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Parasympathetic innervation of cutaneous blood vessels examined by retrograde tracing in the rat lower lip
Journal of the Autonomic Nervous System, 32 (1991) 153-158 © 1991 Elsevier Science Publishers B.V. 0165-1838/91/$03.50
153
JANS 01121
Parasympathetic innervation of cutaneous blood vessels examined by retrograde tracing in the rat lower lip Akira Kaji 1, Toshihiro M a e d a 2 and Shohei W a t a n a b e 1 Departments of ~ Dermatology and 2 Anatomy, Shiga University of Medical Science, Otsu, Japan (Received 29 May 1990) (Revision received and accepted 27 September 1990)
Key words: Rat lip; Cutaneous blood vessel; Parasympathetic nerve fiber; Otic ganglion; Retrograde tracing; Vasoactive intestinal polypeptide Abstract The origin of vasoactive intestinal polypeptide (VIP)-immunoreactive and acetylcholinesterase (AChE)-positive perivascular nerve fibers in the lower lip of rats was investigated using the retrograde tracer, wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase gold complex (WGAapoHRP-Au), in combination with immunohistochemistry and enzyme histochemistry, by comparing the cells of origin of projection to the parotid gland. After the application of the tracer to the lip, small- to medium-sized nerve cells were labelled exclusively in the ipsilateral otic ganglion. Most of them showed moderate VIP-immunoreactivity and AChE activity. In contrast, injection into the parotid gland resulted in labelling of mostly large-sized cells of the otic ganglion which showed intense VIP-immunoreactivity and AChE activity. These results confirmed that the parasympathetic innervation of the rat lip originates from the otic ganglion. It was further suggested that there are at least two subpopulations in the otic ganglion cells, different from each other in size and in VIP-immunoreactivity, which separately innervate the salivary gland and the blood vessels.
Introduction
Sympathetic aminergic fibers innervate cutaneous blood vessels, but it is still unclear whether the parasympathetic nervous system regulates cutaneous blood flow or not. With the development of immunohistochemistry, vasodilator neuropeptides, including vasoactive intestinal polypeptide (VIP), have been observed around cutaneous blood vessels [2,4,5,8-10,24]. Recently, some of the cranial vessels, e.g. cerebral vessels [3,7,22,27,28], meningeal artery [25], superficial
Correspondence: A. Kaji, Department of Dermatology, Shiga University of Medical Science, Seta, Otsu, Japan.
temporal artery [26] and arterioles supplying the tooth pulp [21], have been reported to receive VIP-immunoreactive (IR) nerve supply from parasympathetic ganglia, as revealed by denervation experiments or with retrograde tracers. Our previous denervation study revealed that the VIPIR nerve fibers around blood vessels in the lip skin of rats originate from the parasympathetic otic ganglion [11]. The purpose of this study was to confirm the origin of the perivascular VIP-IR and acetylcholinesterase (AChE)-positive fibers, and to compare the origin cells with the ganglion cells innervating the salivary glands by employing the newly developed gold-labelled retrograde tracer, wheat germ agglutinin conjugated to enzymati-
154 cally inactive horseradish peroxidase gold complex (WGAapoHRP-Au), in combination with VIP-immunohistochemistry.
