NOD-like receptors in the human upper airways: a potential role in nasal polyposis.

BACKGROUND: Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are newly discovered cytosolic receptors belonging to the pattern-recognition receptor family. They detect various pathogen-associated molecular patterns, triggering an immune response. The knowledge about these receptors, and their role in health and disease, is limited. The aim of the present study was to characterize the expression of NOD1, NOD2, and NALP3 in the human upper airways. METHODS: Surgical samples were obtained from patients with tonsillar disease (n = 151), hypertrophic adenoids (n = 9), and nasal polyposis (n = 24). Nasal biopsies were obtained from healthy volunteers (n = 10). The expression of NOD1, NOD2, and NALP3 was analyzed using real-time PCR and immunohistochemistry. RESULTS: Expression of NOD1, NOD2, and NALP3 mRNA and protein were seen in all tissue specimens. The NLR mRNA was found to be higher in nasal polyps than in normal nasal mucosa, and local steroid treatment reduced the NLR expression in polyps. In contrast, tonsillar infection with Streptococcus pyogenes or Haemophilus influenzae did not affect the NLR expression. CONCLUSIONS: The present study demonstrates the presence of NLRs in several upper airway tissues and highlights a potential role of NLRs in chronic rhinosinusitis with polyps.

Nod1, Nod2 and Nalp3 receptors, new potential targets in treatment of allergic rhinitis?

BACKGROUND: Recently, a new set of pattern-recognition receptors, the nucleotide-binding oligomerization domain (Nod)-like receptors (NLRs), have emerged. Their activation, either by allergens or microbes, triggers an inflammatory response. The knowledge about NLRs in human airways is limited. AIM OF THE STUDY: To investigate presence of NLRs in the human nose of healthy individuals and patients with intermittent allergic rhinitis outside and during pollen season. METHODS: The expression of Nod1, Nod2, and Nalp3 in nasal biopsies was determined with real-time RT-PCR and immunohistochemistry. Cultured primary human nasal epithelial cells (HNECs) were analyzed using real-time RT-PCR and flow cytometry to further verify the presence of NLRs in the epithelium. RESULTS: Immunohistochemical analysis revealed presence of Nod1, Nod2, and Nalp3 in the nasal epithelium. This was corroborated in cultured HNECs. Patients suffering from symptomatic allergic rhinitis exhibited lower Nod1 and Nalp3 mRNA levels than both controls and patients during pollen season. Nod2 expression was found in all specimens tested, but no differences were seen between the three groups. CONCLUSION: Nod1, Nod2, and Nalp3 receptors were found to be present in the human nose. The expression of Nod1 and Nalp3 were down-regulated during pollen season among patients with allergic rhinitis. This opens up for new insights and novel therapeutic strategies in inflammatory airway disease.

Neurokinin A and galanin in the thyroid gland: neuronal localization.

The distribution of neurokinin A (NKA) and galanin (GAL) in the thyroid gland of several species was examined with immunocytochemistry. NKA-immunoreactive fibers were observed around blood vessels and follicles in all species examined, whereas GAL-immunoreactive fibers were found in mice and rats only. NKA-containing fibers were more numerous than GAL-containing fibers. All thyroid NKA-containing fibers harbored substance P (SP), and the majority of them stored calcitonin gene-related peptide (CGRP) as well. Most thyroid GAL-immunoreactive fibers contained NKA, SP, and CGRP; in a minor population GAL coexisted instead with vasoactive intestinal peptide. Cervical vagotomy (extirpation of the nodose ganglion) reduced the number of NKA- and GAL-containing fibers in the thyroid by approximately 50%. The jugular ganglion and cervical dorsal root ganglia are fairly rich in GAL-, NKA/SP-, and CGRP-containing cell bodies, which presumably represent the source of GAL-, NKA/SP-, and CGRP-containing fibers in the thyroid. The thyroid ganglion is rich in vasoactive intestinal peptide nerve cell bodies, which presumably project to the thyroid gland; a minor proportion of these cell bodies was found to contain GAL as well. Although the distribution of NKA and GAL fibers in the thyroid suggests that the two peptides are involved in the regulation of local blood flow and follicular cell activity, neither NKA nor GAL had any influence on thyroid hormone release as tested in conscious mice.

Neuropeptide Y in the thyroid gland: neuronal localization and enhancement of stimulated thyroid hormone secretion.

