Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye

The occurrence, distribution, and origin of immunoreactive calcitonin gene-related peptide (CGRP) in nerves of rat, guinea pig, cat, and monkey eyes were investigated by immunocytochemistry, radioimmunoassay, and chromatography. A rich network of CGRP-immunoreactive nerve fibres was noted in the anterior uvea, which was widely distributed in both dilator and constrictor pupillae muscles and extended to the ciliary body and uveal blood vessels. Numerous CGRP-immunoreactive neuronal cells were present in the trigeminal ganglion. The extractable CGRP was 8.6 +/- 1.8 pmoles/gm of tissue in the iris and 44.0 +/- 8.1 pmoles/gm in the trigeminal ganglion. Following damage to the Gasserian ganglion a marked decrease of CGRP immunoreactivity was observed in the anterior uvea (control 11.3 +/- 1.6 pmoles/gm; operated 1.4 +/- 0.1 pmoles/gm) confirming the origin of the immunoreactive fibres from trigeminal primary sensory neurons. The sensory nature of the CGRP-immunoreactive fibres was substantiated by the depletion of CGRP immunoreactivity observed after treatment with capsaicin, which is known to cause selective degeneration of sensory neurons. Comparative studies on the distribution and colocalisation of CGRP and the putative sensory neurotransmitter substance P revealed a closely parallel distribution of the two peptides in certain regions of the uvea and their coexistence in a subpopulation of trigeminal primary sensory neurons. This study suggests that the sensory nervous system in the eye is more heterogeneous in terms of its putative neurotransmitters than previously indicated.     

An immunohistochemically distinct population of cat ciliary ganglion cells.

In the cat ciliary ganglion, 16% and 23% respectively of the neurons are surrounded by nerve fibers immunoreactive to calcitonin gene-related peptide (CGRP) and somatostatin (SOM). One-third of the ganglion cell perikarya are immunoreactive to neuropeptide Y (NPY). Analysis of the coincidence of immunoreactivities shows a striking correlation. Practically all of the ganglion cells surrounded by CGRP-like immunoreactive (LI) nerve fibers also are surrounded by SOM-LI nerve processes and demonstrate NPY-LI perikarya. These observations define a subset of NPY-LI ciliary ganglion cells with a particular peptidergic input and perhaps a specific physiologic function.     

Cholecystokinin-like immunoreactivity occurs in ocular sensory neurons and partially co-localizes with substance P

Based on immunohistochemical analysis of the trigeminal, superior cervical, ciliary and sphenopalatine ganglia and of the eye after sensory denervation and sympathectomy, cholecystokinin (CCK)-like immunoreactive nerves in the guinea pig eye derive from the trigeminal ganglion. Substance P (SP) also occurs in some ocular sensory neurons, suggesting the possible co-localization in this system of CCK- and SP-immunoreactivities. A double-labeling immunofluorescence technique stained 3 types of trigeminal cells and ocular nerve fibers: some immunoreactive for both peptides, some immunoreactive only for CCK and some immunoreactive only for SP.     

Cholecystokinin-like immunoreactivity occurs in ocular sensory neurons and partially co-localized with substance P

Based on immunohistochemical analysis of the trigeminal, superior cervical, ciliary and sphenopalatine ganglia and of the eye after sensory denervation and sympathectomy, cholecystokinin (CCK)-like immunoreactive nerves in the guinea pig eye derive from the trigeminal ganglion. Substance P (SP) also occurs in some ocular sensory neurons, suggesting the possible co-localization in this system of CCK- and SP-immunoreactivities. A double-labeling immunofluorescence technique stained 3 types of trigeminal cells and ocular nerve fibers: some immunoreactive for both peptides, some immunoreactive only for CCK and some immunoreactive only for SP.     

Extraocular muscle proprioception and the descending trigeminal nucleus

Scope of the present investigation was to look for the termination within the brain stem of the central process of the perikarya contained in the semilunar ganglion which provide the extraocular muscle spindles. Unitary responses to stretching single eye muscles were recorded by means of tungsten microelectrodes from the pars oralis of the ipsilateral descending trigeminal tract and nucleus in the lamb. The responses were characterized by a sudden increase in discharge rate of the units followed by a modest adaptation; the firing ceased as soon as the stretch was released. Single-pulse electrical stimulation of the cellular pool of the medial dorsolateral part of the semilunar ganglion which innervate the eye muscle proprioceptors elicited evoked potentials in the same ipsilateral pontine sites responsive to stretching the extraocular muscles. The latency of such evoked potentials was very short: 0.42–0.95 msec. We conclude that the afferent impulses from the eye muscles project to the descending trigeminal nucleus. Thus the central process of the semilunar ganglion cells concerned with the eye muscle proprioception enters the brain stem through the trigeminal sensory root, runs along the ipsilateral descending trigeminal tract, and terminates within the homonymous nucleus.     

