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Distribution and origins of VIP-immunoreactive nerves in the cephalic circulation of the cat
Pepndes. Vol. 5, pp 209--212, 1984 " Ankho Internattonal Inc Printed m the U.S.A
0196-9781/84 $3 00 + 00
Distribution and Origins of VIP-Immunoreactive Nerves in the Cephalic Circulation of the Cat I. L . G I B B I N S , 1 J. E . B R A Y D E N
A N D J. A . B E V A N
D e p a r t m e n t o f Pharmacology, U C L A School o f Medicine, L o s Angeles, CA 90024
GIBBINS, I. L., J. E BRAYDEN AND J. A. BEVAN. Distribution and origins of VIP-immunoreactive nerves in the c'ephaht csrculathm of the cat. PEPTIDES 5(2)209-212, 1984.--VIP-immunoreacuve fiR) nerves were visualized in whole mounts and sections of cephalic arteries and cranial nerves of cats with indirect immunofluorescence. Perivascular VIP-IR nerves were very widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-IR nerves were most abundant in the Circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brain stem and ventral areas of the cerebral cortex. VIP-IR nerves were absent from arterial branches supplying the posterior brain stem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya within mtcroganglia found in the cavernous plexus and external rete. Extracerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya in micrnganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. It seems likely, therefore, that each major segment of the cephalic circulation is supplied by local VIP-IR neurons. VIP-immunoreacuve neurons
C~ts
Indirect immunofluorescence
IN recent years, it has become apparent that neurons which are immunoreactive (IR) for vasoactive intestinal polypeptide (VIP) are widespread in the peripheral autonomic nervous system I l l , 12, 22]. Much work has been directed towards the parasympathetic VIP innervation of cranial tissues, particularly the blood vessels supplying the brain [7, 8, 14, 15], eye [23,26], salivary glands [16, 27, 31], nasal mucosa [17, 25, 28], and, most recently, the tongue [1,18]. However, no systematic study has been made of the detailed distribution of perivascular VIP-IR nerves in the cephalic circulation as a whole. Furthermore, although VIP-IR nerve cell bodies comprise most of the sphenopalatine [l l, 17] and submandibular gland ganglia [16], it is clear that at least some of the cephalic perivascular VIP-IR nerves, such as those in the cerebral circulation, are not derived from these sources [8]. Therefore, in this study, the distribution o f perivascular VIP-IR nerves has been systematically examined throughout the entire cephalic circulation of the cat. In addition, a search for VIP-IR nerve cell bodies has been c a r d e d out along the cranial nerves which might supply those blood vessels. The VIP-IR nerves have been visualized using a fluorescence immunohistochemical method upon whole mounts of blood vessels and nerves [5].
cranial nerves were prepared for immunofluorescence histochemistry following the method o f Costa et al. [5]. Large blood vessels were split open, slightly stretched and pinned to pieces o f dental wax. Small blood vessels and nerve trunks were pinned intact at resting length. Tissue pieces were fixed overnight in 15% saturated picric acid and 2% formaldehyde in 0.1 M phosphate buffer, pH 7.3 at 4-8°C. The pieces of tissue were washed free of picric acid in 80% ethanol, dehydrated through a graded series o f ethanols, cleared in xylene, and rehydrated back to phosphate buffered saline, pH 7.0 (PBS). At this stage, blood vessels were usually carefully stripped of their outer adventitia and associated connective tissue, and cranial nerves were stripped of their perineural connective tissues. The tissue pieces were then incubated in a humid atmosphere for 16 hr at room temperature with VIP antiserum (7913, kindly provided by Dr. J. H. Walsh; for characterization, see [10]), diluted 1:200 with PBS. After three washes in PBS, the tissue pieces were incubated for 1 hr with fluorosceine-isothiocyanate ( F I T C ) - - c o n j u g a t e d sheep antirabbit Ig antiserum (Sigma), diluted 1:30 with PBS. After a further three washes in PBS, the tissue pieces were mounted in carbonate buffered 50% glycerol, pH 8.6, and examined on a Zeiss Universal fluorescence microscope under epi-illumination. Photographs were taken on Ilford XP-1 film rated at ISO 400127 °. Some material was prepared for wax sectioning. The tis-
METHOD Adult cats of either sex were killed with a lethal dose of sodium pentobarbital. Whole mounts of blood vessels and
~Requests for repnnts should be addressed to I. L. Gibbins at his present address: Department of Pharmacology. College of Medicine, Umversity of Vermont. Burhngton. VT 05405
209
210
GIBBINS. BRAYDEN AND BEVAN
sue pieces were fixed and taken through xylene as described above, before being embedded in low melting point wax. Sections were cut at 15 or 20 p.m, mounted on gelatin coated slides, and rehydrated back to PBS. Subsequent treatment with antisera was as described above for the whole mount procedure. No fluorescent nerves were found in tissues treated with VIP antisera which had been incubated overnight with 10-~ M porcine VIP. Terminology for naming the cephalic arteries follows that of Davis and Story [6].
