Cell and Tissue Research
Cell Tissue Res. 198, 43-51 (1979)
9 by Springer-Verlag 1979
A Histochemical Study of the Innervation of the Cerebral Blood Vessels in the Domestic Fowl Takasuke Tagawa, KSichi AndS, and Takeo Wasano Department of Anatomy, School of Medicine, Fukuoka University, Fukuoka, Japan
Summary.Adrenergic and cholinergic nerves innervating the cerebral arteries of the domestic fowl were examined by specific histochemical techniques. The adrenergic nerve plexuses of the cerebral carotid system are markedly denser than those of other vertebrates observed by similar techniques. They form longitudinally elongated meshworks of fine fibres in the vascular wall of the arterial branches. Those innervating the vertebro-basilar system are less dense and more elongated, and, as the size of the artery diminishes, the fibres of the plexus become coarser. In the small pial and parenchymal arteries they are reduced to a few fibres running parallel to, or spiralling around the vascular axis. The cholinergic nerve plexuses are not as dense as the adrenergic system. The acetylcholinesterase activity is very weak, except in the plexuses innervating the cerebral carotid artery and the proximal portion of the anterior and posterior rami. In the vertebro-basilar system, a few thick nerve bundles run alongside the blood vessels of the vertebral and basilar arteries. Cholinergic nerves enter the cranial cavity along the internal carotid, the vertebral and possibly the cerebroethmoidal arteries. Intracerebral capillaries and some arterioles are not innervated with cholinergic and adrenergic fibres of peripheral origin, but with ones arising from parenchymal nerve cells. Key words: Cerebral blood vessels (domestic fowl) - Adrenergic nerves Cholinergic nerves - Capillary innervation - Neurogenic vascular control.
Histochemical and electron microscopic studies have shown that the cerebral blood vessels in mammals are innervated by both adrenergic and cholinergic nerves (Nelson and Rennels, 1970; Motavkin and Dovbish, 1971; Peerless and Yasargil, 1971 ; Owman et al., 1974; Edvinsson, 1975). Information about their innervation in Prof. T. Tagawa, M.D., Department of Anatomy, School of Medicine, Fukuoka University, Fukuoka 814, Japan
Send offprint requests to:
0302-766X/79/0198/0043/$01.80
T. Tagawa et al.
44
1
.'-:. 9
\!'~'f'~/~
:.
'" ~ k ( .
~
~
- - k i .":: ' . . ' : " :
Fig. 1. Diagram of the arterial supply to the ventral surface of the brain in the domestic fowl, a cerebral carotid artery; b anterior ramus; c posterior cerebral artery; d middle cerebral artery; e anterior cerebral artery; f cerebro-ethmoidal artery; g posterior ramus; h posterior superior cerebellar artery; i basilar artery; j posterior inferior cerebellar artery; k vertebral artery
Figs. 2-13. Adrenergic nerve plexuses in cerebral blood vessels demonstrated by fluorescent histochemistry Fig. 2. The adrenergic innervation in a whole stretched preparation of the anterior ramus, x 67 Fig. 3. The posterior ramus. • 67 Fig. 4. The posterior cerebral artery which arises from the anterior ramus. • 67 Fig. 5. The middle cerebral artery which is a branch of the anterior ramus, x 67 Fig. 6. The cerebro-ethmoidal artery, x 67 Fig. 7. The small branches of the middle cerebral artery. Arrow indicates an adrenergic nerve running straight along the axis of a very small artery, x 67 Fig. 8. The basilar artery, x 67 Pig. 9. The confluence of the vertebral arteries (k) and basilar artery (0. • 67 Fig. 10. Cross section of the internal carotid artery in the carotic canal. Long arrow indicates the adrenergic plexuses in the adventitia closely adjacent to the media, and short arrow indicates the autofluorescences of the elastic fibres. Arrowhead indicates a large stem nerve which contains fluorescent fibres. • 67 Fig, 11. The adrenergic plexuses in a cross section of anterior ramus. • 67 Fig. 12. Cross section of the basilar artery, x 67 Fig. 13. Fluorescent photomicrograph of a cross-sectioned intracerebral capillary (asterisk). Arrowhead indicates an adrenergic fibre of brain parenchymal nerve cell origin. • 268
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s u b m a m m a l i a n vertebrates, however, is sparse. I n o u r l a b o r a t o r y , a series o f c o m p a r a t i v e h i s t o c h e m i c a l studies is being carried o u t on the a u t o n o m i c i n n e r v a t i o n o f the cerebral b l o o d vessels o f v a r i o u s v e r t e b r a t e g r o u p s ( W a s a n o et al., 1975; I i j i m a et al., 1977; Iijima, 1977; T a g a w a et al., 1979), a n d the present study on the d o m e s t i c fowl is p a r t o f this series.