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.     

A quantitative correlation of substance P-, calcitonin gene-related peptide- and cholecystokinin-like immunoreactivity with retrogradely labeled trigeminal ganglion cells innervating the eye

To evaluate the intraganglionic organization of ocular sensory neurons in the guinea pig, we studied the retrograde axonal transport from the eye to the trigeminal ganglion of cholera toxin B subunit and then applied immunohistochemistry for substance P, calcitonin gene-related peptide and cholecystokinin. Retrogradely labeled cells were observed only in the anteromedial portion of the ipsilateral ganglion. We observed no somatotopical organization to trigeminal neurons containing any of these three peptides, either for cells projecting to the eye or for the ganglion as a whole. The relative proportion of neurons immunoreactive for each of these three peptides was similar among the population of neurons retrogradely labeled with cholera toxin B and among the population of neurons without direct projections to the eye.     

Nasal mucosa sensitization with toluene diisocyanate (TDI) increases preprotachykinin A (PPTA) and preproCGRP mRNAs in guinea pig trigeminal ganglion neurons.

Toluene diisocyanate (TDI) induces respiratory allergy in mammals. Using immunohistochemistry and in situ hybridization histochemistry, the present study examined effects of nasal mucosa sensitization by TDI on the immunoreactivity for substance P (SP) and calcitonin gene-related peptide (CGRP) and on the expression of their mRNAs in guinea pig trigeminal ganglion and their terminals. Single intranasal application of TDI (acute experiment) did not induce nasal allergy-like behaviours and failed to cause changes of SP and CGRP immunoreactivity and in the expression of preprotachykinin A (PPTA) mRNA and preproCGRP mRNA coding for SP and CGRP respectively in the trigeminal ganglion neurons. However, repeated application of TDI (chronic experiment) caused a dramatic increase of SP and CGRP immunoreactivity in peripheral neurites of sensory nerves in the nasal mucosa but a slight increase in the spinal trigeminal nucleus, a decrease of the same immunoreactivities in the cell bodies of the trigeminal ganglion neurons, and an increase of the expression of PPTA and preproCGRP mRNA in the same neurons. These findings suggest that chronic exposure of the nasal mucosa to TDI apparently causes enhancement of both the biosynthesis of SP and CGRP and their axonal transport in the trigeminal system.     

Some cholecystokinin-8 immunoreactive fibers in large pial arteries originate from trigeminal ganglion

Cholecystokinin-8 immunoreactive (CCK8I) nerve fibers were demonstrated in whole mount preparations and cross-sections of pial blood vessels in the cat, guinea pig and rat using a specific antiserum and the avidin-biotin-peroxidase complex method. Positive fibers were present in nearly all pial arteries examined, and were located in the adventitial layer and at the junction of the adventitia and media. In general, CCK8I fibers were less abundant than substance P immunoreactive (SPI) fibers visualized in the same vessels. A marked depletion of CCK8I was noted in large cerebral arteries following treatment of adult guinea pigs with capsaicin, a drug shown previously to deplete CCK8 in some primary sensory neurons. The density of CCK8I-containing fibers was also decreased in the ipsilateral vessels of the cat circle of Willis following unilateral trigeminal ganglionectomies. These results indicate that CCK8I is contained in afferent fibers within large pial arteries of Willis' circle which project from neurons in the ipsilateral trigeminal ganglion. Whether CCK8 coexists with SP in these fibers remains to be determined.     