Materials and Methods
Tracer injection Colloidal-gold-labelled W G A a p o H R P was used as a retrograde tracer and was prepared according to Basbaum and Menetrey [1]. Eleven Sprague-Dawley male rats (150-250 g) were divided into three groups, After anesthesia with sodium pentobarbital (50 m g / k g ; i.p.), 3 /~1 of W G A a p o H R P - A u were injected into the following sites: left side of the lower lip in six rats of the first group, left parotid gland in two rats of the second group, and left side of three different sites of the hairy skin (submandibular, subocular, and preauricular in three rats each) of the third group. Tissue processing All the rats were perfused 48 h following the injection. The perfusion via the heart was carried out as follows. Blood was first washed out with 100 ml of 0.01 M phosphate-buffered saline. Next 200 ml of an ice-cold fixative solution containing 2% paraformaldehyde and 0.3% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4, was used. The otic ganglia, the superior cervical ganglia, the trigeminal ganglia, all from both sides, and the injected tissues, were excised, immersed in a post-fixative solution containing 2% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, p H 7.4, for a day and stored in 15% sucrose in 0.1 M phosphate buffer, p H 7.4, until sectioning. Twenty-/~m thick sections were cut with a cryotome. The free-floating sections were then placed in phosphate-buffered saline, pH 7.4. Histochernistry and immunohistochemistry The sections were silver-enhanced in a physical developer in the dark for 60 min at room temperature according to Menetrey [17] or in the silver enhancement reagent (IntenSE, Janssen) for 20 rain at room temperature in normal daylight. After
the silver-intensification procedure, some sections were stained by Tago's method for AChE activity [23]. The reaction was detected by the appearance of brown polymerized diaminobenzidine (DAB) product to discriminate AChE activity from silver particles implying the uptake of the tracer. The other sections were stored in 0.3% Triton-X in phosphate-buffered saline for at least three days for VIP-immunohistochemistry which was carried out as previously described [11]. The sections were incubated with VIP antiserum (1 : 10 000, Calbiochem) for 5 days at 4°C, followed by biotinylated lgG for 2 h and ABC for 1 h at room temperature. For the detection of peroxidase activity, 0.025% DAB solution and 0,001% hydrogen peroxidase solution were used.
Analysis of the labelled otic ganglion cells Sixteen sections of otic ganglia taken from six rats in which the tracer were injected into the lower lips were used for cell size measurements. Eighty-seven labelled cells in these sections were measured along their long axis by a scale mounted in the microscope eyepiece. The same measurements were carried out on 63 labelled cells in 18 sections from two rats in which the tracer were injected into the parotid glands. Cell size distribution histograms representing the overall proportions of labelled cells of both groups were made from these measurements.
Results
Tracer injection into the lip In the sections of the W G A a p o H R P - A u injected lip, particles visualized by silver intensification were localized among the vessels and were diffusely scattered between collagen bundles and in phagocytic cells. Retrograde labelling was found exclusively in the ipsilateral otic ganglion, where a small number of nerve cell bodies, less than 5% of the total population, were labelled. No labelling was found in the contralateral ganglion. The labelled cells were evenly distributed in the ganglion without an apparent pattern. The labelling was seen in the cytoplasm of the perikarya a n d / o r in dendrites.
155 I~J I n j e c t i o n into the L o w e r ( x =17.0, SD=3.7, N=87) []
30]
Lip
I n j e c t i o n into the P a r o t i d Gland (x=35.3, SD=7.8. N=63)
ii ii
z z z ii
//
10-15 1 5 - 2 0 2 0 - 2 5 2 5 - 3 0 30-35 35-4(
IR
40-45 45-50" IJm
The labelled cells were small to medium in size, ranging from 12 to 25 g m in diameter with a mean of 17.0 g m (Fig. 1), and the larger cells in the ganghon displayed no labelling. Immunohistochemical analysis for VIP showed moderate to weak staining intensity in the labelled cell bodies (Fig. 2A). AChE activity in these cells was also weak to moderate (Fig. 2B). HRP-labelled cells were also found both in the ipsilateral superior cervical ganglion and in the ipsilateral trigeminal ganglion. However VIP-immunoreactivity was not detected in any of the labelled cells. No labelled cell was found in the contralateral ganglia.
Diameter of Labeled C e l l s
Fig. 1. Cell size histogram of labelled neurons of the otic ganglion by WGAapoHRP-Au injection into the lower lip (hatched bars) and the parotid gland (open bars). ,X, mean; SD, standard deviation; N, number of cells.
Tracer injection into the parotid gland Labelled ganglion cells in the otic ganglion were mainly large in size, with a range from 15 to
Fig. 2. Otic ganglion. Combination of retrograde tracing with W G A a p o H R P - A u and VIP-immunohistochemistry (A) or AChE histochemistry (B). WGAapoHRP-Au was injected into the lower lip of rats and was silver enhanced. In A, WGAapoHRP-Au-labelled cells (arrowheads) displayed a moderate intensity of VIP-immunoreactivity. In B, five small labelled cells (arrows) show weak AChE activity. No labelling was found in the large nerve cell bodies with intense AChE activity. Bar = 10 gm.