m small groups associated with the lingual nerve as well as being in close association with small blood vessels supplying striated muscle in the distal half of the tongue. Additional single VIP-IR nerve cell bodies and microganglia containing 15 to 100 VIP-IR cell bodies were consistently found along the chorda tympani nerve (Fig. 5). the Vidian nerve, the tympanic ramus of the glossopharyngeal nerve (Fig. 6). within the cavernous plexus and within the external rete. Occasional VIP-IR nerve cell bodies occurred within the greater superficml petrosal nerve, the deep petrosal nerve and the inferior carotico-tympanic nerve.
RESULTS
DISCUSSION
Perivascular VIP-IR nerves were very widely distributed throughout the cephalic arterial tree. The only major arteries which were not supplied with VIP-IR axons were the external carotid artery proximal to the external fete; the occipital, posterior auricular and superficial temporal arteries, and the extra-cranial portions of the vertebral and ascending pharyngeal arteries. The most densely innervated major distributing arteries were the lingual artery (Fig. 1) and the internal maxillary artery, distal to the external rete: these arteries were surrounded by a dense, irregularly oriented plexus of varicose VIP-IR axons concentrated at the adventitio-medial junction. A somewhat less dense plexus of VIP-IR axons surrounded the external maxillary artery. The external fete itself was well supplied with both varicose and non-varicose VIP-IR axons. All the arteries arising from the rete, including the internal and external ethmoidal arteries, ciliary and lacrimal arteries, and the anastomosing artery (connecting the fete to the Circle of Willis) received a dense VIP-IR innervation. Terminal branches of extracerebral arteries as small as 15 /~m diameter were generally well supplied with varicose VIP-IR axons. Arteries and arterioles supplying the parotid, submandibular, sublingual, molar and zygomatic salivary glands, the lacrimal gland, and glands in the palatine mucosa were all well innervated. Similarly, arterioles supplying the lingual musculature and most of the anterior facial musculature, including the buccinator, labial, and anterior parts of the masseter (Fig. 2) and temporal muscles were generally well innervated by varicose VIP-IR axons. Within the cerebral circulatioa, VIP-IR axons were largely restricted to the Circle of Willis and its major branches: the anterior, middle and posterior cerebral arteries, the superior cerebellar artery, the basilar artery and the intracranial portions of the vertebral arteries. The density of VIP-IR axons decreased markedly proximo-distally along the length of the cerebral arteries. Although arterioles as small as 15 tim diameter were commonly innervated by VIP-IR axons on the ventral areas of the cerebral hemispheres and anterior brain stem (Fig. 3), arterioles on the parietal and frontal areas of the cerebral cortex were rarely innervated (Fig. 4). None of the major branches of the bas/lar or vertebral arteries were innervated to any extent, and the VIP-IR innervation of the superior cerebellar artery ceased at the level of the cerebellar cortex. In confirmation of previous reports, the sphenopalatine and submandibular ganglia were found to consist almost entirely of VIP-IR nerve cell bodies. Nearly all the nerve cell bodies of the sublingual ganglia were also VIP-IR. However. within the otic ganglia only some of the nerve cell bodies were VIP-IR, whilst no VIP-IR neurons occurred within the ciliary ganglion. VIP-IR ganglion cells were found
The results of the present study demonstrate that VIP-IR nerves are much more widely distributed throughout the cephalic arterial tree of the cat than had been thought previously. VIP-IR nerve cell bodies are predominant within the major parasympathetic ganglia of the facial nerve. In addition, microganglia containing VIP-IR nerve cell bodies occur along all the major parasympathetic outflows of the facial nerve i.e., the Vidian nerve, lingual nerve and chorda tympani. VIP-IR nerve cell bodies are also associated with the parasympathetic outflow of the glossopharyngeal nerve m the otic ganglion and the microganglia of the tympanic plexus. The VIP-IR microganglia of the cavernous plexus and external rete in principle could be part of the facial, glossopharyngeal