peptide-containing neurons projecting to the vocal cords of the rat: retrograde tracing and...

Journal of the Autonomic Nervous System, 30 (1990) 179-192 179 Elsevier

JANS 01064

Peptide-containing neurons projecting to the vocal cords of the rat" retrograde tracing and immunocytochemistry

Anders Luts 1, Rolf U d d m a n 2, Torsten Grundi tz 2 and Frank Sundler 1

l Department of Medical Cell Research, Lund, Sweden, 2 Department of Otolaryngology, General Hospital, University of Lund, Malm6, Sweden

(Received 2 October 1989) (Revision received and accepted 16 February 1990)

Key words: R e t r o g r a d e t rac ing ; N e u r o p e p t i d e s ; I m m u n o c y t o c h e m i s t r y ; L a r y n x ; Voca l cords

Abstract

The distribution and origin of neuropeptide Y-, vasoactive intestinal peptide- and calcitonin gene-related peptide-containing nerve fibers and adrenergic (dopamine-fl-hydroxylase-containing) fibers in the rat larynx were studied by retrograde tracing and selective denervations in combination with immunocytochemistry. An injection of the retrograde tracer True Blue to the right vocal cord resulted in the appearance of labelled nerve cell bodies in the ipsi- and contralateral superior cervical and stellate ganglia, the thyroid ganglia, the jugular-nodose ganglionic complexes, in the ipsilateral trigeminal and dorsal root ganglia at levels C 2 and C 3 and in local tracheal ganglia. Judging from the number of labelled nerve cell bodies, the jugular-nodose ganglionic complexes, dorsal root ganglia and superior cervical ganglia provide the greater part of the vocal cord innervation. Most of the True Blue-labelled nerve cell bodies in the superior cervical and stellate ganglia contained neuropeptide Y. In the thyroid ganglia the majority of labelled nerve cell bodies contained vasoactive intestinal peptide. In the jugular-nodose ganglionic complex and the dorsal root ganglia the majority of the labelled nerve cell bodies stored calcitonin gene-related peptide. Retrograde tracing and denervation studies revealed that all noradrenaline- and the majority of neuropeptide Y-containing nerve fibers emanate from the superior cervical and stellate ganglia. A minor population of neuropeptide Y-containing nerve fibers originate in local tracheal ganglia. The vasoactive intestinal peptide-containing nerve fibers originate in the thyroid ganglion and local tracheal ganglia, whereas calcitonin gene-related peptide-containing nerve fibers emanate from the dorsal root ganglia (C2-C3), the trigeminal ganglia and the jugular-nodose ganglia.

Introduction

The resp i ra to ry t ract is i n n e r v a t e d by ne rve f ibers of sympa the t i c , p a r a s y m p a t h e t i c a n d of

/ ' .

sensory nature. The major neuropeptides m the airways are neuropeptide Y (NPY), vasoactive intestinal pep t ide (VIP) a n d the coex is t ing n e u r o - pep t ide pep t i de h i s t id ine i so leuc ine (PHI ) , sub-

Correspondence: A. Luts, Department of Medical Cell Re- search, Biskopsgatan 5, S-223 62 Lund, Sweden.

s t ance P (SP) a n d the coex is t ing n e u r o p e p t i d e n e u r o k i n i n A ( N K A ) a n d c a l c i t o n i n gene- re l a t ed pep t i de ( C G R P ) [26]. Some of the f ibers are k n o w n to der ive f rom ne rve cell bod ies in the super io r cervical , j u g u l a r - n o d o s e , a n d dorsa l roo t gang l i a at the cervical a n d thorac ic level [14,15]. In the l a r y n x ne rve f ibers are k n o w n to be n u m e r o u s [1,12,16,20]. The o r ig in of these f ibers is n o t k n o w n .

The p r e sen t s t u d y descr ibes the d i s t r i b u t i o n a n d or ig in of N P Y - , VIP- a n d C G R P - c o n t a i n i n g ne rve f ibers as well as ad rene rg i c ne rve f ibers in the vocal cords of the ra t revealed b y re t rograde

0165-1838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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tracing and selective denervations in combination with immunocytochemistry.