Proprioceptive reflex connections of head musculature and the mesencephalic trigeminal nucleus in the carp

The location of the sensory cells concerned with the proprioception of respiratory and extraocular musculature in the carp was studied by retrograde transport of horseradish peroxidase. Sensory cell labeling after intramuscular HRP injection was exclusively found in the trigeminal-facial-anterior lateral line ganglion complex. The muscles innervated by the trigeminal system are represented in the more rostral ganglion areas, the muscles innervated by the facial system in the more caudal ganglion parts. Nearly all labeled cells were situated on the ipsilateral side. Sensory cells labeled after extraocular muscle injection were also found all over the V-VII ganglion, however, to a considerable degree also on the contralateral side. All muscle injections failed to give mesencephalic trigeminal cell labeling. The results of intranerve HRP injections in peripheral trigeminal nerve branches strongly suggests a perioral mechanoreceptive function for the mes.V neurons. A bisynaptic proprioceptive reflex model is described for respiratory musculature consisting of a sensory cranial ganglion component connected to the descending trigeminal nucleus, which on its turn links the proprioceptive ganglion cells to the trigeminal and facial motorneurons. Monosynaptic proprioceptive reflex circuits are discussed on neurophysiological grounds.     

Gene expression of histamine H1 receptor in guinea pig primary sensory neurons: a relationship between H1 receptor mRNA-expressing neurons and peptidergic neurons

Pharmacological studies have suggested that a subgroup of primary sensory neurons is responsive to histamine via the histamine H1 receptor. We addressed this issue using in situ hybridization histochemistry with a cRNA probe for the guinea pig H1 receptor gene. About 15% of the trigeminal and lumber dorsal root ganglion (DRG) neurons, but none of nodose ganglion neurons, were intensely labeled with this probe. The H1 receptor mRNA-positive neurons were exclusively small in size, and were demonstrated to give rise to unmyelinated fibers by ultrastructural analysis of isolectin B4-labeling. However, the H1 receptor mRNA-expressing DRG neurons were not immunoreactive to substance P (SP) and calcitonin gene-related peptide (CGRP). A marked increase in the number of mRNA-positive DRG neurons were observed 1-5 days after a crush injury of the sciatic nerve (3-4-fold of the control value). These neurons turned mRNA-positive after the nerve crush were also mainly small-sized. The mRNA signals were detected in many peptidergic (SP/CGRP) neurons, in contrast to the normal state. On the other hand, in the neurons which showed intense labeling in the normal condition, the mRNA signals were down-regulated. These results suggest that primary sensory neurons include two kinds of H1 receptor-expressing sensory neurons, one expressing H1 receptor mRNAs in the normal state and the other up-regulating the mRNAs following the peripheral nerve damage.     

The motor nuclei and sensory neurons of the IIIrd, IVth, and VIth cranial nerves in the monitor lizard, Varanus exanthematicus

The motor nuclei of the oculomotor, trochlear, and abducens nerves of the reptile Varanus exanthematicus and the neurons that subserve the sensory innervation of the extraocular muscles were identified and localized by retrograde and anterograde transport of horseradish peroxidase (HRP). The highly differentiated oculomotor nuclear complex, located dorsomedially in the tegmentum of the midbrain, consists of the accessory oculomotor nucleus and the dorsomedial, dorsolateral, intermediate, and ventral subnuclei. The accessory oculomotor nucleus projects ipsilaterally to the ciliary ganglion. The dorsomedial, dorsolateral, and intermediate subnuclei distribute their axons to the ipsilateral orbit, whereas the ventral subnucleus, which innervates the superior rectus muscle, has a bilateral, though predominantly contralateral projection. The trochlear nucleus, which rostrally overlaps the oculomotor nuclear complex, is for the greater part a comma-shaped cell group situated lateral, dorsal, and medial to the medial longitudinal fasciculus. Following HRP application to the trochlear nerve, almost all retrogradely labeled cells were found in the contralateral nucleus. The nuclear complex of the abducens nerve consists of the principal and accessory abducens nuclei, both of which project ipsilaterally. The principal abducens nucleus is located just beneath the fourth ventricle laterally adjacent to the medial longitudinal fasciculus and innervates the posterior rectus muscle. The accessory abducens nucleus has a ventrolateral position in the brainstem in close approximation to the ophthalmic fibers of the descending trigeminal tract. It innervates the retractor bulbi and bursalis muscles. The fibers arising in the accessory abducens muscles form a loop in or just beneath the principal abducens nucleus before they join the abducens nerve root. The afferent fibers conveying sensory information from the extraocular muscles course in the oculomotor nerve and have their perikarya in the ipsilateral trigeminal ganglion, almost exclusively in its ophthalmic portion.     