or even cranial sympathetic outflow [19, 20, 21]. Since blood vessels in the salivary glands are innervated by VIP-IR nerves arising in local ganglia [16] and VIP-IR nerves supplying blood vessels in the nasal mucosa arise from the relatively nearby sphenopalatine ganglion [17,28], it seems likely that the micropngiia described here supply VIP-IR axons to vascular beds in their vicinity. Thus the lingual artery and its branches are most probably innervated by VIP-IR axons arising from the intraling.aal nerve cell bodies. Similarly, the VIP-IR ganglion cells of the cavernous plexus and external fete are well situated to be the origins of the VIP-IR perivascular plexus associated with the cerebral arteries. These ganglion cells are probably homologous with those previously described near the carotid plexus of the monkey [4] and rat [30]. If the perivascular VIP-IR plexus represents a functional vasodilator innervation [9,16], then most of the cephalic arterial tree in the cat potentially can be under active neurogenic vasodllatory control. In this case each segment of the vascular bed could well be regulated by a particular vasodflator pathway associated with a group of VIP-IR ganglion ceils located relatively nearby. Three features of the distribution of the perivascular VIP-IR nerves, which may have important functional consequences, deserve comment: (I) The presence of an extemely dense plexus of varicose VIP-IR axons around relatively large distributing arteries, such as the lingual and internal maxillary arteries and the major cerebral arteries, suggests that varying degrees of stimulation of those nerves could effect a major redistribution of blood within the head, for example, under conditions of heat stress [2]. (2) Varicose VIP-IR axons are commonly found around arterioles in the nasal and palatine mucosae, the salivary and lacrimal glands. as well as in the striated muscles of the face and tongue. Such innervation provides the possibility for neurogenic vasodilatory control of resistance-sized vessels in these tissues. However. most vessels of this size in the brain are not innervated by VIP-IR axons: the supply of VIP-IR nerves to
FIG. 1. Bundles of VIP-IR perivascular axons in the lingual artery. In this area, the axon bundles have a somewhat iongitndiuni orientation. Whole mount, x525. Scale bar represents 20 p.m. FIG. 2. Terminal branches of the masseteric artery within the masseter muscle. Varicose VIP-IR axons closely follow the blood vessels. Whole mount, x525. Scale bar represents 20 p.m. FIG. 3. Small branch of the anterior cerebral artery supplying the rostrai brain stem. It has a prominent perivascular plexus. Whole mount. x525. Scale bar represents 20 p.m. FIG. 4. Small dorsal branches of the middle cerebral artery on the suprasylvian gyms. Only a single varicose VIP-IR axon accompanies one arteriole. Whole mount, x525. Scale bar represents 20 p.m. FIG. 5. Part of a VIP-IR microganglion on the chorda tympani nerve. Whole mount, x525. Scale bar represents 20 p.m. FIG. 6. VIP-IR cells in a microganglion on the tympanic ramus of the glossopharyngeal nerve. Whole mount, x640. Scale bar represents 20 p,m.
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GIBBINS, BRAYDEN AND BEVAN
arterioles within the cerebral circulation is mainly restricted to the more ventral regions of the cerebral cortex and the anterior brain stem. This apparent lack o f potential for neurogenic vasodilator influence upon most o f the cerebral arterioles may be reflected in the commonly observed predominance of autoregulatory responses in the control o f cerebral blood flow [3, 13, 24]. (3) The presence o f a dense plexus o f VIP-IR axons around a r t e r i o l e - s i ~ d vessels supplying striated muscles o f the face and tonlgue is unusual when compared with o th e r skeletal muscle vascular beds [7,29]. While VIP.IR axons have been reported to surround arteries supplying other skeletal muscles, the innervation is generally sparse [7,29]. Indeed, in the present study, VIP-IR
nerves were found to be absent from arterial trees supplying striated muscles o f the neck and back of the head. There is as yet no functional information available which might explain why perivascular VIP-IP axons are present in some skeletal muscle vascular beds and are absent in others.
