling immunofluorescence to detect immunoreactivity to tyrosine hydroxylase and neuropeptide Y. Nearly all neurons in the ganglion contained tyrosine hydroxylase immunoreactivity, but only about 50% of them also contained immunoreactivity to neuropeptide Y. The maximum diameter of cells with immunoreactivity to neuropeptide Y was significantly smaller than that of cells without it. Terminal axons containing immunoreactivity to both neuropeptide Y and tyrosine hydroxylase occurred around blood vessels supplying most cranial tissues, including the skin. Axons with immunoreactivity to tyrosine hydroxylase but not to neuropeptide Y innervated the piloerector muscles and the acini of the salivary glands. After injection of Fast blue into the skin or the submandibular salivary gland, populations of vasomotor, pilomotor and secretomotor neurons could be distinguished by soma size and by neuropeptide Y immunoreactivity. Neurons projecting to the salivary glands were the largest (mean diameter: 32 microns) and lacked immunoreactivity to neuropeptide Y; neurons projecting to cutaneous blood vessels were the smallest (mean diameter: 19 microns) and contained immunoreactivity to neuropeptide Y; neurons projecting to piloerector muscles were intermediate in size (mean diameter: 23 microns) and lacked neuropeptide Y immunoreactivity. A cluster analysis procedure confirmed that soma size and peptide content together identify major functional populations of neurons in the superior cervical ganglia of mice.     

Presence of a substance P-like immunoreactive neurone system from the parabrachial area to the central amygdaloid nucleus of the rat with reference to coexistence with calcitonin gene-related peptide

An ascending neurone system containing substance P-like immunoreactivity (SPI) from the lateral parabrachial nucleus (PBL) to the central amygdaloid nucleus (AC) was detected. Destruction of the external subdivision of the PBL resulted in a marked ipsilateral reduction of SPI fibres in the AC, which suggests that SPI neurones project mainly ipsilaterally to the AC. This was supported by the findings that injection of biotin-wheatgerm agglutinin into the AC labelled many neurones in the ipsilateral external subdivision of the PBL. Simultaneous staining with antiserum showed that some of these neurones contain SP. Immunohistochemical double-staining revealed that almost all of the SPI neurones in the external subdivision of the PBL contained calcitonin gene-related peptide.