Materials and Methods The formaldehyde fluorescence techniques (Falck, 1962) and the direct colouring thiocholine method (Karnovsky and Roots, 1964) were used to demonstrate adrenerglc and cholinerglc nerves respectively. Thirty domestic fowls, Gallus domesticus, were decapitated and each brain quickly dissected from the skull and placed in Ringer's solution. The pial arteries were carefully removed from the brain, where some parenchymal arteries were still continuous with the pial ones. For the fluorescence technique the vessels were stretched on glass slides and immediately transferred to a desiccator to be dried in vacuo over phosphorus pentoxide for 1 h. The specimens were then treated with the paraformatdehyde gas (RH = 47 ~) for 1 h at 80~ and mounted for fluorescence microscopy. For the thiocholine method the vessels were fixed for 30min in 4% formaldehyde buffered to pH 7.4, washed thoroughly with distilled water, transferred into Karnovsky's medium (acetylthiocholine iodide-free) for 30 min at 4~C, and then incubated for 1 h at 20~ in medium containing 2 • 10-4M iso-OMPA (tetraisopropylpyrophosphoramide)as an inhibitor of the non-specific cholinesterase. After washing with 50 % ethanol, they were stretched on glass slides, dried in air, dehydrated, and finally mounted in balsam. For sectioning, small blocks (3-4 mm 3) of the brain were frozen rapidly in dry-ice iso-pentane, and dried in vacuo for six days at -45~ then two days at -20~ and one day at room temperature. They were then treated initially with paraformaldehyde gas (RH = 47 %) for 1 h at 80~ and infiltrated in vacuo with paraffin for 10 rain at 60~C. The blocks were sectioned (8-10 ~tmthick), and examined with a fluorescence microscope. For the acetylchofinesterase (ACHE)reaction, frozen sections (15-30 Inn thick) were made from other specimens of brain, fixed with 4 % formaldehyde, and then treated as described above.
Results A s c h e m a t i c illustration o f the arterial s u p p l y to the fowl b r a i n is given in Fig. 1. A l t h o u g h the stem vessels are the cerebral c a r o t i d a n d the v e r t e b r o - b a s i l a r systems, the l a t t e r is e x t r e m e l y u n d e r d e v e l o p e d , a n d the arterial b l o o d s u p p l y to the b r a i n seems m o s t l y to be p r o v i d e d b y the former. T h e cerebral c a r o t i d a r t e r y is divided into a n t e r i o r a n d p o s t e r i o r rami, a n d t h o u g h the p o s t e r i o r r a m u s connects with the b a s i l a r artery, an a n t e r i o r c o m m u n i c a t i n g a r t e r y is lacking, so t h a t the Willis ring is incomplete.
Adrenergic lnnervation. The a d r e n e r g i c nerve plexuses in the cerebral c a r o t i d artery are c o m p o s e d o f thin fibres, o r g a n i z e d into e l o n g a t e d m e s h w o r k s a l o n g the arterial axis. A s the size o f the a r t e r y decreases, the plexus fibres b e c o m e thinner a n d the m e s h w o r k s m o r e elongated. Plexuses in the a n t e r i o r ramus, the m i d d l e cerebral a n d the c e r e b r o - e t h m o i d a l arteries are m o r p h o l o g i c a l l y similar to those o f the cerebral c a r o t i d a r t e r y (Figs. 2, 4, 5, 6), b u t in the p o s t e r i o r r a m u s they are less dense a n d their m e s h w o r k o f fibres is m o r e e l o n g a t e d (Fig. 3). The nerve fibres in the b a s i l a r a n d the v e r t e b r a l arteries are finer t h a n those in the cerebral c a r o t i d system, a n d are inclined to r u n p a r a l l e l with the arterial axis (Figs. 8, 9). In the small pial arteries, the
Cerebral Blood Vessel Innervation in the Domestic Fowl
47