ACKNOWLEDGEMENTS This work was supported by grants from the National Heart Foundation of Australia (I.L.G.), the American Heart Association (J.E.B.) and USPHS HL 15805 (J.A.B.). We would like to thank Irene Jones for preparing the wax-embedded material and Dr. J. L. Morris for comments on the manuscript.
REI~RENCES 1. Bneker, B., N. Yanalhara and W. G. Forsmann. VlP innervation of the tongue in vertebrates. Anat Embryol 167: 173-189, 1983. 2. Baker, M. A. and J. N. Hayward. Carotid rete and brain temperature of cat. Nature 216: 1:39-141, 1967. 3. Berms, R. M., H. R. Winn and R, Rubio. Metabolic regulation of cerebral blood flow. In: Vasodilatation, ecfited by P. M. Vanhoutte and I. Leusen. New York: Raven Press, 1981, pp. 231241. 4. Chorobski, J. and W. Peng'wld. Cerebral vasodilator nerves and their pathway from the medulla obionl|ata, with observations on the pial and intra~rebrul vascular plexus. Arch Neurol Psychiatry 28: 1257-1289, 1932. 5. Costa, M., R. Buffa, J. B. Furness and E. Solcia. Immunohistochemical localization of polypel~ides in peripheral autonomic nerves using whole mount preparations. Histochemistry 65: 157-165, 1980. 6. Davis, D. D. and H. E. Story. The carotid circulation of the domestic cat. Field Mus Nat Hist Chicago Zool Ser 28: 1--47. 1943. 7. Della, N. G., R. E. Papka, J. B. Furness and M. Costa. Vasoactire intestinal pcptide-like immunoreactivity in nerves associ, ated with the cardiovascular system of guinea pigs. Neuroscience 9:. 605-619, 1983. 8. Edvinsson, L., J. Fahrenkrus, J. Hanko, C. Owman, F. Sundier and R. Uddman. VIP (vasoactive intestinal polyl~,,ptid¢)containing nerves of intracranial arteries in mammals. Cell Tissue Res 208" 135-142, 1980. 9. Fahrenkrug, J. Vasoactive intestinal polypepude: measurement, distribution and putative neurotransmitter function. Digestaon 19: 149--169, 1979. 10. Furness, J. B.. M. Costa and J. H. Walsh. Evidence for and significance of the projection of VIP neurons from the myenteric plexus to the tnenia coil in the guinea-pig. GastroenterohJgy SO: 1557-1561, 1981. I 1. HAkanson, R., F. Sundler and R. Uddman. Distribution and topography of peripheral VIP nerve fibers: functional implications. In: Vasoac rive Intestinal Peptide. edited by S. Said. New York: Raven Press, 1982. pp. 121-144. 12 Hokfelt, T., M. Schultzberg, J. M. Lundberg. K. Fuxe. V. Mutt. J. Fahrenkrug and S. I. Said. Distribution of vasoacttve intestinal polypeptide in the central and peripheral nervous systems as revealed by immunocytochemistry. In: Vasoac tire Intestinal Pepttde, edited by S. ! Said New York: Raven Pres,, 1982. pp. 65-90 13 Kontos. H A. Regulation of the cerebral clrculatton Ann Rev Phy~tol 43: 397-407. 1981. 14 Larsson, L.-I . L. Edvmsson, J. Fahrenkrug, R. H~tkanson and C. Owman lmmunohtstochemtcal localization ofa vasoddatory polypeptide (VIP) m cerebrovascular nerves. Br~,n Re~ 113: 400--404, 1976. 15 Lmdvall, M., J. Alumets, L. Edvmsson, J. Fahrenkrug. R. H~tkan,son, J. Hanko, C. Owman, O. B. Schaffalitzky de Muckadell and F. Sundler. Pepttdergtc (VIP) nerves m the mammahan chorold plexus Neuro~ i Lett 9: 77-82, 1978.