Nerve fibers displaying neuropeptide Y (NPY) immunoreactivity occurred around blood vessels and follicles in the thyroid gland of several mammals, including man. Removal of the superior cervical ganglia or chemical sympathectomy (6-hydroxydopamine treatment) markedly reduced the number of NPY-containing nerve fibers in the thyroid and eliminated norepinephrine-containing fibers. NPY-immunoreactive nerve cell bodies were numerous in the superior cervical ganglia of mouse, rat, and guinea pig. Not unexpectedly, therefore, immunocytochemistry involving sequential staining with antibodies against dopamine-beta-hydroxylase (an enzyme marker for adrenergic neurons) and NPY revealed that most of the NPY fibers were adrenergic. As tested in mice in vivo, NPY did not affect basal or norepinephrine-stimulated thyroid hormone secretion, but enhanced isoprenaline-, TSH-, and vasoactive intestinal polypeptide-induced iodothyronine release. These findings suggest a modulatory role for NPY in the control of thyroid hormone secretion.

Calcitonin gene-related peptide in thyroid nerve fibers and C cells: effects on thyroid hormone secretion and response to hypercalcemia.

Calcitonin gene-related peptide (CGRP) in the thyroid has a dual localization to nerve fibers around blood vessels and follicles and to parafollicular (C) cells. CGRP was found to coexist with substance P (SP) in most of the nerve fibers; a few CGRP fibers seemed to lack SP, and a few SP fibers seemed to be devoid of CGRP. In the C cells, CGRP coexisted with calcitonin (CT). Cervical vagotomy (extirpation of the nodose ganglion) eliminated approximately 50% of the CGRP/SP fibers in the thyroid without any overt influence on CGRP/CT in the C cells. Removal of the superior cervical ganglion or chemical sympathectomy (6-hydroxydopamine treatment) affected neither thyroid CGRP/SP nerve fibers nor CGRP/CT-storing C cells. CGRP nerve cell bodies were numerous in the jugular-nodose ganglionic complex (notably in the jugular portion); in many of them, CGRP coexisted with SP. A few scattered CGRP nerve cell bodies also occurred in the laryngeal ganglion, whereas none was found in the thyroid ganglion. Hypercalcemia evoked by vitamin D2 treatment, which is known to degranulate thyroid C cells, reduced the thyroid content of both CGRP and CT. As tested in mice in vivo, CGRP and SP alone or together had no effect on basal or TSH- or isoprenaline-induced thyroid hormone secretion. Vasoactive intestinal peptide-stimulated iodothyronine release, on the other hand, was enhanced by CGRP, but not by SP. SP had no effect on combined vasoactive intestinal peptide-CGRP-stimulated iodothyronine release. These findings suggest that CGRP participates in the control of thyroid hormone secretion and that, like CT, CGRP in the C cells is under control of the serum calcium level.

Peptide histidine isoleucine amide stimulates thyroid hormone secretion and coexists with vasoactive intestinal polypeptide in intrathyroid nerve fibers from laryngeal ganglia.

Peptide histidine isoleucine amide (PHI) and vasoactive intestinal polypeptide (VIP) are fragments of the same precursor molecule, prepro-VIP, and coexistence of the two peptides is, therefore, to be expected. Nerve fibers displaying PHI and VIP immunoreactivity occurred around blood vessels and follicles in the thyroid gland of several species. Sequential staining with antibodies against PHI and VIP revealed coexistence of the two peptides in the same population of nerve cell bodies in ganglia situated along the laryngeal nerves and in intrathyroid nerve fibers. Chemical sympathectomy (6-hydroxydopamine treatment), surgical sympathectomy (removal of the superior cervical ganglia), and unilateral cervical vagotomy (removal of the nodose ganglion) failed to affect the number and distribution of PHI/VIP fibers in the thyroid gland. Taken together, the findings suggest that both the perivascular and interfollicular PHI/VIP fibers originate in laryngeal ganglia. PHI weakly stimulated basal thyroid hormone secretion in mice in vivo, but did not influence the response to TSH or VIP. PHI had no effect on calcitonin secretion in rats. Like VIP, PHI may play a physiological role in the regulation of thyroid hormone secretion.

Neuropeptides in the thyroid gland: distribution of substance P and gastrin/cholecystokinin and their effects on the secretion of iodothyronine and calcitonin.