156
Fig. 3. Otic ganglion. Combination of retrograde tracing with W G A a p o H R P - A u and VIP-immunohistochemistry (A) or A C h E histochemistry (B). The tracer was injected into the parotid gland. In A, two large labelled cells (arrowheads) show VIP-irnmunoreactivity. In B, two AChE active cells of large size (arrows) are labelled. Bar = 10 ~tm.
47.5/~m in diameter and a mean of 35.3/xm. They were usually larger in size than those demonstrated in the otic ganglion of lip-injected rats (Fig. 1). VIP-immunoreactivity was seen clearly in all labelled cell bodies (Fig. 3A). AChE staining showed moderate to intense reactivity (Fig. 3B).
Tracer injection into the hairy skin of the face No labelling was found in any otic ganglion cell bodies of the three rats, in which W G A a p o H R P Au was injected into one of three areas of the facial skin.
Discussion
The nerve fibers supplying the blood vessels in the skin of the lip in rats are stained by VIP-immunohistochemistry and AChE enzyme histochemistry [5,11]. In the present study, apphcation
of the retrograde tracer, WGAapoHRP-Au, to the lip, resulted in labelling of the ipsilateral otic ganglion and most of the labelled cells showed VIP-immunoreactivity. As the injection site has no other structures innervated with VIP-IR fibers and the tracer was localized within the injection site with minimal spread, as previously reported [1], the labelling in the VIP-IR ganglion nerve cell bodies is considered to be the result of the tracer uptake from the VIP-IR perivascular fibers. This implies that the perivascular VIP-IR fibers in the lower lip originate from the ipsilateral otic ganglion. An additional finding in the present study supports this interpretation. No notable VIP-immunoreactivity was shown in labelled cells either in the superior cervical ganglion or in the trigeminal ganglion; indeed no VIP-immunoreactivity was detected in these ganglia under the conditions used in this study. Accordingly, the present tracer experiment confirmed our previous study that the
157
lip vessels in the rat are supplied by the parasympathetic otic neuron, as evidenced by the finding that VIP-IR and AChE-positive perivascular fibers disappear after the removal of the otic ganglion [11]. Since neurons in the otic ganglion of the rat are homogeneous in their levels of choline acetyltransferase immunoreactivity [12], it is likely that both large and small neurons are cholinergic despite the variable intensity of AChE staining. It is almost certain therefore that some of the cutaneous vessels are supplied with ACh- and VIPcontaining parasympathetic nerve fibers, as is known to be the case in the salivary gland [14] and nasal mucosa [15]. In this context, of interest is the difference in size and in VIP-immunoreactivity between the ganglion cells innervating the cutaneous vessels and those supplying the parasympathetic nerve fibers to the parotid gland. When the tracer was applied to the lip, small-sized and moderately to weakly VIP-IR ganglion cell bodies were labelled, while when it was applied to the parotid gland, mainly large-sized and intensely VIP-IR ganglion cells were labelled. This implies that the same single neuron in the otic ganglion does not project to the lip cutaneous vessels and to the parotid gland, but there are two ganglion cell populations which are at least different from each other in cell size and in VIP-immunoreactivity as well as in target organ. It is likely that the VIP which itself is contained in these two different postganglionic neuronal system functions in different ways in the target organ. VIP in the perivascular parasympathetic nerve fibers in the lip may exert direct vasodilative effects as manifested on other blood vessels [6,19,20], in contrast to its function as a neuromodulator in the secretory response of the salivary glands [13,16]. Thus, the dual sympathetic and parasympathetic innervation of the cutaneous vessels in the lip [11] is further supported in the present study. However, it remains to be elucidated whether a dual innervation of cutaneous vessels is present also in the skin of the head or not. In the cat, extracerebral vessels such as the branches of the external carotid artery are supplied by VIP-IR fibers of varying fluorescence intensity [5]. In the rat, the meningeal and superficial temporal arteries
are shown supplied by parasympathetic nerve fibers originating from the pterygopalatine ganglion and the otic ganglion, to which VIP-IR neurons contribute [25,26]. In the guinea-pig, arterioles supplying the tooth pulp are innervated by otic ganglion [21]. In man, AChE-positive perivascular fibers in the face were observed more than a quarter of a century ago [18]. In addition, VIP-IR nerve fibers are frequently found around the cutaneous blood vessels in the human face (unpublished data). However, the tracer experiment of the present study could not demonstrate parasympathetic perivascular innervation in the hairy facial skin of the rat, where neither intense AChE activity nor notable VIP-immunoreactivity was found. It is likely that parasympathetic innervation is sparse or absent in the peripheral vessels of the facial skin of the rat. If the intense AChE activity accompanied by VIP-immunoreactivity in the human cutaneous blood vessels of the face represents parasympathetic innervation, the species difference may be due to an increase of the complexity in the control of cutaneous blood flow in the face of humans, by comparison with the hairy skin of the rat. This question remains to be elucidated.