Figs. 14-19. Cholinergic nerve plexuses in cerebral blood vessels demonstrated by the thiocholine technique Fig. 14. The cholinergic innervation in a whole stretched preparation of the cerebral carotid artery (a), the anterior (b) and the posterior (g) rami. Arrow indicates a stem nerve. • 64 Fig. 15. The cholinergic plexuses in the anterior ramus. Arrow indicates cholinergic stem nerve, x 64 Fig. 16. The posterior cerebral artery. Arrow indicates a stem nerve, x 64 Fig. 17. The middle cerebral artery, x 64 Fig. 18. The cerebro-ethmoidal artery, x 64 Fig. 19. Small branches of the middle cerebral artery. • 64
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Figs. 20--25. Cholinergic nerve plexuses in the cerebral blood vessels Fig. 20. The basilar artery. Arrowhead indicates a cholinergic nerve cell. • 64 Fig. 21. The confluenceof the vertebral arteries (k) to the basilar artery (0. Arrow indicatesa stem nerve. • 64 Fig. 22. Cross section of the internal carotid artery in the carotic canal. Arrow indicates the adrenergic plexuses in the adventitia-media border, and the arrowhead indicates a large stem nerve which contains cholinergic fibres, x 64 Fig. 23. Cross section of the anterior ramus. Arrows indicate the cholinergic fibres, x 64 Fig. 24. Cross section of the vertebral arteries. Arrows indicate the cholinergic nerve bundles. • 64 Fig. 25. A cholinergicnerve fibre (arrow) whichoriginates from a cholinergicnervecell(arrowhead) in the brain and terminates in a capillary wall (asterisk). x 320
fibres m a y be straight or spiral (Fig. 7). I n the carotic canal, the d i s t r i b u t i o n of nerve fibres in the i n t e r n a l carotid artery is less dense t h a n in the cerebral carotid artery, a n d a large fluorescent nerve b u n d l e runs parallel with the artery (Fig. 10). I n cross sections, these adrenergic fibres are located in the vascular wall in the t u n i c a adventitia, adjacent to the tunica media, a n d their density of distribution closely c o r r e s p o n d s with the degree of density shown by the plexus m e s h w o r k in the whole m o u n t p r e p a r a t i o n s (Figs. 11, 12). O n l y a few nerve fibres o f peripheral origin are observed in the p a r e n c h y m a l arterial wall, a n d in some p a r e n c h y m a l capillaries a n d arterioles, fibres c a n n o t be detected. Instead, nerve fibres originating from the intracerebral nerve cells are seen, a n d such fibres are c o m m o n in the d i e n c e p h a l o n (Fig. 13).
Cerebral BloodVesselInnervationin the DomesticFowl
49
Cholinergic Innervation. In general, the AChE-positive nerve fibres in the cerebral arteries are considerably less numerous than adrenergic ones, and the ACHEactivity is generally weak. The cerebral carotid artery plexuses are composed of fine fibres, in elongated meshworks along the arterial axis (Fig. 14). Their density and AChE-activity rapidly diminishes toward the distal portion of the anterior and posterior rami and their peripheral branches (Figs. 15, 16, 17, 19). However, in the cerebro-ethmoidal artery, which is the end branch of the anterior ramus and anastomoses with the ethmoidal artery, the density and AChE-activity increases again (Fig. 18). In the distal portion of the basilar artery, which connects with the posterior ramus, the density and the AChE-activity of the nerve plexuses are approximately similar to the distal portion of the anterior ramus (Fig. 20). In the proximal portion of the small basilar artery arising from the vertebral artery, one or two large nerve bundles with a high AChE-activity are constantly observed (Fig. 21), but in the small pial and parenchymal arteries they are not commonly seen. In the carotic canal, on the other hand, a large AChE-positive nerve bundle runs parallel with the internal carotid artery and is identical with that observed by fluorescence microscopy (Figs. 10, 22). It is of interest to note that isolated cholinergic nerve cells occur scattered throughout the nerve plexuses of the large cerebral arteries (Fig. 20). Like the adrenergic nerves, in the cross sections of the blood vessels, the cholinergic fibres are similarly located in the tunica adventitia (Figs. 23, 24), though in the small pial arteries the AChE-positive fibres can scarcely be seen. Cholinergic nerves of peripheral origin are not evident in the small parenchymal artery, arterioles and capillaries, and these vessels appear to be directly innervated by nerve fibres arising from cholinergic nerve cells in the brain, especially in the diencephalon (Fig. 25). It is interesting to note that the parenchymal capillaries and arterioles show a strongly positive AChE-activity in their walls. Discussion
It has been established that the mammalian cerebral arteries have a dual innervation, and both adrenergic and cholinergic nerve plexuses coexist in the vascular walls (Schenk and Badawi, 1968; Edvinsson, 1975; Wasano et al., 1975). A dual innervation is also found in reptiles, and in the cerebral arteries of a snake, the innervation is essentially similar to that of mammals, except that adrenergic plexuses are more prominent than cholinergic ones (Iijima et al., 1977). In the turtle, however, the cholinergic plexuses are significantly less well developed, and more closely resemble the amphibian pattern where, in the bullfrog at least, the adrenergic fibres are well developed as in reptiles, but cholinergic nerves cannot be detected (Tagawa et al., 1979). In the present study, the adrenergic nerve plexuses of the cerebral blood vessels of the fowl are denser than those in mammals and the other vertebrates that have so far been studied, but the cholinergic plexuses are less dense and show weaker AChE-activity than in mammals and snakes. It has previously been reported that the cerebrovascular nerves of mammals (Edvinsson, 1975) and turtles (Iijima, 1977) enter the cranial cavity along the internal carotid and the vertebral arteries, and in the snake along the cerebral
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c a r o t i d a n d o p h t h a l m i c arteries (Iijima et al., 1977). In the present study, however, the cholinergic nerves enter the cranial cavity chiefly a l o n g the internal c a r o t i d a n d p a r t l y a l o n g the v e r t e b r a l a n d e t h m o i d a l arteries. In the case o f the adrenergic nerves, they m o s t likely enter the cranial cavity along the internal c a r o t i d a n d p a r t l y a l o n g the v e r t e b r a l arteries. I n rats, p a r e n c h y m a l arteries are i n n e r v a t e d directly by n o n - s y m p a t h e t i c a d r e n e r g i c fibres o r i g i n a t i n g f r o m the i n t r a c e r e b r a l cell bodies ( F a l c k et al., 1965, 1968; H a r t m a n et al., 1972; E d v i n s s o n et al., 1973a, b), a n d in their electron m i c r o s c o p i c study, Rennels a n d N e l s o n (1975) have p r o v i d e d conclusive evidence o f the existence o f a x o n terminals in capillaries o f the cat h y p o t h a l a m u s . In o u r p r e v i o u s h i s t o c h e m i c a l studies on the b r a i n s o f the snake, turtle a n d bullfrog, we have similarly n o t e d t h a t the i n t r a c e r e b r a l capillaries a n d arterioles are dually i n n e r v a t e d b y p a r e n c h y m a l nerve cells (Iijima, 1977; Iijima et al., 1977; T a g a w a et al., 1979), a n d in the present s t u d y this central i n n e r v a t i o n is also evident in the c e r e b r a l vessels o f the fowl. T h e vessel walls o f the i n t r a c e r e b r a l arterioles a n d capillaries have an ACHEpositive activity in the fowl, as has also been r e c o r d e d in m a m m a l i a n ( F l u m e r f e l t et al., 1973), reptilian (Iijima, 1977; Iijima et al., 1977) a n d a m p h i b i a n species (Shen et al., 1955; B r i g h t m a n a n d Albers, 1959; Contestabile, 1976; T a g a w a et al., 1979).