Previously, vasoactive intestinal polypeptide was localized to intrathyroidal nerve fibers. It stimulates iodothyronine secretion in mice. In the present study two populations of nerve fibers containing substance P and gastrin/cholecystokinin (CCK)-like immunoreactivity, respectively, were demonstrated in the thyroid gland of several mammals. The substance P fibers occurred around blood vessels and follicles, whereas the gastrin/CCK fibers occurred mainly around follicles. In the chicken thyroid and ultimobranchial glands only substance P-containing fibers could be demonstrated. Such fibers were particularly numerous in the ultimobranchial gland. CCK-4, CCK-8, and substance P did not increase thyronine secretion measured as release of radioiodine into the circulation of mice pretreated with Na125I and T4. The TSH-induced release of radioiodine was also unaffected. Calcitonin secretion in rats was stimulated by CCK-4, CCK-8, substance P, and vasoactive intestinal polypeptide.

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.

Nerve fibres containing gastrin-releasing peptide around pial vessels.

Nerve fibres containing immunoreactive gastrin-releasing peptide (GRP) were demonstrated around pial blood vessels of cat, guinea pig, rat, and mouse. A sparse supply was found around spinal cord blood vessels, whereas the choroid plexus seemed to be devoid of GRP fibres. Sympathectomy did not affect the number or distribution of the GRP fibres. The administration of neither GRP nor its closely related analogue, bombesin, contracted or dilated feline pial arteries in vitro.

Perivascular substance P: occurrence and distribution in mammalian pial vessels.

Nerve fibres containing immunoreactive substance P (SP) were demonstrated in the wall of cerebral blood vessels of several mammalian species. Pial arteries of cat and guinea-pig were richly supplied with SP nerve fibres, while those of rat, rabbit, pig, and man had a moderate number. SP fibres were more numerous in pial vessels belonging to the rostral parts of the circle of Willis as compared to more caudally located blood vessels. In cat and guinea-pig, blood vessels in the choroid plexus were surrounded by few SP nerve fibres; also spinal cord blood vessels of cat contained few such fibres.

Cerebral veins: fluorescence histochemistry, electron microscopy, and in vitro reactivity.

Pial veins, choroid plexus veins, and the cerebri magna vein were investigated with regard to their ultrastructural organization, adrenergic nerve supply, and in vitro reactivity. The vessel walls consisted of a continuous layer of endothelial cells, large amounts of collagenous material, and occasional pericytes. Smooth muscle cells were observed only in a few specimens from the cerebri magna vein. All veins were surrounded by adrenergic nerve fibres. Potassium (124 mM) and noradrenaline (10(-5) - 10(-4) M) induced small contractions (0.2-0.5 mN) of isolated veins during in vitro conditions. The magnitude of these responses was less than one-tenth of that obtained in small pial arteries.

Tachykinins (substance P, neurokinin A, neuropeptide K, and neurokinin B) in the cerebral circulation: vasomotor responses in vitro and in situ.

The vasomotor responses of tachykinins have been studied in the cerebral vasculature of human, pig, cat, and guinea pig. Substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and neuropeptide K (NPK) induced concentration-dependent relaxations of precontracted cerebral arteries in all species when examined by a sensitive in vitro technique. In addition, the relaxant responses to SP, NKA, and NKB were studied in cat pial arterioles by peptide microapplication in situ. In human pial vessels, the order of relaxant potency was SP greater than NKB greater than NKA greater than NPK; in the pig middle cerebral artery, there was no difference in potency between the tachykinins; in the cat middle cerebral artery, SP = NKB greater than NKA = NPK; and in the guinea pig basilar artery, SP much greater than NPK = NKA greater than NKB. Responses induced by SP, NKA, and NKB in the cat were comparable in vitro and in situ. Removal of the endothelium abolished relaxation induced by all four tachykinins. The relaxant responses of guinea pig basilar arteries to SP, NKA, and NPK were competitively antagonized by the SP antagonist Spantide. However, Spantide lowered the Imax of the NKB concentration-response curve without any rightward shift, suggesting action at a different site than the other tachykinins. In the guinea pig basilar artery, the relaxation seems to be exerted via a NK-1 receptor subtype while the receptor subtype is more unclear in cerebral arteries from human, cat, and pig.(ABSTRACT TRUNCATED AT 250 WORDS)

PACAP, a VIP-like peptide: immunohistochemical localization and effect upon cat pial arteries and cerebral blood flow.