Secretin mRNA in the Subdivision of Primary Sensory Neurons in the Trigeminal Ganglion of Rats

The primary sensory neurons use glutamate as a major neurotransmitter. Several neuropeptides are also found in these neurons. In our laboratory we demonstrated secretin-like immunoreactivity in primary sensory neurons of several species including human, rat and cat. In the present experiment utilizing in situ hybridization, we have demonstrated for the first time that secretin is not only immunostained but is also expressed in the primary sensory neurons of the trigeminal ganglion of male rats. In intact rats, secretin mRNA was not observed; we had to use intracerebroventricular colchicine administration to induce the expression of secretin. Secretin was expressed in about 5% of the cells in all the three subdivisions of the trigeminal ganglion. The secretin-synthetizing cells were large and medium sized, and their mean diameter was about 50 μm. When we compared the percentage and the size of secretin to that of calcitonin gene-related peptide (CGRP), substance-P (SP) and vasoactive intestinal polypeptide (VIP) cells, it was found that CGRP, SP and VIP are present in about 15–20% of the cells and their mean diameter is about 20–25 μm. The morphometric data indicate that secretin is present in a subdivision of neurons that is different from the subdivision of the CGRP, SP and VIP cells. It is suggested that secretin may modulate the function of the primary neurotransmitter.     

Colocalization of CGRP with 5-HT1B/1D receptors and substance P in trigeminal ganglion neurons in rats

Vasodilatation in the dura mater has been implicated in migraine pathogenesis. Anti-migraine triptan drugs block vasodilatation by binding to 5-HT1B/1D receptors localized on the peripheral sensory terminals and dural blood vessel smooth muscles. Previous studies suggest that calcitonin gene-related peptide (CGRP) released from Adelta-fibres plays a more important role than substance P (SP) released from C-fibres in inducing dural vasodilatation and that one of the antimigraine mechanisms of triptan drugs is inhibiting CGRP release. In the present study, the relationship between CGRP and 5-HT1B/1D receptors, and between CGRP and SP in the trigeminal ganglion neurons in rats was examined by double immunohistochemical staining. CGRP, 5-HT1B, 5-HT1D and SP-positive trigeminal ganglion neurons were all predominantly small and medium-sized. In the trigeminal ganglia, approximately 50% of CGRP-positive neurons were 5-HT1B positive. Similarly, approximately 55% of CGRP-positive neurons were 5-HT1D immunoreactive. Approximately 50% of CGRP-positive neurons were SP-positive, while 93% of SP-positive neurons were CGRP-positive, suggesting that nearly all SP-positive neurons also contain CGRP. The fibre types of the 5-HT1B- and 5-HT1D-positive neurons were further investigated with an antibody against the A-fibre marker 200-kDa neurofilaments (NF200). Approximately 46% of the 5-HT1B-positive and 43% of the 5-HT1D-positive trigeminal ganglion neurons were also NF200 positive, indicating that many A-fibre trigeminal neurons express 5-HT1B or 5-HT1D receptors. These results support the hypothesis that one important action of antimigraine drugs is the inhibition of CGRP release and that Adelta-fibres may play an important role in migraine pathogenesis.     

Retrograde neuronal labelling and double-staining immunohistochemistry of tachykinin- and calcitonin gene-related peptide-immunoreactive pathways in the carotid sinus nerve of the guinea pig

The origin of tachykinin- and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibres in the guinea pig carotid body and carotid sinus was determined by retrograde labelling of the carotid sinus nerve with Fluoro-gold and immunohistochemical double staining with fluorescein- and rhodamine-conjugated second antisera. Fluoro-gold-labelled perikarya with characteristic features of primary sensory neurones were numerous in the glossopharyngeal (petrosal) ganglion and occurred rarely in the closely attached superior vagal (jugular) ganglion. An efferent pathway from the brainstem could not be detected. Co-existence of tachykinin- and CGRP-LI was observed in 25-47% of labelled sensory neurones; less than 1% of Fluoro-gold-containing perikarya were exclusively stained by CGRP antiserum. Co-existence of tachykinin- and CGRP-LI was also demonstrated in nerve fibres of the carotid body and carotid sinus. Somatostatin-, cholecystokinin- and dynorphin-LI did not co-exist with tachykinin-LI in these fibres. Thus, tachykinin/CGRP-LI fibres in the carotid presso- and chemoreceptive areas exhibit a peptide pattern being generally characteristic for sensory fibres supplying great vessels in the guinea pig. In view of the present findings doubt is raised as to a primary involvement of these fibres in presso- or chemoreception, although a modulatory influence on these specific functions appears to be likely.     