References Brightman, M.W., Albers, R.W.: Species differences in the distribution of extraneuronal cholinesterase within the vertebrate central nervous system. J. Neurochem. 4, 244-250 (1959) Contestabile, A.: Histochemical characterization of cholinesterase activity in the frog brain with special reference to its localization on the wall of blood vessels. Histochem. J. 8, 513-521 (1976) Edvinsson, L.: Neurogenic mechanisms in the cerebrovascular bed. Autonomic nerves, amine receptors and their effects on cerebral blood flow. Acta Physiol. Scand. (Suppl.) 427, 1-35 (1975) Edvinsson, L., Lindvall, M., Nielsen, K.C., Owman, Ch.: Are brain vessels innervated also by central (non-sympathetic) adrenergic neurones? Brain Res. 63, 496-499 (1973a) Edvinsson, L., Nielsen, K.C., Owrnan, Ch., West, K.A.: Evidence of vasoconstrictor sympathetic nerves in brain vessels of mice. Neurol. 23, 73-77 (1973b) Falck, B.: Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta Physiol. Scand. (Suppl. 197) 56, 1-25 (1962) Falck, B., Mchedlishvili, G.I., Owman, Ch.: Histochemical demonstration of adrenergic nerves in cortex-pia of rabbit. Acta Pharmacol. Toxicol. 23, 133-142 (1965) Falck, B., Nielsen, K.C., Owman, Ch.: Adrenergic innervation of the pial circulation. Scand. J. Cli. Lab. Invest. (Suppl.) 102, 96-98 (1968) Flumerfelt, B.A., Lewis, P.R., Gwyn, D.G.: Cholinesterase activity of capillaries in the rat brain. A light and electron microscopic study. Histochem. J. 5, 67-77 (1973) Hartman, B.K., Zide, D., Udenfriend, S.: The use of dopamine fl-hydroxylase as a marker for the central noradrenergic nervous system in rat brain. Proc. Natl. Acad. Sci. (U.S.A.) 69, 2722-2726 (1972) Iijima, T.: A histochemical study of the innervation of cerebral blood vessels in the turtle. J. Comp. Neur. 176, 307-314 (1977) Iijima, T., Wasano, T., Tagawa, T., AndS, K.: A histochemical study of the innervation of cerebral blood vessels in the snake. Cell Tissue Res. 179, 143-155 (1977) Karnovsky, M.I., Roots, L.: A "direct-coloring" thiocholine method for cholinesterases. J. Histochem. Cytochem. 12, 219-221 (1964) Motavkin, P.A., Dovbish, T.V.: Histochemical characteristics of acetylcholinesterase of the nerves innervating the brain vessels. Acta Morphol. Acad. Sci. Hung. 19, 159-173 (1971) Nelson, E., Rennels, M.: Innervation of intracranial arteries. Brain 93, 475-490 (1970)
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Owman, Ch., Edvinsson, L., Nielsen, K.C.: Autonomic neuroreceptor mechanisms in brain vessels. Blood Vessels 11, 2-31 (1974) Peerless, S_I., Yasargil, M.G.: Adrenergic innervation of the cerebral blood vessels in the rabbit. J. Neurosurg. Psychiatry 35, 148-154 (1971) Rennels, M.L., Nelson, E.: Capillary innervation in the mammalian central nervous system: An electron microscopic demonstration (1). Am. J. Anat. 144, 233-241 (1975) Schenk, E.A., Badawi, A.EI.: Dual innervation of arteries and arterioles. A histochemical study. Z. 'Zellforsch. 91, 170-177 (1968) Shen, S.C., Greenfield, P., Boe11, E.J.: The distribution of cholinesterase in the frog brain. J. Comp. Neurol. 102, 717-743 (1955) Tagawa, T., AndS, K., Wasano, T., Iijima, T.: A histochemical study of the innervation of cerebral blood vessels in the bullfrog. J. Comp. Neurol. 183, 25-32 (1979) Wasano, T., Tagawa, T., Iijima, T., AndS, K.: Histochemical studies on the adrenergic and cholinergic nerves innervating the cerebral arteries of vertebrates. Proc. 10th Int. Cong. Anat. Tokyo p. 144 (1975) Accepted January 22, 1979
Cell Tissue Res. 198, 43-51 (1979)
9 by Springer-Verlag 1979
A Histochemical Study of the Innervation of the Cerebral Blood Vessels in the Domestic Fowl Takasuke Tagawa, KSichi AndS, and Takeo Wasano Department of Anatomy, School of Medicine, Fukuoka University, Fukuoka, Japan
Summary.Adrenergic and cholinergic nerves innervating the cerebral arteries of the domestic fowl were examined by specific histochemical techniques. The adrenergic nerve plexuses of the cerebral carotid system are markedly denser than those of other vertebrates observed by similar techniques. They form longitudinally elongated meshworks of fine fibres in the vascular wall of the arterial branches. Those innervating the vertebro-basilar system are less dense and more elongated, and, as the size of the artery diminishes, the fibres of the plexus become coarser. In the small pial and parenchymal arteries they are reduced to a few fibres running parallel to, or spiralling around the vascular axis. The cholinergic nerve plexuses are not as dense as the adrenergic system. The acetylcholinesterase activity is very weak, except in the plexuses innervating the cerebral carotid artery and the proximal portion of the anterior and posterior rami. In the vertebro-basilar system, a few thick nerve bundles run alongside the blood vessels of the vertebral and basilar arteries. Cholinergic nerves enter the cranial cavity along the internal carotid, the vertebral and possibly the cerebroethmoidal arteries. Intracerebral capillaries and some arterioles are not innervated with cholinergic and adrenergic fibres of peripheral origin, but with ones arising from parenchymal nerve cells. Key words: Cerebral blood vessels (domestic fowl) - Adrenergic nerves Cholinergic nerves - Capillary innervation - Neurogenic vascular control.