Material and Methods

Retrograde neuronal tracing and immunocytochemistry

Nine female rats, weighing 200-250 g were used. A suspension of the retrograde tracer True Blue (TB) (Prof. D. Dann, Institut fiir Pharmazie und Lebensmittelchemie der Friedrich-Alexander Universit~it, Erlangen, F.R.G.) (2-3%, w/v) in distilled water was stored frozen and thawed im- mediately before use. The larynx was dissected free under chloralose anesthesia. TB (0.05-0.1 ~tl) was slowly injected via a Hamilton syringe into the laryngeal wall, deposing TB into one of the vocal cords. Two different routes of administra- tion were used: (1) In four rats the right vocal cord was injected from the serosal surface through the wall of the larynx. (2) In five other rats the larynx was opened by a longitudinal midline inci- sion and the fight vocal cord was reached for injection from the lumen. After injection the wounds were sutured and the rats were left to recover. They were killed 3 weeks after the injection. The larynx was dissected out in order to localize the TB injection site and to exclude leakage of tracer. The following ganglia were dissected out for examination of TB-labelled nerve cell bodies:

the superior cervical, the stellate (only after injection from the lumen), the otic, the sphenopalatine, the trigeminal, the jugular-nodose ganglionic complexes and the dorsal root ganglia at level C2-Th 2. To study the laryngeal, local tracheal and thyroid ganglia (c.f. [8]) the larynx-thyroid region was sectioned serially.

The specimens were fixed by immersion in a mixture of 2% formaldehyde and 0,2% picric acid solution in 0.1 M phosphate buffer (pH 7.2) over- night and thoroughly rinsed in Tyrode solution containing 10% sucrose. They were frozen on dry ice and serially sectioned at 15/~m thickness in a cryostat. The sections were then examined for tracer fluorescence. Sections harbouring labelled nerve cell bodies were processed for immunocytochemistry, reexamined and photographed in an epi-illumination fluorescence microscope fitted with appropriate filter settings for viewing TB (excitation 365 nm, emission 420 nm) and fluo- rescein isothiocyanate (FITC) (excitation 490 nm, emission 520 nm) fluorescence alternately. Anti- bodies to the following neuropeptides were used: NPY, VIP and CGRP. Dopamine-/3-hydroxylase (DBH) antibodies were used to identify adrenergic nerves [17]. Details on the antisera are given in Table I. The sections were exposed to the primary antiserum for 24 h in a moist chamber. The site of the antigen-antibody reaction was revealed by application of FITC-labelled antibodies against immunoglobulin G (Milab, Maim/3, Sweden) in a

TABLE I

Details of the antisera used

Antigen Code Raised against Raised in Working dilution Source Reference

DBH 2012 Bovine adrenomedullary Rabbit 1 : 320 ETI, Allendale, 17 DBH USA

NPY 8404 Protein conjugated Rabbit 1 : 640 Milab, Malm~5, 5 synthetic porcine NPY Sweden

VIP 7852 Unconjugated pure Rabbit l : 640 Milab, Malmr, 6 natural porcine VIP Sweden

VIP 8701 Unconjugated pure Guinea-pig 1 : 640 Milab, MaimS5, 7 natural porcine V| P Sweden

CGRP 8427 Protein conjugated Rabbit 1 : 1280 Milab, MalmiS, 23 synthetic rat C GR P Sweden

CGRP 8513 Protein conjugated Guinea-pig 1 : 1280 Milab, MalmtS, 8 synthetic rat CGRP Sweden

dilution of 1 :320 for 1 h at room temperature. Control sections were exposed to antiserum that had been preabsorbed with an excess amount of the antigen (10-100/~g of synthetic or pure natural peptide per ml diluted antiserum). Addition- ally, each of the peptide antisera was tested for cross-reaction with the other peptides examined (10-100/xg of peptide per ml diluted antiserum). No such cross-reaction was found. Cross-reactions with still other peptides or proteins containing amino acid sequences recognized by the different antisera cannot be excluded. It is appropriate, therefore, to refer to the immunoreactive material as NPY-like, VIP-like and so forth. For brevity, however, the shorter terms NPY, VIP etc will be used henceforth.