16. Lundberg, J. M. Evidence for the co-existence of vasoactwe intestinal polypeptide (VIP) and acetylcholine in neurons of cat exocrine glands. Morphological, biochemical and functional studies. Acta Physiol S t a n d Suppl 496: i-57, 1981 17. Lundberg, J. M., A. Anggird, P. Emson, J. Fahrenkrug and T. H6kfeit. Vasoactive intestinal polypeptide and cholinergic mechanisms in cat nasal mucosa: studies on choline acetyltransferase and release of vasoactive intestinal polypeptide. Proc Natl Acad Sci USA 78: 5255-5259, 1981. 18. Lundberg, J. M., A. A n i ~ , J. Fahrenkrng and T. Hokfelt. Vasoactive intestinal polypeptide in cholinergic neurons of exocrine glands. In: Vasoactive Intestinal Peptide. edited by S. Said. New York: Raven Press, 1982, pp. 373--390. 19. Mitchell, G. A. G. The cranial extremities of the sympathetic trunks. Acta Anat 18: 195-201. 1953. 20. Mitchell, G. A. G. Anatomy o f the Autonomic Nervous System. Edinburgh and London: E and S Ltvingston, 1953. 21. Mitchell, G. A. G. The autonomic nerve supply of the throat, nose and ear. J Laryngol ~ 495-516, 1954. 22. Polak, J. R. and S. R. Bloom. Distribution and tissue Iocaltzatton of VIP in the central nervous system and in seven peripheral organs. In: Vasoactrve Intestinal Peptide. edited by S. Said. New York: Raven Press, 1982, pp. 107-120. 23. Terenghi, G., J. M. Poink, L. Probert, G. P. McGregor, G. L Fern, M. A. Blank, J M. Butler. W. G. Unger, S. Zhang. D F Cole and S. R. Bloom. Mapping, quantttative distribution and origin of substance P- and VIP-containing nerves m the urea of guinea-pig eye. Histothemtstrv 75: 399-417, 1982. 24. Traystman, R. J. Control of cerebral blood flow. In: Va~oddatatum, edited by P. M. Vanhoutte and 1. Leusen. New York Raven Press, 1981. pp. 39--48. 25. Uddman, R., J. Aiumets, O. Densert, R. HAkanson and F. Sundler. Occurrence and distribution of VIP nerves in the nasal mucosa and tracheobronchial wall. Acta Otolarvngol 85: 443448. 1978 26. Uddman, R.. J. Alumets, B. Ehinger, R. HAkanson, 1. Loren and F. Sundler. Vasoacttve intestinal peptide nerves m ocular and orbttal structures of the cat. Int.e~t Ophthmol Vt~ Stt 19: 878--885, 1980 27. Uddman, R., J. Fahrenkrug, L. Maim, J. Alumets, R. Hhkanson and F. Sundler Neuronal VIP in salivary glands: distributton and release. Acta Physiol S t a n d ll0: 31-38. 1980 28. Uddman. R., L. Maim and F. Sundler. The origin of vasoacttve intestinal polypepttde (V1P) nerves in the feline nasal mucosa. A~ ta Otolarvngol 89:152-156. 1980. 29 Uddman, R., J Alumets, L Edvmsson, R. H~tkanson and F Sundler VIP nerve fibers around peripheral blood vessels. At ta Phv~tol 5t and 112: 65-70, 1981 30. Vasquez, J. and M. J. Purves The cholmergtc pathway to cerebral blood vessels. 1, Morphological studies. Pfluger,~ Art h 379: 157-163. 1979 31 Wharton, J , J. M. Polak, M G Bryant, S van Noorden, S R. Bloom and A G. E Pearse Vasoacttve intestinal polypepttde (VIP)-hke tmmunoreactlwty m salivary glands Ltte S¢l 25: 273-280, 1979
0196-9781/84 $3 00 + 00
Distribution and Origins of VIP-Immunoreactive Nerves in the Cephalic Circulation of the Cat I. L . G I B B I N S , 1 J. E . B R A Y D E N
A N D J. A . B E V A N
D e p a r t m e n t o f Pharmacology, U C L A School o f Medicine, L o s Angeles, CA 90024
GIBBINS, I. L., J. E BRAYDEN AND J. A. BEVAN. Distribution and origins of VIP-immunoreactive nerves in the c'ephaht csrculathm of the cat. PEPTIDES 5(2)209-212, 1984.--VIP-immunoreacuve fiR) nerves were visualized in whole mounts and sections of cephalic arteries and cranial nerves of cats with indirect immunofluorescence. Perivascular VIP-IR nerves were very widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-IR nerves were most abundant in the Circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brain stem and ventral areas of the cerebral cortex. VIP-IR nerves were absent from arterial branches supplying the posterior brain stem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya within mtcroganglia found in the cavernous plexus and external rete. Extracerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya in micrnganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. It seems likely, therefore, that each major segment of the cephalic circulation is supplied by local VIP-IR neurons. VIP-immunoreacuve neurons
