Rolf Uddman

Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: Trigeminal origin and co-existence with substance P

The presence of a population of nerve fibers containing immunoreactive calcitonin gene-related peptide (CGRP) has been demonstrated around cerebral arteries of the cat with immunocytochemistry and radioimmunoassay. CGRP immunoreactivity in the feline cerebral vasculature, as characterized by high-performance liquid chromatography, is similar to authentic rat CGRP. Numerous perikarya containing CGRP are present in the trigeminal ganglia, and surgical lesions of the trigeminal ganglia significantly reduce the levels of CGRP in the cerebral vasculature, suggesting that this cranial nerve is the principal origin of these cerebrovascular nerve fibers. As demonstrated by sequential immunocytochemistry, CGRP coexists with substance P both in the trigeminal ganglion and nerve fibers around cerebral blood vessels. The presence of CGRP in the cerebrovascular trigeminal innervation provides further versatility and complexity for this sensory afferent system putatively involved in the transmission of intracranial pain.

Neuropeptide Y: Cerebrovascular innervation an vasomotor effects in the cat ☆

Avian pancreatic polypeptide (APP) has been proposed to be a neurotransmitter in a subpopulation of sympathetic nerves. Here, we present immunocytochemical and pharmacological evidence that the structurally related peptide, neuropeptide Y (NPY), is likely to be the biologically active material in these nerves. Cerebral arteries from cats are invested with a dense network of NPY-containing nerve fibres, as demonstrated by immunocytochemistry. This immunoreaction is abolished by prior removal of the superior cervical ganglion. NPY causes strong contractions of cerebral arteries in vitro whereas APP has small effects on the vasomotor reactivity. The NPY-induced contractions were not inhibited by the alpha 2-adrenoceptor antagonist rauwolscine (10(-7) M) or the 5-hydroxytryptamine antagonist ketanserin (10(-7) M). The contractions were, however, sensitive to calcium removal or to the calcium antagonist diltiazem (10(-4) M).

Pituitary adenylate cyclase activating peptide is a sensory neuropeptide: Immunocytochemical and immunochemical evidence

Pituitary adenylate cyclase activating peptide (PACAP) is a vasoactive intestinal peptide-like hypothalamic peptide occurring as two variants, PACAP-27 and the C-terminally extended PACAP-38. Immunoreactive PACAP has also been demonstrated in the enteric nervous system and in the innervation of the respiratory tract. We have examined the possibility that PACAP occurs in the sensory nervous system of the rat. Immunocytochemistry revealed PACAP in numerous nerve fibres in the superficial layer of the dorsal horns of the spinal cord, in nerve cell bodies (most of them of small size) of the spinal ganglia and trigeminal ganglia and in nerve fibres running close to and within the surface epithelium in the skin of the nose, the tongue, the larynx-trachea, and the urinary bladder as well as around the ducts of the submandibular gland. In all locations, PACAP co-existed with calcitonin gene-related peptide and substance P, the PACAP-immunoreactive fibres and cell bodies constituting a subpopulation of those storing substance P and/or calcitonin gene-related peptide. Additional PACAP-immunoreactive fibres not associated with epithelia seemed to lack calcitonin gene-related peptide and substance P. Capsaicin treatment reduced the density of PACAP- and calcitonin gene-related peptide/substance P-immunoreactive fibres in the tissues examined. On the whole, the immunocytochemical results agreed with those obtained by radioimmunoassay for PACAP and CGRP. The data favour a role for PACAP in primary sensory neurons.

Retrograde Tracing of Nerve Fibers to the Rat Middle Cerebral Artery with True Blue: Colocalization with Different Peptides:

The origin of nerve fibers to the rat middle cerebral artery was studied by retrograde tracing with the fluorescent tracer True Blue (TB) in combination with immunocytochemistry to known perivascular peptides. Application of TB to the middle cerebral artery labeled nerve cell bodies in the ipsilateral superior cervical ganglion, the otic ganglion, the sphenopalatine ganglion, the trigeminal ganglion, and the cervical dorsal root ganglion at level C2. A few labeled nerve cell bodies were seen in contralateral ganglia. Judging from the number and intensity of the labeling, the superior cervical ganglion and the trigeminal ganglion and dorsal root ganglion at level C2 contributed most to the innervation. A moderate number of nerve cell bodies were labeled in the sphenopalatine and otic ganglia. The TB-labeled nerve cell bodies were further examined for the presence of neuropeptides. For that purpose antibodies raised against neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) were used. A considerable portion of the TB-labeled nerve cell bodies in the superior cervical ganglion contained NPY. About half of the labeled nerve cell bodies in the sphenopalatine and otic ganglia contained VIP. In the trigeminal ganglion and in the dorsal root ganglion at level C2, one-third of the TB-labeled nerve cell bodies were CGRP-immunoreactive, while only few nerve cell bodies contained SP. The study provides direct evidence for the origin of cerebrovascular peptidergic nerve fibers and demonstrates that not only ipsilateral but also contralateral ganglia contribute to the innervation of the cerebral circulation.

Messenger molecules and receptor mRNA in the human trigeminal ganglion

The presence and distribution of neuromessenger molecules and receptor mRNA in human trigeminal ganglion was studied with immunocytochemical, in situ hybridisation and RT-PCR techniques. Immunofluorescence staining revealed that calcitonin gene-related peptide (CGRP) immunoreactive (-ir) neurons occurred in high numbers, constituting 36-40% of all nerve cell bodies in the ganglion. Accordingly, in situ hybridisation demonstrated CGRP mRNA in a large portion of the trigeminal neurons. A small number of the nerve cell bodies showed substance P (SP)-ir, (18%), nitric oxide synthase (NOS)-ir (15%), and pituitary adenylate cyclase activating peptide (PACAP)-ir (20%). Double immunostaining revealed that only few CGRP-ir neurons also were NOS-ir (less than 5%). The C-terminal flanking peptide of neuropeptide Y, C-PON, was not visible in any of the nerve cell bodies studied. Agarose gel electrophoresis of the RT-PCR products from the ganglia demonstrated the presence of mRNA corresponding to CGRP1, NPY Y1 and Y2, and VIP1 receptors. These results suggest both sympathetic and parasympathetic influence on the activity in the trigeminal ganglion.

VIP (vasoactive intestinal polypeptide)-containing nerves of intracranial arteries in mammals.

SummaryImmunohistochemical and radioimmunochemical investigations have shown, in various species, the occurrence of numerous nerve fibres containing vasoactive intestinal polypeptide (VIP) in connection with blood vessels of the central nervous system. Pial arteries from pig, cat, and rat have the richest supply of VIP nerve fibres; those of cow, dog, guinea pig and hamster have an intermediary number of nerves, while only few are found in pial arteries from the monkey, rabbit, gerbil, and mouse. The regional variation in VIP-nerve density follows the order: cerebral arteries > cerebellar arteries > basilar > vertebral > spinal cord arteries. Unilateral extirpation of either the pterygopalatine or the superior cervical ganglia does not affect the amount or distribution of VIP fibres in the wall of brain vessels of the ipsilateral side.Measurement of the VIP content by radioimmunoassay shows mean concentrations in the pial arteries varying between 19 and 82 pmol/g tissue wet weight. Regional and species variations in measured VIP levels are similar to the variations in distribution of immunoreactive nerve fibres.

Neuropeptide Y-like immunoreactivity in perivascular nerve fibres of the guinea-pig

The distribution of perivascular nerve fibres displaying neuropeptide Y-like immunoreactivity was studied in the guinea-pig. Generally, neuropeptide Y fibres were numerous around arteries and moderate in number around veins. In the heart, immunoreactive fibres were numerous in the auricles and the atria (epi- and endocardium) whereas the ventricles had a more scarce supply. The coronary vessels were richly supplied with fibres. Around large elastic and muscular arteries the fibres formed well developed plexuses. Small arteries in the respiratory tract, the gastrointestinal tract and the genito-urinary tract received a particularly rich supply. In the liver, spleen and kidney only few perivascular fibres were seen. Since immunoreactive fibres around blood vessels disappeared upon surgical or chemical sympathectomy, and sequential immunostaining with antisera against dopamine-beta-hydroxylase (a marker for adrenergic neurons) and against neuropeptide Y revealed their co-existence, it is concluded that neuropeptide Y fibres around blood vessels are sympathetic and adrenergic.