References 1 Basbanm, A.I. and Menetrey, D., Wheat germ agglutininapo HRP gold: a new retrograde tracer for fight- and electron-microscopic single- and double-label studies, J. Comp. Neurol., 261 (1987) 306-318. 2 Dalsgaad, C.J., Jonsson, C.E., Ht~kfelt, T. and Cuello, A.C., Localization of substance P-immunoreactive nerve fibers in the human digital skin, Experientia, 39 (1983) 1018-1020. 3 Edvinsson, L., Hara, H. and Uddman, R., Retrograde tracing of nerve fibers to the rat middle cerebral artery with true blue: colocalization with different peptides, J. Cereb. Blood Flow Metab., 9 (1989) 212-218. 4 Furness, J.B., Papka, R.E., Della, N.G., Costa, M. and Eskay, R.L., Substance P-like immunoreactivity in nerves associated with the vascular system of Guinea-Pigs, Neuroscience, 7 (1982) 447-459. 5 Gibbins, I.L., Brayden J.E. and Bevan J.A., Perivascular nerves with immunoreactivity to vasoactive intestinal polypeptide in cephalic arteries of the cat: distribution, possible origins and functional implications, Neuroscience, 13 (1984) 1327-1346. 6 Goadsby, P.J. and Macdonald, G.J., Extracranial vasodila-
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tation mediated by vasoactive intestinal polypeptide (VIP), Brain Res., 329 (1985) 285-288. Hara, H., Hamill, G.S. and Jacobowitz, D.M., Origin of cholinergic nerves to the rat major cerebral arteries: coexistence with vasoactive intestinal polypeptide, Brain Res. Bull., 14 (1985) 179-188. Hartschuh, W., Weihe, E. and Reinecke, M., Peptidergic (neurotensin, VIP, substance P) nerve fibers in the skin. Immunohistochemical evidence of an involvement of neuropeptides in nociception, pruritus and inflammation, Br. J. Dermatol., 109 (1983) suppl 25, 14-17. Hartschuh, W., Reinecke, M., Weihe, E. and Yanaihara, N., VIP-immunoreactivity in the skin of various mammals: immunohistochemical, radioimmunological and experimental evidence for a dual localization in cutaneous nerves and Merkel cells, Peptides, 5 (1984) 239-245. Johansson, O., Evidence for PHI-immunoreactive nerve fibers in the human skin: coexistence with VIP?, Med. Biol., 64 (1986) 67-73. Kaji, A., Shigematsu, H., Fujita, K., Maeda, T. and Watanabe, S., Parasympathetic innervation of cutaneous blood vessels by vasoactive intestinal polypeptide-irnmunoreactive and acetylcholinesterase-positive nerves: histochemical and experimental study on rat lower lip, Neuroscience, 25 (1988) 353-362. Leblanc, G.G., Trimmer, B.A. and Landis, S.C. Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: Coexistence with vasoactive intestinal peptide and choline acetyltransferase, Proc. Natl. Aead. Sci. U.S.A., 84 (1987) 3511-3515. Lundberg, J.M., HSkfelt, T., Schultzberg, M., Uvn~is-Wallensten, K., K~hler, C. and Said, S.I., Occurrence of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in certain cholinergic neurons of the cat: evidence from combined immunohistochemistry and acetylcholinesterase staining, Neuroscience, 4 (1979) 1539-1559. Lundberg, J., Angg~rd, A., Fahrenkrug, J., HOkfelt, T. and Mutt, V., Vasoactive intestinal polypeptide in cholinergic neurons of exocrine glands: functional significance of coexisting transmitters for vasodilation and secretion, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1651-1655. Lundberg, J.M., A.ngg~rd, A., Emson, P., Fahrenkrug, J. and HSkfelt, T., Vasoactive intestinal polypeptide and cholinergic mechanisms in cat nasal mucosa: studies on choline acetyltransferase and release of vasoactive intestinal polypeptide, Proc. Natl. Acad. Sci. U S . A , 78 (1981) 52555259. Lundberg, J.M., Hedlund, B. and Bartfai, T., Vasoactive intestinal polypeptide enhances muscarinic ligand binding in cat submandibular salivary gland, Nature (Lond.), 295 (1982) 147-149.