Calcitonin gene-related peptide and cerebral blood vessels: distribution and vasomotor effects

The innervation of cerebral blood vessels by nerve fibers containing calcitonin gene-related peptide (CGRP) and the vasomotor effects of this peptide are described for a number of different mammalian species. CGRP-immunoreactive nerve fibers were present in the adventitia of cerebral arteries in all species examined (guinea pig, cat, rabbit, rat, and mouse). Numerous perikarya containing CGRP immunoreactivity are demonstrable in the trigeminal ganglion of all species. In the cerebral perivascular nerve fibers and in trigeminal perikarya, CGRP is often colocalized with substance P and neurokinin A. Marked interspecies differences exist both in the density of CGRP-immunoreactive nerve fibers and in the cerebrovascular levels measured with radioimmunoassay. The highest concentrations were observed in cerebral vessels from guinea pigs, the lowest concentration in rabbit vessels, and intermediate levels in the feline and human cerebral vasculature. CGRP is a potent dilator of cerebral arteries in all species examined (human pial, feline middle cerebral, rabbit, guinea pig and rat basilar arteries). The concentration of CGRP eliciting half-maximal responses ranged from 0.4 nM (human pial artery) to 3 nM (rat and rabbit basilar arteries). Pretreatment of cerebral arteries with low concentrations of either substance P (0.1 nM) or neurokinin A (3 nM) attenuated slightly the CGRP-induced relaxations of guinea pig basilar arteries. Calcitonin was found to be a very weak dilator of cerebral arteries from human and guinea pig. Thus, cardiovascular nerve fibers containing CGRP appear to be present in all mammalian species (although to varying degrees) and CGRP is invariably a potent dilator of the cerebral arteries for all species.     

Neurotensin-like immunoreactivity in presumptive baroreceptor neurons innervating the guinea pig carotid sinus

This study was carried out to determine the ultrastructure and origin of previously described neurotensin-like immunoreactive (NT-LI) nerve fibres in the wall of the carotid sinus of the guinea pig. In our degeneration experiments, these NT-LI axons were unaffected by surgical sympathectomy but disappeared in response to transection of the carotid sinus nerve, thus suggesting a sensory origin. This assumption could be supported by combined retrograde tracing and immunohistochemistry. Primary afferent neurons of the sensory glossopharyngeal (petrosal) ganglion projecting to the carotid sinus were identified by injecting the retrograde fluorescent tracer, Fluoro-Gold, into the sinus wall, and some of these neurons displayed NT-LI. Within the carotid sinus, the terminals of these NT-LI sensory neurons exhibited ultrastructural features characteristic of baroreceptor endings, i.e. axonal swellings filled with mitochondria and closely associated to elastin. However, many endings also fulfilling the ultrastructural criteria for baroreceptors were devoid of immunolabelling. Thus, we conclude that the NT-LI terminals constitute a subgroup rather than the entire population of baroreceptor endings within the guinea pig carotid sinus. With respect to the established pharmacological effects of NT in guinea pig, we propose an involvement of NT-LI fibres in the modulation of baroreception at the peripheral level.     

Histamine H1 receptor mRNA is expressed in capsaicin-insensitive sensory neurons with neuropeptide Y-immunoreactivity in guinea pigs

Histamine H1 receptor mRNA-expressing sensory neurons in guinea pigs are unmyelinated and are not immunoreactive to substance P and calcitonin gene-related peptide (CGRP) [Mol. Brain Res. 66 (1999) 24], which are implicated in the nociceptive transmission of the primary sensory system. In this study, we examined whether these H1 receptor mRNA-expressing neurons are sensitive to capsaicin by using in situ hybridization histochemistry. Of lumbar dorsal root ganglion (DRG) neurons in control animals, 17% were positive for CGRP. In guinea pigs neonatally treated with capsaicin (50 mg/kg), few CGRP-immunoreactive neurons were seen in the DRGs. However, the percentages of H1 receptor mRNA-expressing neurons (15-20%) and the intensity of the mRNA signals in these neurons were not affected by neonatal capsaicin treatment. We also revealed the presence of both capsaicin-sensitive and insensitive neuropeptide Y (NPY)-immunoreactive neurons in the DRGs. These neurons were exclusively small. H1 receptor mRNA was expressed in NPY-immunoreactive neurons in naive guinea pig DRGs. These results suggest that H1 receptor mRNA is expressed in capsaicin-insensitive DRG neurons with NPY-immunoreactivity in guinea pigs.