Pituitary adenylate cyclase activating polypeptide expression in sensory neurons

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel vasoactive intestinal peptide (VIP)-like peptide isolated from ovine hypothalami. The presence of PACAP-like immunoreactivity was recently demonstrated in nerve cell bodies of sensory ganglia in the rat. Since PACAP belongs to a large family of chemically related neuropeptides, we have, in the present study, tried to establish the synthesis of PACAP in neurons of sensory ganglia, using in situ hybridization with a 35S-labelled oligonucleotide probe complementary to PACAP mRNA. The expression of PACAP was compared to that of calcitonin gene-related peptide (CGRP) using a radiolabelled CGRP oligonucleotide probe. The PACAP probe labelled small to medium-sized neurons in the trigeminal ganglion and dorsal root ganglia at different levels, indicating the presence of PACAP mRNA. The CGRP probe labelled nerve cell bodies of varying size, outnumbering those labelled by the PACAP probe. In dorsal root ganglia, cells expressing PACAP constituted c. 10% and those expressing CGRP 46% of the total number of nerve cell bodies. Expression of PACAP was seen in a small subpopulation of cells expressing CGRP. We conclude that PACAP is synthesized in a subpopulation of neurons of sensory ganglia in the rat. Therefore, the recently described effects of PACAP--cutaneous vasodilation, potentiation of oedema formation and depression of nociceptive spinal reflexes--may be physiological and related to neurogenic inflammation and modulation of pain transmission.

Nerve Fibers Containing Neuropeptide Y in the Cerebrovascular Bed: Immunocytochemistry, Radioimmunoassay, and Vasomotor Effects

Perivascular nerve fibers containing neuropeptide Y (NPY)-like immunoreactivity were identified around cerebral blood vessels of human, cat, guinea pig, rat, and mouse. The major cerebral arteries were invested by dense plexuses; veins, small arteries, and arterioles were accompanied by few fibers. Removal of the superior cervical ganglion resulted in a reduction of NPY-like material in pial vessels and dura mater. Pretreatment with 6-hydroxydopamine or reserpine reduced the number of visible NPY fibers and the concentration of NPY in rat cerebral vessels. Sequential immuno-staining with antibodies toward dopamine-β-hydroxylase (DBH) (an enzyme involved in the synthesis of noradrenaline) and NPY revealed an identical localization of DBH and NPY in nerve cell bodies in the superior cervical ganglion and in perivascular fibers of pial blood vessels, suggesting their coexistence. Administration of NPY in vitro resulted in concentration-dependent contractions that were not modified by a sympathectomy. The contractions induced by noradrenaline, 5-hydroxytryptamine, and prostaglandin F2α and the dilator responses to calcitonin gene-related peptide were not modified by NPY in rat cerebral arteries. However, the constrictor response to NPY was reduced by 70% in the presence of the calcium entry blocker nifedipine, and abolished following incubation in a calcium-free buffer. These data suggest an interaction of NPY at a postsynaptic site, which for induction of contraction may open calcium channels in the sarcolemma of cerebral arteries.

Pituitary adenylate cyclase-activating peptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract

SummaryPituitary adenylate cyclase-activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers displaying PACAP immunoreactivity were found in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. A moderate supply of PACAP-immunoreactive fibers was seen in the nasal mucosa of guinea pigs. Few to moderate numbers of PACAP-containing fibers occurred in the tracheo-bronchial wall of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. The fibers were distributed beneath the epithelium, around blood vessels and seromucous glands, and among bundles of smooth muscle. In the lungs, the immunoreactive fibers were observed close to small bronchioli. A few PACAP-immunoreactive nerve cell bodies were seen in the sphenopalatine and otic ganglia of guinea pigs. Simultaneous double immunostaining of the respiratory tract of sheep and ferrets revealed that all PACAP-containing nerve fibers stored VIP. We suggest that neuronal PACAP may take part in the regulation of smooth muscle tone and glandular secretion.