Pituitary adenylate cyclase activating peptide (PACAP) is a vasoactive intestinal polypeptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers containing PACAP immunoreactivity were present in the adventitia and the adventitia-media border of cat cerebral arteries. Double immunostaining revealed that PACAP-immunoreactive nerve fibers constituted a subpopulation of the VIP-containing fibers. PACAP effected a concentration-dependent relaxation of feline middle cerebral arteries that had been precontracted with prostaglandin F2 alpha. The maximum relaxation, 24 and 34% of precontraction, was achieved with PACAP-38 and PACAP-27, respectively, at a concentration of 10(-6) M. In cats anesthetized with alpha-chloralose, intracerebral microinjection of PACAP effected a moderate increase in cerebral blood flow. The maximal increase (18.6 +/- 6%) was observed following the injection of 5 micrograms PACAP.

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.

Helospectin-like peptides: immunochemical localization and effects on isolated cerebral arteries and on local cerebral blood flow in the cat.

Helospectin I and II and helodermin are nonamidated, vasoactive intestinal peptide (VIP)-like peptides, isolated from the salivary gland venom of the lizards Heloderma suspectum and Heloderma horridum. Helospectin I has 38 amino acid residues and differs from helospectin II in that it has an additional serine residue at the C-terminus. Numerous nerve fibers containing helospectin-like immunoreactivity (LI) and a few fibers containing helodermin-LI were present in the adventitia and at the adventitia-media border of cat cerebral arteries. In the sphenopalatine ganglion, numerous nerve cell bodies containing helospectin-LI were seen. Double immunostaining revealed that helospectin-LI nerve cell bodies coexisted with VIP-containing cell bodies. Radioimmunoassay showed high levels of helospectin-LI in extracts of cerebral vessels from the circle of Willis (27.4 pg/mg [wt/wt]). Helospectin I and II and helodermin (10(-10) to 10(-6) mol/L) produced concentration-dependent relaxations of feline middle cerebral arteries amounting to 50% to 80% of precontraction induced by U46619. The maximum effects and the potency were similar to that of VIP. Neither of these peptides elicited endothelium-dependent relaxations. Intracerebral microinjection of helospectin and helodermin produced a moderate concentration-dependent increase of the cerebral blood flow of alpha-chloralose anesthetized cats. The maximum increase (21 +/- 5%) was observed after the injection of 5 microg helodermin, whereas 16 +/- 7% was seen with helospectin I and 19 + 5% with helospectin II. The results suggest that helospectin/helodermin-like peptides co-localize with VIP in perivascular nerve fibers originating in the sphenopalatine ganglion. They seem to have strong and potent vasodilator effects.

Involvement of perivascular sensory fibers in the pathophysiology of cerebral vasospasm following subarachnoid hemorrhage.

The involvement of perivascular sensory fibers containing substance P (SP) and calcitonin gene-related peptide (CGRP) in the events occurring in conjunction with subarachnoid hemorrhage (SAH) has been studied in a rat model. Two days after blood injection, the time point at which maximum vasoconstriction is occurring in this model, immunocytochemistry and radioimmunoassay showed a reduction in SP- and CGRP-like immunoreactivity (LI). The quantitative measurements revealed a significant 50% reduction of CGRP-LI and a slight reduction of SP-LI in SAH as compared to controls. This partial reduction in neurotransmitter content (denervation) caused no change in the sensitivity of the rat basilar artery to SP or CGRP as studied using a sensitive in vitro method. However, the maximum relaxant response to CGRP was increased from 52 to 81% (p less than 0.05), while there was no change in the maximum SP-induced relaxations. It is suggested that not only a pre-, but also a postsynaptic modulation of perivascular sensory fibers may occur in experimental SAH.

Acetylcholine and vasoactive intestinal peptide in cerebral blood vessels: effect of extirpation of the sphenopalatine ganglion.

The innervation of cerebral blood vessels by nerve fibers containing acetylcholinesterase (AChE) and vasoactive intestinal peptide (VIP) and the vasomotor effects of the two neurotransmitters have been analyzed in the rat following the uni- or bilateral removal of the sphenopalatine ganglion (SPG), which is thought to be the major origin of this innervation. Histochemistry of AChE-positive nerve fibers and the immunoreactivity toward VIP revealed only a 30% reduction in the innervation pattern of the rostral part of the cerebral circulation following the operation. At approximately 4 weeks postoperatively, the original nerve network was restored. Quantitative measurements of cholineacetyltransferase activity and VIP revealed similar reductions in the levels of collected large cerebral arteries at the base of the brain and in small pial vessels overlying the cerebral cortex at the various postoperative times following uni- or bilateral removal of the SPG. The two techniques thus complemented each other. Vasomotor reactivity to acetylcholine (ACh) and VIP was examined in proximal segments of the middle cerebral artery at the various postoperative times. Generally, the removal of the SPG had no effect on the responses to ACh or VIP. The evidence indicates that only approximately one-third of the cholinergic/VIP innervation of the rostral part of the cerebral circulation originates in the SPG.