To reveal coexistence of neuropeptides in the same neuron a simultaneous double immunostain- ing method was used [3,23]. This technique utilizes primary antibodies raised in different species and secondary antibodies labelled with different fluo- rophores. In the present study, peptide antibodies raised in guinea-pigs (Table I) were used in combination with antibodies raised in rabbits, the labels being FITC and tetramethyl rhodamine isothiocyanate (TRITC), respectively. The sections were first incubated with one of the pair of peptide antibodies and then with FITC-labelled secondary antibodies. The sections were then incubated with the other peptide antibody. The latter antibodies were visualized by using TRITC-labelled secondary antibodies. The sections were examined in a fluorescence microscope fitted with appropriate filter settings for viewing FITC and TRITC fluorescence alternately (Zeiss No. 09 and 15, respectively). The frequency of immunoreactive nerve fibers was estimated semiquantitatively by two independent observers and graded as few, moderate in number and numerous. For quantification of TB-labelled nerve cell bodies ganglia were sectioned serially and labelled nerve cell bodies were counted in every third section of each ganglion. The results were expressed as the number of labelled cell bodies per ganglion.

Denervations Female rats (150-200 g; at least four in each

group) were subjected to one of the following

181

denervations under chloralose anesthesia: (1) bilateral removal of the superior cervical ganglia; (2) bilateral removal of the superior cervical ganglia and the right thyroid ganglion; (3) bilateral removal of the superior cervical ganglia and the thyroid ganglia; (4) unilateral cervical vagotomy - removal of the right nodose ganglion including vagal branches to the larynx.

Five age-matched (sham-operated) animals served as controls.

The animals were killed 1 week after denervation and the larynx was dissected out for im- munocytochemical examination. The elimination of adrenergic nerve fibers from the larynx was verified by using antiserum against DBH.

R ~

Peptide-containing nerve fibers at vocal cord level The larynx at the vocal cord level was ex-

amined for the presence of nerve fibers storing NPY, VIP and CGRP (Fig. 1). The NPY-containing nerve fibers were few in number around blood vessels and few to moderate in number among bundles of muscle fibers and in the subepithelial layer. Fibers were not detected within the surface epithelium or around seromucous glands. VIP- containing nerve fibers had a widespread distribution. They were numerous around seromucous glands and blood vessels; moderate numbers were seen in the subepithelial layer and among bundles of muscle fibers. VIP-containing nerve fibers were not detected within the epithelium. In deeper layers of the larynx a few VIP-containing nerve cell bodies were seen. A particularly rich supply of CGRP-containing nerve fibers was found within the epithelium and in the subepithelial layer. A few additional fibers were seen to run among bundles of muscle fibers and close to blood vessels.

Adrenergic (DBH-containing) fibers at vocal cord level

DBH-containing nerve fibers were moderate in number among bundles of muscle and around blood vessels; only a few were found around seromucous glands. In the subepithelial layer only

182

Fig. 1. Sections from laryngeal wall at the vocal cord level immunosta ined for CGRP (a), NPY (b), and VIP (c and d). Nerve fibers containing CGRP are abundant within the epithelium and in the subepithelial layer (a). NPY-containing fibers are few to moderate in number around blood vessels (arrow), among bundles of muscle and in the subepithelial layer (b). VIP-containing fibers are moderate in number in the subepithelial layer (e). A few VIP-containing nerve cell bodies together with scattered VIP-containing

fibers are found in a nerve trunk (nt) in deeper layers of the larynx (d). ( × 300). (Publisher's magnification x 0.88)

183

Fig. 2. Injection site of True Blue (TB) in the fight vocal cord applied from the serosal surface. A minor leakage is seen through the injection canal. ( × 150).

a few fibers were detected, while no fibers were seen in the epithelium.

Retrograde TB-labelling After the injection of TB, either from the serosal

or luminal surface, an accumulation of intensely bright blue fluorescence marking the injection site was seen in the submucosa of the vocal cord (Fig. 2). When injected from the serosal surface there were also small deposits of TB in the injection canal. Three weeks after the TB injection a bright blue fluorescence had appeared in nerve cell bodies in several of the examined ganglia. Often, the labelled cell bodies were not randomly distributed but restricted to one region of the ganglion. All animals examined harboured markedly higher numbers of TB-labelled cell bodies in ipsilateral ganglia than in contralateral ones.