17 Menetrey, D., Retrograde tracing of neural pathways with a protein-gold complex. 1: light microscopic detection after silver intensification, Histoehemistry, 83 (1985) 391-395. 18 Montagna, W., Yun, J., Ore, B., Formisano, V, and Ri, P., Histology and cytochemistry of human skin XXX. Cholinesterase-containing nerves in the face, J. Invest. Dermatol., 90 (1964) 526-529. 19 Piper, P.J., Said, S.I. and Vane, J.R., Effects on smooth muscle preparations of unidentified vasoactive peptides from intestine and lung, Nature (Lond.), 225 (1970) 11441146. 20 Sata, T., Misra, H.P., Kubota, E. and Said, S.I., Vasoactive intestinal polypeptide relaxes pulmonary artery by an endothelium-independent mechanism, Peptides, 7 suppl. 1 (1986) 225-227. 21 Segade, L.A.G. and Suarez-Quintanilla, D, Otic ganglion parasympathetic neurons innervate the pulp of the mandibular incisor of the guinea pig, Neurosci. Letr, 90 (1988) 33-38. 22 Suzuki, N., Hardebo, J.E. and Owman, C., Origins and pathways of cerebrovascular vasoactive intestinal polypeptide-positive nerves in rat, J. Cereb. Blood Flow Metab., 8 (1988) 697-712. 23 Tago, H., Kimura, H. and Maeda, T. Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure, J. Histochem. Cytochem., 34 (1986) 1431-1438. 24 Uddman, R., Edvinsson, L., Jansen, I., Stiernholm, P., Jensen, K., Olesen, J. and Sundler, F., Peptide-containing nerve fibers in human extracranial tissue: a morphological basis for neuropeptide involvement in extracranial pain?, Pain, 27 (1986) 391-399. 25 Uddman, R., Hara, H. and Edvinsson, L., Neuronal pathways to the rat middle meningeal artery revealed by retrograde tracing and immunocytochemistry, J. Auton. NerL,. Syst., 26 (1989) 69-75. 26 Uddman, R., Edvinsson, L. and Hara, H., Axonal tracing of autonomic fibers to the superficial temporal artery in the rat, Cell Tissue Res., 256 (1989) 559-565. 27 Uemura, Y., Sugimoto, T., Kikuchi, H. and Mizuno, N., Possible origins cerebrovascular nerve fibers showing vasoactive intestinal polypeptide-like immunoreactivity: an immunohistochemical study in the dog, Brain Res., 448 (1988) 98-105. 28 Walters, B.B., Gillespie, S.A. and Moskowitz, M.A., Cerebrovascular projections from the sphenopalatine and otic ganglia to the middle cerebral artery of the cat, Stroke, 17 (1986) 488-494.