C~ts
Indirect immunofluorescence
IN recent years, it has become apparent that neurons which are immunoreactive (IR) for vasoactive intestinal polypeptide (VIP) are widespread in the peripheral autonomic nervous system I l l , 12, 22]. Much work has been directed towards the parasympathetic VIP innervation of cranial tissues, particularly the blood vessels supplying the brain [7, 8, 14, 15], eye [23,26], salivary glands [16, 27, 31], nasal mucosa [17, 25, 28], and, most recently, the tongue [1,18]. However, no systematic study has been made of the detailed distribution of perivascular VIP-IR nerves in the cephalic circulation as a whole. Furthermore, although VIP-IR nerve cell bodies comprise most of the sphenopalatine [l l, 17] and submandibular gland ganglia [16], it is clear that at least some of the cephalic perivascular VIP-IR nerves, such as those in the cerebral circulation, are not derived from these sources [8]. Therefore, in this study, the distribution o f perivascular VIP-IR nerves has been systematically examined throughout the entire cephalic circulation of the cat. In addition, a search for VIP-IR nerve cell bodies has been c a r d e d out along the cranial nerves which might supply those blood vessels. The VIP-IR nerves have been visualized using a fluorescence immunohistochemical method upon whole mounts of blood vessels and nerves [5].
cranial nerves were prepared for immunofluorescence histochemistry following the method o f Costa et al. [5]. Large blood vessels were split open, slightly stretched and pinned to pieces o f dental wax. Small blood vessels and nerve trunks were pinned intact at resting length. Tissue pieces were fixed overnight in 15% saturated picric acid and 2% formaldehyde in 0.1 M phosphate buffer, pH 7.3 at 4-8°C. The pieces of tissue were washed free of picric acid in 80% ethanol, dehydrated through a graded series o f ethanols, cleared in xylene, and rehydrated back to phosphate buffered saline, pH 7.0 (PBS). At this stage, blood vessels were usually carefully stripped of their outer adventitia and associated connective tissue, and cranial nerves were stripped of their perineural connective tissues. The tissue pieces were then incubated in a humid atmosphere for 16 hr at room temperature with VIP antiserum (7913, kindly provided by Dr. J. H. Walsh; for characterization, see [10]), diluted 1:200 with PBS. After three washes in PBS, the tissue pieces were incubated for 1 hr with fluorosceine-isothiocyanate ( F I T C ) - - c o n j u g a t e d sheep antirabbit Ig antiserum (Sigma), diluted 1:30 with PBS. After a further three washes in PBS, the tissue pieces were mounted in carbonate buffered 50% glycerol, pH 8.6, and examined on a Zeiss Universal fluorescence microscope under epi-illumination. Photographs were taken on Ilford XP-1 film rated at ISO 400127 °. Some material was prepared for wax sectioning. The tis-
METHOD Adult cats of either sex were killed with a lethal dose of sodium pentobarbital. Whole mounts of blood vessels and
~Requests for repnnts should be addressed to I. L. Gibbins at his present address: Department of Pharmacology. College of Medicine, Umversity of Vermont. Burhngton. VT 05405
209
210
GIBBINS. BRAYDEN AND BEVAN
sue pieces were fixed and taken through xylene as described above, before being embedded in low melting point wax. Sections were cut at 15 or 20 p.m, mounted on gelatin coated slides, and rehydrated back to PBS. Subsequent treatment with antisera was as described above for the whole mount procedure. No fluorescent nerves were found in tissues treated with VIP antisera which had been incubated overnight with 10-~ M porcine VIP. Terminology for naming the cephalic arteries follows that of Davis and Story [6].