An injection from the serosal surface resulted in the appearance of numerous moderately to intensely TB-labelled nerve cell bodies in the ipsi- and contralateral superior cervical ganglia and jugular-nodose ganglionic complexes (Table II; Figs. 3 and 4). A large number of moderately TB-labelled cell bodies occurred in the thyroid ganglia, whereas TB-labelling could not be detected in the laryngeal ganglia (Fig. 3). In the ipsilateral otic ganglion a few weakly TB-labelled cells could be seen. In the ipsilateral trigeminal ganglion a moderate number of weakly to moderately labelled nerve cell bodies occurred; they were few in the contralateral ganglion (Fig. 5). In the ipsilateral dorsal root ganglia at levels C2-C 6 a moderate to rich number of intensely TB-labelled cell bodies could be detected (Fig. 5). A few weakly labelled nerve cell bodies were found at

184

Fig. 3. The superior cervical ganglion (a and b) and the thyroid ganglion (e and d). Concomitant visualization of TB (a) and NPY (h); and TB (e) and VIP (d). TB- and peptide-containing cell bodies are indicated by arrows. ( x 250).

TABLE II

The mean number of labelled nerve cell bodies in various ganglia after application of True Blue (TB) to the right vocal cord from the serosal surface

Ganglia Mean number of TB-labeUed cell bodies

Right Left

Superior cervical 94 70 Thyroid 20 10 Nodose 103 41 Jugular 120 33 Otic 3 0 Trigeminal 18 7 Dorsal root C 2 26 6

C a 22 8 C 4 15 2 C 5 17 2 C 6 13 5 C 7 5 1 TH 1 2 0 TH2 1 0

levels C7-Th2; l abe l l ed n e r v e cel l b o d i e s w e r e n o t

seen fu r the r cauda l ly . I n do r sa l r o o t gang l i a o f t he

o p p o s i t e s ide a few w e a k l y T B - l a b e l l e d cel l b o d i e s

o c c u r r e d at levels C 2 - C 7.

T B in j ec t ed f r o m the l u m i n a l su r f ace r e su l t ed

in a lower n u m b e r o f T B - l a b e l l e d cel l b o d i e s in

the e x a m i n e d gang l i a ( T a b l e I I I ) . A s t rong T B - l a -

be l l ing was o b s e r v e d in a m o d e r a t e to h igh n u m -

be r s o f ne rve cel l b o d i e s in the ipsi- a n d con-

TABLE III

The mean number of labelled nerve cell bodies in various ganglia after application of True Blue (TB) to the right vocal cord from the luminal surface

Ganglia Mean number of TB-labelled cell bodies

Right Left

Superior cervical 30 4 Stellate 8 0 Thyroid 15 8 Nodose 80 11 Jugular 103 15 Otic 0 0 Trigeminal 4 0 Dorsal root C 2 10 0

C 3 1 0

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TABLE IV

The approximate percentage of True Blue (TB)-labelled cell bodies that displayed NPY, VIP and CGRP immanoreactivity, after an injection of TB into the right vocal cord from the serosal surface

Ganglia Approximate percentage of TB-labelled cell bodies containing

NPY VIP CGRP

Superior cervical 20 0 0 Thyroid 0 40 0 Nodose 0 0 10 Jugular 0 0 20 Otic 15 0 0 Trigeminal 0 0 45 Dorsal root C2 0 0 25

C 3 0 0 25 (7-4 0 0 25 C 5 0 0 20 C 6 0 0 25 C 7 0 0 25 TH 1 0 0 30 TH 2 0 0 25

t r a l a t e ra l s u p e r i o r ce rv i ca l gangl ia , the j u g u l a r -

n o d o s e g a n g l i o n i c c o m p l e x e s a n d the t h y r o i d

gangl ia . L o c a l l a r y n g e a l a n d t r achea l gang l i a

h a r b o u r e d few T B - l a b e l l e d cel l bodies . I n the

ips i l a t e ra l t r i g e m i n a l g a n g l i o n a few w e a k l y to

m o d e r a t e l y l abe l l ed n e r v e cel l b o d i e s occu r red .