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Parasympathetic innervation of cutaneous blood vessels examined by retrograde tracing in the rat lower lip Akira Kaji 1, Toshihiro M a e d a 2 and Shohei W a t a n a b e 1 Departments of ~ Dermatology and 2 Anatomy, Shiga University of Medical Science, Otsu, Japan (Received 29 May 1990) (Revision received and accepted 27 September 1990)
Key words: Rat lip; Cutaneous blood vessel; Parasympathetic nerve fiber; Otic ganglion; Retrograde tracing; Vasoactive intestinal polypeptide Abstract The origin of vasoactive intestinal polypeptide (VIP)-immunoreactive and acetylcholinesterase (AChE)-positive perivascular nerve fibers in the lower lip of rats was investigated using the retrograde tracer, wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase gold complex (WGAapoHRP-Au), in combination with immunohistochemistry and enzyme histochemistry, by comparing the cells of origin of projection to the parotid gland. After the application of the tracer to the lip, small- to medium-sized nerve cells were labelled exclusively in the ipsilateral otic ganglion. Most of them showed moderate VIP-immunoreactivity and AChE activity. In contrast, injection into the parotid gland resulted in labelling of mostly large-sized cells of the otic ganglion which showed intense VIP-immunoreactivity and AChE activity. These results confirmed that the parasympathetic innervation of the rat lip originates from the otic ganglion. It was further suggested that there are at least two subpopulations in the otic ganglion cells, different from each other in size and in VIP-immunoreactivity, which separately innervate the salivary gland and the blood vessels.
Introduction
Sympathetic aminergic fibers innervate cutaneous blood vessels, but it is still unclear whether the parasympathetic nervous system regulates cutaneous blood flow or not. With the development of immunohistochemistry, vasodilator neuropeptides, including vasoactive intestinal polypeptide (VIP), have been observed around cutaneous blood vessels [2,4,5,8-10,24]. Recently, some of the cranial vessels, e.g. cerebral vessels [3,7,22,27,28], meningeal artery [25], superficial
Correspondence: A. Kaji, Department of Dermatology, Shiga University of Medical Science, Seta, Otsu, Japan.
temporal artery [26] and arterioles supplying the tooth pulp [21], have been reported to receive VIP-immunoreactive (IR) nerve supply from parasympathetic ganglia, as revealed by denervation experiments or with retrograde tracers. Our previous denervation study revealed that the VIPIR nerve fibers around blood vessels in the lip skin of rats originate from the parasympathetic otic ganglion [11]. The purpose of this study was to confirm the origin of the perivascular VIP-IR and acetylcholinesterase (AChE)-positive fibers, and to compare the origin cells with the ganglion cells innervating the salivary glands by employing the newly developed gold-labelled retrograde tracer, wheat germ agglutinin conjugated to enzymati-
154 cally inactive horseradish peroxidase gold complex (WGAapoHRP-Au), in combination with VIP-immunohistochemistry.
Materials and Methods
Tracer injection Colloidal-gold-labelled W G A a p o H R P was used as a retrograde tracer and was prepared according to Basbaum and Menetrey [1]. Eleven Sprague-Dawley male rats (150-250 g) were divided into three groups, After anesthesia with sodium pentobarbital (50 m g / k g ; i.p.), 3 /~1 of W G A a p o H R P - A u were injected into the following sites: left side of the lower lip in six rats of the first group, left parotid gland in two rats of the second group, and left side of three different sites of the hairy skin (submandibular, subocular, and preauricular in three rats each) of the third group. Tissue processing All the rats were perfused 48 h following the injection. The perfusion via the heart was carried out as follows. Blood was first washed out with 100 ml of 0.01 M phosphate-buffered saline. Next 200 ml of an ice-cold fixative solution containing 2% paraformaldehyde and 0.3% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4, was used. The otic ganglia, the superior cervical ganglia, the trigeminal ganglia, all from both sides, and the injected tissues, were excised, immersed in a post-fixative solution containing 2% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, p H 7.4, for a day and stored in 15% sucrose in 0.1 M phosphate buffer, p H 7.4, until sectioning. Twenty-/~m thick sections were cut with a cryotome. The free-floating sections were then placed in phosphate-buffered saline, pH 7.4. Histochernistry and immunohistochemistry The sections were silver-enhanced in a physical developer in the dark for 60 min at room temperature according to Menetrey [17] or in the silver enhancement reagent (IntenSE, Janssen) for 20 rain at room temperature in normal daylight. After
the silver-intensification procedure, some sections were stained by Tago's method for AChE activity [23]. The reaction was detected by the appearance of brown polymerized diaminobenzidine (DAB) product to discriminate AChE activity from silver particles implying the uptake of the tracer. The other sections were stored in 0.3% Triton-X in phosphate-buffered saline for at least three days for VIP-immunohistochemistry which was carried out as previously described [11]. The sections were incubated with VIP antiserum (1 : 10 000, Calbiochem) for 5 days at 4°C, followed by biotinylated lgG for 2 h and ABC for 1 h at room temperature. For the detection of peroxidase activity, 0.025% DAB solution and 0,001% hydrogen peroxidase solution were used.