m small groups associated with the lingual nerve as well as being in close association with small blood vessels supplying striated muscle in the distal half of the tongue. Additional single VIP-IR nerve cell bodies and microganglia containing 15 to 100 VIP-IR cell bodies were consistently found along the chorda tympani nerve (Fig. 5). the Vidian nerve, the tympanic ramus of the glossopharyngeal nerve (Fig. 6). within the cavernous plexus and within the external rete. Occasional VIP-IR nerve cell bodies occurred within the greater superficml petrosal nerve, the deep petrosal nerve and the inferior carotico-tympanic nerve.
RESULTS
DISCUSSION
Perivascular VIP-IR nerves were very widely distributed throughout the cephalic arterial tree. The only major arteries which were not supplied with VIP-IR axons were the external carotid artery proximal to the external fete; the occipital, posterior auricular and superficial temporal arteries, and the extra-cranial portions of the vertebral and ascending pharyngeal arteries. The most densely innervated major distributing arteries were the lingual artery (Fig. 1) and the internal maxillary artery, distal to the external rete: these arteries were surrounded by a dense, irregularly oriented plexus of varicose VIP-IR axons concentrated at the adventitio-medial junction. A somewhat less dense plexus of VIP-IR axons surrounded the external maxillary artery. The external fete itself was well supplied with both varicose and non-varicose VIP-IR axons. All the arteries arising from the rete, including the internal and external ethmoidal arteries, ciliary and lacrimal arteries, and the anastomosing artery (connecting the fete to the Circle of Willis) received a dense VIP-IR innervation. Terminal branches of extracerebral arteries as small as 15 /~m diameter were generally well supplied with varicose VIP-IR axons. Arteries and arterioles supplying the parotid, submandibular, sublingual, molar and zygomatic salivary glands, the lacrimal gland, and glands in the palatine mucosa were all well innervated. Similarly, arterioles supplying the lingual musculature and most of the anterior facial musculature, including the buccinator, labial, and anterior parts of the masseter (Fig. 2) and temporal muscles were generally well innervated by varicose VIP-IR axons. Within the cerebral circulatioa, VIP-IR axons were largely restricted to the Circle of Willis and its major branches: the anterior, middle and posterior cerebral arteries, the superior cerebellar artery, the basilar artery and the intracranial portions of the vertebral arteries. The density of VIP-IR axons decreased markedly proximo-distally along the length of the cerebral arteries. Although arterioles as small as 15 tim diameter were commonly innervated by VIP-IR axons on the ventral areas of the cerebral hemispheres and anterior brain stem (Fig. 3), arterioles on the parietal and frontal areas of the cerebral cortex were rarely innervated (Fig. 4). None of the major branches of the bas/lar or vertebral arteries were innervated to any extent, and the VIP-IR innervation of the superior cerebellar artery ceased at the level of the cerebellar cortex. In confirmation of previous reports, the sphenopalatine and submandibular ganglia were found to consist almost entirely of VIP-IR nerve cell bodies. Nearly all the nerve cell bodies of the sublingual ganglia were also VIP-IR. However. within the otic ganglia only some of the nerve cell bodies were VIP-IR, whilst no VIP-IR neurons occurred within the ciliary ganglion. VIP-IR ganglion cells were found
The results of the present study demonstrate that VIP-IR nerves are much more widely distributed throughout the cephalic arterial tree of the cat than had been thought previously. VIP-IR nerve cell bodies are predominant within the major parasympathetic ganglia of the facial nerve. In addition, microganglia containing VIP-IR nerve cell bodies occur along all the major parasympathetic outflows of the facial nerve i.e., the Vidian nerve, lingual nerve and chorda tympani. VIP-IR nerve cell bodies are also associated with the parasympathetic outflow of the glossopharyngeal nerve m the otic ganglion and the microganglia of the tympanic plexus. The VIP-IR microganglia of the cavernous plexus and external rete in principle could be part of the facial, glossopharyngeal or even cranial sympathetic outflow [19, 20, 21]. Since blood vessels in the salivary glands are innervated by VIP-IR nerves arising in local