T h e ips i l a t e ra l s te l la te g a n g l i o n a n d dorsa l r o o t

TABLE V

The approximate percentage of True Blue (TB)-labelled cell bodies that displayed NP Y, VIP and CGRP immunoreactivity, after an injection of TB into the right vocal cord from the luminal surface

Ganglia Approximate percentage of TB-labelled cell bodies containing

NPY VIP CGRP

Superior cervical 30 0 0 Stellate 20 0 0 Thyroid 0 40 0 Nodose 0 0 15 Jugular 0 0 15 Otic 0 0 0 Trigeminal 0 0 30 Dorsal root C2 0 0 30

C 3 0 0 30

~ ~

• H

?

~ i,

~

L

¸¸

~ •

ganglia at levels C 2 and C 3 harboured a few to a moderate number of labelled cell bodies (Fig. 5). In the cervical root ganglia at the lower levels and in the thoracic root ganglia TB-positive cell bodies could not be detected. Furthermore, TB-labelled cell bodies were not found in the otic ganglia, the sphenopalatine ganglia or in the laryngeal ganglia.

Peptides in TB-labelled nerve cell bodies Ganglia harbouring TB-labelled nerve cell bod-

ies were examined for the presence of NPY, VIP and CGRP (Figs. 3, 4 and 5; Tables IV and V). The main population of TB-labelled nerve cell bodies in the superior cervical ganglia and the stellate ganglia were found to contain NPY (Fig. 3). None of these TB-labelled nerve cell bodies stored CGRP or VIP. In the thyroid ganglia the TB-labelled nerve cell bodies harboured VIP but neither NPY nor CGRP (Fig. 3). The few weakly labelled nerve cell bodies in the otic ganglia contained NPY but neither VIP nor CGRP. TB- labelled cell bodies in small local laryngeal and tracheal ganglia stored NPY and VIP. In the jugular-nodose ganglionic complexes, the trigeminal ganglia and the dorsal root ganglia, a proportion of TB-labelled nerve cell bodies contained CGRP (Figs. 4 and 5). None of the labelled cell bodies in these ganglia contained NPY or VIP.

Denervations Bilateral sympathectomy eliminated all adren-

ergic nerve fibers in the larynx and a major proportion of the NPY-containing fibers. Most of the NPY-containing fibers still present did also store VIP (Fig. 6). The CGRP-containing nerve fibers were unaffected.

Bilateral sympathectomy and extirpation of the right thyroid ganglion resulted in an elimination of the adrenergic nerve fibers and a marked reduction of VIP- and NPY-containing fibers on the right side. Only a few scattered NPY-containing fibers were found and they did also store VIP. The CGRP-containing nerve fibers were not affected.

187

Also on the left side there was a reduction of VIP-containing fibers, albeit to a lesser extent.

Bilateral sympathectomy and extirpation of the thyroid ganglia caused an elimination of adrenergic nerves and an almost total disappearance of VIP- and NPY-containing nerve fibers. In the very few remaining fibers NPY was found to coexist with VIP.

Cervical vagotomy did not effect the frequency of NPY, VIP- or CGRP-containing nerve fibers in the laryngeal wall.

Discussion

Previous studies have revealed a dense supply of nerve fibers in the larynx [2,12,13,16,18,24]. In the guinea-pig, adrenergic fibers, originating in cervical sympathetic ganglia, are distributed in the connective tissue, around blood vessels and among acini of seromucous glands [1,9,10]. Denervation experiments on the monkey have revealed that the larynx receives its sensory innervation from the internal branch of the superior laryngeal nerve and to a minor extent from the recurrent nerve and the external branch of the superior laryngeal nerve [11,25]. In the canine larynx SP-immunoreactive nerve fibers, of presumably sensory origin, have been observed close to small blood vessels and around the submucous glands [20]. Retrograde tracing with horseradish peroxidase of the superior laryngeal nerve of the cat labelled nerve cell bodies in the nodose ganglion, the jugular ganglion and the superior cervical ganglion. Thus, although the superior laryngeal nerve is composed primarily of sensory and parasym- pathetic fibers it also seems to contain a small population of sympathetic fibers [14]. Injection of TB into the tracheal wall of the rat has revealed labelled nerve cell bodies in the nodose and jugular ganglia and in dorsal root ganglia. A proportion of these nerve cell bodies contained SP and CGRP [22]; the combination of these peptides is

Fig. 4. The jugular (a and b) and the nodose (e and d) ganglia. Concomitant visualization of TB (a and c) and CGRP (b and d). TB- and peptide-containing cell bodies are indicated by arrows. ( × 300).