Analysis of the labelled otic ganglion cells Sixteen sections of otic ganglia taken from six rats in which the tracer were injected into the lower lips were used for cell size measurements. Eighty-seven labelled cells in these sections were measured along their long axis by a scale mounted in the microscope eyepiece. The same measurements were carried out on 63 labelled cells in 18 sections from two rats in which the tracer were injected into the parotid glands. Cell size distribution histograms representing the overall proportions of labelled cells of both groups were made from these measurements.
Results
Tracer injection into the lip In the sections of the W G A a p o H R P - A u injected lip, particles visualized by silver intensification were localized among the vessels and were diffusely scattered between collagen bundles and in phagocytic cells. Retrograde labelling was found exclusively in the ipsilateral otic ganglion, where a small number of nerve cell bodies, less than 5% of the total population, were labelled. No labelling was found in the contralateral ganglion. The labelled cells were evenly distributed in the ganglion without an apparent pattern. The labelling was seen in the cytoplasm of the perikarya a n d / o r in dendrites.
155 I~J I n j e c t i o n into the L o w e r ( x =17.0, SD=3.7, N=87) []
30]
Lip
I n j e c t i o n into the P a r o t i d Gland (x=35.3, SD=7.8. N=63)
ii ii
z z z ii
//
10-15 1 5 - 2 0 2 0 - 2 5 2 5 - 3 0 30-35 35-4(
IR
40-45 45-50" IJm
The labelled cells were small to medium in size, ranging from 12 to 25 g m in diameter with a mean of 17.0 g m (Fig. 1), and the larger cells in the ganghon displayed no labelling. Immunohistochemical analysis for VIP showed moderate to weak staining intensity in the labelled cell bodies (Fig. 2A). AChE activity in these cells was also weak to moderate (Fig. 2B). HRP-labelled cells were also found both in the ipsilateral superior cervical ganglion and in the ipsilateral trigeminal ganglion. However VIP-immunoreactivity was not detected in any of the labelled cells. No labelled cell was found in the contralateral ganglia.
Diameter of Labeled C e l l s
Fig. 1. Cell size histogram of labelled neurons of the otic ganglion by WGAapoHRP-Au injection into the lower lip (hatched bars) and the parotid gland (open bars). ,X, mean; SD, standard deviation; N, number of cells.
Tracer injection into the parotid gland Labelled ganglion cells in the otic ganglion were mainly large in size, with a range from 15 to
Fig. 2. Otic ganglion. Combination of retrograde tracing with W G A a p o H R P - A u and VIP-immunohistochemistry (A) or AChE histochemistry (B). WGAapoHRP-Au was injected into the lower lip of rats and was silver enhanced. In A, WGAapoHRP-Au-labelled cells (arrowheads) displayed a moderate intensity of VIP-immunoreactivity. In B, five small labelled cells (arrows) show weak AChE activity. No labelling was found in the large nerve cell bodies with intense AChE activity. Bar = 10 gm.
156
Fig. 3. Otic ganglion. Combination of retrograde tracing with W G A a p o H R P - A u and VIP-immunohistochemistry (A) or A C h E histochemistry (B). The tracer was injected into the parotid gland. In A, two large labelled cells (arrowheads) show VIP-irnmunoreactivity. In B, two AChE active cells of large size (arrows) are labelled. Bar = 10 ~tm.
47.5/~m in diameter and a mean of 35.3/xm. They were usually larger in size than those demonstrated in the otic ganglion of lip-injected rats (Fig. 1). VIP-immunoreactivity was seen clearly in all labelled cell bodies (Fig. 3A). AChE staining showed moderate to intense reactivity (Fig. 3B).