ganglia [16] and VIP-IR nerves supplying blood vessels in the nasal mucosa arise from the relatively nearby sphenopalatine ganglion [17,28], it seems likely that the micropngiia described here supply VIP-IR axons to vascular beds in their vicinity. Thus the lingual artery and its branches are most probably innervated by VIP-IR axons arising from the intraling.aal nerve cell bodies. Similarly, the VIP-IR ganglion cells of the cavernous plexus and external fete are well situated to be the origins of the VIP-IR perivascular plexus associated with the cerebral arteries. These ganglion cells are probably homologous with those previously described near the carotid plexus of the monkey [4] and rat [30]. If the perivascular VIP-IR plexus represents a functional vasodilator innervation [9,16], then most of the cephalic arterial tree in the cat potentially can be under active neurogenic vasodllatory control. In this case each segment of the vascular bed could well be regulated by a particular vasodflator pathway associated with a group of VIP-IR ganglion ceils located relatively nearby. Three features of the distribution of the perivascular VIP-IR nerves, which may have important functional consequences, deserve comment: (I) The presence of an extemely dense plexus of varicose VIP-IR axons around relatively large distributing arteries, such as the lingual and internal maxillary arteries and the major cerebral arteries, suggests that varying degrees of stimulation of those nerves could effect a major redistribution of blood within the head, for example, under conditions of heat stress [2]. (2) Varicose VIP-IR axons are commonly found around arterioles in the nasal and palatine mucosae, the salivary and lacrimal glands. as well as in the striated muscles of the face and tongue. Such innervation provides the possibility for neurogenic vasodilatory control of resistance-sized vessels in these tissues. However. most vessels of this size in the brain are not innervated by VIP-IR axons: the supply of VIP-IR nerves to
FIG. 1. Bundles of VIP-IR perivascular axons in the lingual artery. In this area, the axon bundles have a somewhat iongitndiuni orientation. Whole mount, x525. Scale bar represents 20 p.m. FIG. 2. Terminal branches of the masseteric artery within the masseter muscle. Varicose VIP-IR axons closely follow the blood vessels. Whole mount, x525. Scale bar represents 20 p.m. FIG. 3. Small branch of the anterior cerebral artery supplying the rostrai brain stem. It has a prominent perivascular plexus. Whole mount. x525. Scale bar represents 20 p.m. FIG. 4. Small dorsal branches of the middle cerebral artery on the suprasylvian gyms. Only a single varicose VIP-IR axon accompanies one arteriole. Whole mount, x525. Scale bar represents 20 p.m. FIG. 5. Part of a VIP-IR microganglion on the chorda tympani nerve. Whole mount, x525. Scale bar represents 20 p.m. FIG. 6. VIP-IR cells in a microganglion on the tympanic ramus of the glossopharyngeal nerve. Whole mount, x640. Scale bar represents 20 p,m.
212
GIBBINS, BRAYDEN AND BEVAN
arterioles within the cerebral circulation is mainly restricted to the more ventral regions of the cerebral cortex and the anterior brain stem. This apparent lack o f potential for neurogenic vasodilator influence upon most o f the cerebral arterioles may be reflected in the commonly observed predominance of autoregulatory responses in the control o f cerebral blood flow [3, 13, 24]. (3) The presence o f a dense plexus o f VIP-IR axons around a r t e r i o l e - s i ~ d vessels supplying striated muscles o f the face and tonlgue is unusual when compared with o th e r skeletal muscle vascular beds [7,29]. While VIP.IR axons have been reported to surround arteries supplying other skeletal muscles, the innervation is generally sparse [7,29]. Indeed, in the present study, VIP-IR
nerves were found to be absent from arterial trees supplying striated muscles o f the neck and back of the head. There is as yet no functional information available which might explain why perivascular VIP-IP axons are present in some skeletal muscle vascular beds and are absent in others.
ACKNOWLEDGEMENTS This work was supported by grants from the National Heart Foundation of Australia (I.L.G.), the American Heart Association (J.E.B.) and USPHS HL 15805 (J.A.B.). We would like to thank Irene Jones for preparing the wax-embedded material and Dr. J. L. Morris for comments on the manuscript.
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