188

Fig. 5. The trigerninal (a and b) and the cervical dorsal root (c and d) ganglial Concomitant visualization of TB (a and c) and CGRP (b and d). One of the TB-labelled cells does not contain CGRP but has a surrounding network of CGRP (d). TB- and

peptide-containing cell bodies are indicated by arrows. ( x 250).

i

189

b

i

d Fig. 6. The laryngeal wall after surgical bilateral sympathectomy. Double imrnunostaining for NPY (a and e) and VIP (b and d). A few NPY-containing nerve fibers around a blood vessel (bv = blood vessel) (a) and close to bundles of muscle (c) contain VIP (b and

d). ( x 250). (Publisher's magnification x 0.88)

charac ter i s t ica l ly found in sensory neurons (c.f. [23]). Toge ther these f indings suggest that several d i f ferent gangl ia con t r ibu te to the la ryngeal sensory input .

In the present s tudy, the vocal cords of the ra t were injected with TB in order to trace the or igin

of the very rich a u t o n o m i c nervous supply to this region. The use of TB as a t racer is well docu- men ted (c.f. [8,21]). TB is t r anspor t ed f rom the nerve t e rmina l s to the nerve cell bodies wi thout any leakage or t ransgangl ion ic passage [21]. The c o m b i n a t i o n of TB- and F I T C - i m m u n o f l u o r e s -

190

otic

jugu lar ggl. t r igeminal g0, ~ II / gg,

nodose ggl, ~,

ggL

supe r i o r , ~ dorsa l c e r v i c a l roo t

ggl. - - r ' ggl.

Fig. 7. Schematic outline of the contributions made by the different cervical ganglia to the vocal cord innervation. The extent of each contribution is reflected in the thickness of the

arrows.

innervation of the vocal cords [1,10]. Thus, surgical sympathectomy eliminated all DBH-containing nerve fibers and a great proportion of the NPY-containing nerve fibers. It has been shown previously that the majority of the NPY-containing nerve fibers in the airways are adrenergic and thus store also DBH (c.f. [26]), while a minor population harbours VIP instead (Luts, unpub- lished observation); these latter fibers were not eliminated by sympathectomy. The majority of the NPY/VIP-containing fibers seem to originate in the thyroid ganglia; after removal of the thyroid ganglia only very few scattered NPY/VIP-containing fibers could be detected. The present study thus adds the thyroid ganglia as another source of neurons projecting to the vocal cord level.

cence has been described previously [8,19,22,27]. In the present study, nerve cell bodies containing TB were found in several ganglia, indicating mul- tiple sources for the innervation of the vocal cords (Fig. 7).

The superior cervical ganglia, the jugular ganglia, the nodose ganglia and the cervical dorsal root ganglia provide major contributions, while the trigeminal ganglia and stellate ganglia contrib- ute less. A difference in numbers of TB-labelled cells was observed between the two injection methods. The less traumatic technique of injecting TB from the serosal side of the larynx gave rise on the whole to a greater number of TB-labelled cells. This injection also labelled - albeit weakly - cell bodies in the otic ganglia and in the dorsal root ganglia at the thoracic level. It cannot be excluded that injection by this route causes some leakage through the injection canal thereby giving rise to a more widespread labelling (Fig. 1). An injection of TB from the luminal surface of the larynx, on the other hand, did not cause labelling of cell bodies in the otic ganglia or in the dorsal root ganglia at the thoracic level. Conceivably, the more restricted localization achieved with injection from the luminal side reflects tracing more selective for the vocal cords.

The results of the present study and previous denervation experiments support the view that the superior cervical ganglia provide the adrenergic

Acknowledgements

The authors received Grant support from the Swedish Medical Research Council (projects no 4499, 6859), and from the Medical Faculty, Uni- versity of Lund.

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peptide-containing neurons projecting to the vocal cords of the rat: retrograde tracing and...

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