Tracer injection into the hairy skin of the face No labelling was found in any otic ganglion cell bodies of the three rats, in which W G A a p o H R P Au was injected into one of three areas of the facial skin.
Discussion
The nerve fibers supplying the blood vessels in the skin of the lip in rats are stained by VIP-immunohistochemistry and AChE enzyme histochemistry [5,11]. In the present study, apphcation
of the retrograde tracer, WGAapoHRP-Au, to the lip, resulted in labelling of the ipsilateral otic ganglion and most of the labelled cells showed VIP-immunoreactivity. As the injection site has no other structures innervated with VIP-IR fibers and the tracer was localized within the injection site with minimal spread, as previously reported [1], the labelling in the VIP-IR ganglion nerve cell bodies is considered to be the result of the tracer uptake from the VIP-IR perivascular fibers. This implies that the perivascular VIP-IR fibers in the lower lip originate from the ipsilateral otic ganglion. An additional finding in the present study supports this interpretation. No notable VIP-immunoreactivity was shown in labelled cells either in the superior cervical ganglion or in the trigeminal ganglion; indeed no VIP-immunoreactivity was detected in these ganglia under the conditions used in this study. Accordingly, the present tracer experiment confirmed our previous study that the
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lip vessels in the rat are supplied by the parasympathetic otic neuron, as evidenced by the finding that VIP-IR and AChE-positive perivascular fibers disappear after the removal of the otic ganglion [11]. Since neurons in the otic ganglion of the rat are homogeneous in their levels of choline acetyltransferase immunoreactivity [12], it is likely that both large and small neurons are cholinergic despite the variable intensity of AChE staining. It is almost certain therefore that some of the cutaneous vessels are supplied with ACh- and VIPcontaining parasympathetic nerve fibers, as is known to be the case in the salivary gland [14] and nasal mucosa [15]. In this context, of interest is the difference in size and in VIP-immunoreactivity between the ganglion cells innervating the cutaneous vessels and those supplying the parasympathetic nerve fibers to the parotid gland. When the tracer was applied to the lip, small-sized and moderately to weakly VIP-IR ganglion cell bodies were labelled, while when it was applied to the parotid gland, mainly large-sized and intensely VIP-IR ganglion cells were labelled. This implies that the same single neuron in the otic ganglion does not project to the lip cutaneous vessels and to the parotid gland, but there are two ganglion cell populations which are at least different from each other in cell size and in VIP-immunoreactivity as well as in target organ. It is likely that the VIP which itself is contained in these two different postganglionic neuronal system functions in different ways in the target organ. VIP in the perivascular parasympathetic nerve fibers in the lip may exert direct vasodilative effects as manifested on other blood vessels [6,19,20], in contrast to its function as a neuromodulator in the secretory response of the salivary glands [13,16]. Thus, the dual sympathetic and parasympathetic innervation of the cutaneous vessels in the lip [11] is further supported in the present study. However, it remains to be elucidated whether a dual innervation of cutaneous vessels is present also in the skin of the head or not. In the cat, extracerebral vessels such as the branches of the external carotid artery are supplied by VIP-IR fibers of varying fluorescence intensity [5]. In the rat, the meningeal and superficial temporal arteries
are shown supplied by parasympathetic nerve fibers originating from the pterygopalatine ganglion and the otic ganglion, to which VIP-IR neurons contribute [25,26]. In the guinea-pig, arterioles supplying the tooth pulp are innervated by otic ganglion [21]. In man, AChE-positive perivascular fibers in the face were observed more than a quarter of a century ago [18]. In addition, VIP-IR nerve fibers are frequently found around the cutaneous blood vessels in the human face (unpublished data). However, the tracer experiment of the present study could not demonstrate parasympathetic perivascular innervation in the hairy facial skin of the rat, where neither intense AChE activity nor notable VIP-immunoreactivity was found. It is likely that parasympathetic innervation is sparse or absent in the peripheral vessels of the facial skin of the rat. If the intense AChE activity accompanied by VIP-immunoreactivity in the human cutaneous blood vessels of the face represents parasympathetic innervation, the species difference may be due to an increase of the complexity in the control of cutaneous blood flow in the face of humans, by comparison with the hairy skin of the rat. This question remains to be elucidated.
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