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 immunostaining with antibodies toward dopamine-beta-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 F2 alpha 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.

NOS-containing neurons in the rat gut and coeliac ganglia.

Nitric oxide (NO) is considered an important inhibitory neurotransmitter in the gut. Nitric oxide synthase (NOS)-containing neurons were visualized by immunocytochemistry using antibodies against neuronal NOS in the oesophagus, the gastrointestinal tract and the coeliac ganglion of rat. NOS-containing nerve cell bodies were numerous in the myenteric but fewer in the submucous ganglia all along the gut. Synthesis of NOS in enteric nerve cell bodies was confirmed by in situ hybridization, demonstrating the presence of NOS mRNA. Varicose nerve fibers formed extensive networks in the circular smooth muscle and the myenteric ganglia. The pyloric sphincter contained abundant NOS-containing nerve fibers. NOS-containing nerve terminals were frequently found around the Brunner glands in the duodenum; scattered nerve terminals occurred in the gastric and colonic mucosa and around blood vessels in the submucosa all along the gut. The neuronal cell bodies in the coeliac ganglion were non-immunoreactive but frequently surrounded by baskets of NOS-immunoreactive nerve fibers. Double staining for NOS and neuropeptides in oesophagus, stomach and small and large intestine revealed that a small subpopulation of the NOS-containing nerve cell bodies stored in addition vasoactive intestinal peptide (VIP), and in oesophagus, stomach and small intestine also neuropeptide Y (NPY). However, NOS-containing nerve terminals, particularly those in the circular muscle of the gut, frequently contained VIP throughout the gut; in the oesophagus, stomach and the small intestine they contained also NPY.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal pathways to the rat thyroid revealed by retrograde tracing and immunocytochemistry.

The distribution and origin of the nerve fibres innervating the rat thyroid were studied by immunocytochemistry, retrograde tracing and denervation experiments. Immunocytochemistry revealed nerve fibres containing noradrenaline, neuropeptide Y, vasoactive intestinal peptide, peptide histidine-isoleucine, galanin, substance P, neurokinin A and calcitonin gene-related peptide around blood vessels and follicles. Many of these transmitter candidates were found to co-exist with each other in different combinations in different subpopulations of neurons. Sympathectomy eliminated all noradrenaline- and noradrenaline/neuropeptide Y-containing fibres in the thyroid. Cervical vagotomy eliminated about 50% of the galanin-, substance P- and calcitonin gene-related peptide-containing fibres. Local denervation (removal of the thyroid ganglion and the thyroid nerve) eliminated all galanin- and substance P-immunoreactive fibres and the majority of noradrenaline-, noradrenaline/neuropeptide Y-, vasoactive intestinal peptide- and calcitonin gene-related peptide-containing fibres in the thyroid gland. Injection of True Blue into the thyroid gland labelled cell bodies in the thyroid ganglion, the laryngeal ganglion, the superior cervical ganglion, the jugular-nodose ganglionic complex, the dorsal root ganglia (C2-C5) and the trigeminal ganglion. Judging from the number of labelled nerve cell bodies, the superior cervical ganglion and the thyroid ganglion contribute most to the thyroid innervation, while the laryngeal ganglion and the trigeminal ganglion contribute least. The True Blue-labelled ganglia were examined for the presence of various populations of nerve cell bodies (only major populations are listed). The thyroid ganglion harboured neuropeptide Y, vasoactive intestinal peptide and galanin/vasoactive intestinal peptide cell bodies (in order of predominance); the laryngeal ganglion galanin/vasoactive intestinal peptide, vasoactive intestinal peptide and calcitonin gene-related peptide cell bodies; the superior cervical ganglion noradrenaline/neuropeptide Y and noradrenaline cell bodies; the jugular ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies; the nodose ganglion vasoactive intestinal peptide and vasoactive intestinal peptide/galanin cell bodies; the dorsal root ganglia (C2-C5) and the trigeminal ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)