Histochemical and electron microscopic studies have shown that the cerebral blood vessels in mammals are innervated by both adrenergic and cholinergic nerves (Nelson and Rennels, 1970; Motavkin and Dovbish, 1971; Peerless and Yasargil, 1971 ; Owman et al., 1974; Edvinsson, 1975). Information about their innervation in Prof. T. Tagawa, M.D., Department of Anatomy, School of Medicine, Fukuoka University, Fukuoka 814, Japan
Send offprint requests to:
0302-766X/79/0198/0043/$01.80
T. Tagawa et al.
44
1
.'-:. 9
\!'~'f'~/~
:.
'" ~ k ( .
~
~
- - k i .":: ' . . ' : " :
Fig. 1. Diagram of the arterial supply to the ventral surface of the brain in the domestic fowl, a cerebral carotid artery; b anterior ramus; c posterior cerebral artery; d middle cerebral artery; e anterior cerebral artery; f cerebro-ethmoidal artery; g posterior ramus; h posterior superior cerebellar artery; i basilar artery; j posterior inferior cerebellar artery; k vertebral artery
Figs. 2-13. Adrenergic nerve plexuses in cerebral blood vessels demonstrated by fluorescent histochemistry Fig. 2. The adrenergic innervation in a whole stretched preparation of the anterior ramus, x 67 Fig. 3. The posterior ramus. • 67 Fig. 4. The posterior cerebral artery which arises from the anterior ramus. • 67 Fig. 5. The middle cerebral artery which is a branch of the anterior ramus, x 67 Fig. 6. The cerebro-ethmoidal artery, x 67 Fig. 7. The small branches of the middle cerebral artery. Arrow indicates an adrenergic nerve running straight along the axis of a very small artery, x 67 Fig. 8. The basilar artery, x 67 Pig. 9. The confluence of the vertebral arteries (k) and basilar artery (0. • 67 Fig. 10. Cross section of the internal carotid artery in the carotic canal. Long arrow indicates the adrenergic plexuses in the adventitia closely adjacent to the media, and short arrow indicates the autofluorescences of the elastic fibres. Arrowhead indicates a large stem nerve which contains fluorescent fibres. • 67 Fig, 11. The adrenergic plexuses in a cross section of anterior ramus. • 67 Fig. 12. Cross section of the basilar artery, x 67 Fig. 13. Fluorescent photomicrograph of a cross-sectioned intracerebral capillary (asterisk). Arrowhead indicates an adrenergic fibre of brain parenchymal nerve cell origin. • 268
46
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s u b m a m m a l i a n vertebrates, however, is sparse. I n o u r l a b o r a t o r y , a series o f c o m p a r a t i v e h i s t o c h e m i c a l studies is being carried o u t on the a u t o n o m i c i n n e r v a t i o n o f the cerebral b l o o d vessels o f v a r i o u s v e r t e b r a t e g r o u p s ( W a s a n o et al., 1975; I i j i m a et al., 1977; Iijima, 1977; T a g a w a et al., 1979), a n d the present study on the d o m e s t i c fowl is p a r t o f this series.
Materials and Methods The formaldehyde fluorescence techniques (Falck, 1962) and the direct colouring thiocholine method (Karnovsky and Roots, 1964) were used to demonstrate adrenerglc and cholinerglc nerves respectively. Thirty domestic fowls, Gallus domesticus, were decapitated and each brain quickly dissected from the skull and placed in Ringer's solution. The pial arteries were carefully removed from the brain, where some parenchymal arteries were still continuous with the pial ones. For the fluorescence technique the vessels were stretched on glass slides and immediately transferred to a desiccator to be dried in vacuo over phosphorus pentoxide for 1 h. The specimens were then treated with the paraformatdehyde gas (RH = 47 ~) for 1 h at 80~ and mounted for fluorescence microscopy. For the thiocholine method the vessels were fixed for 30min in 4% formaldehyde buffered to pH 7.4, washed thoroughly with distilled water, transferred into Karnovsky's medium (acetylthiocholine iodide-free) for 30 min at 4~C, and then incubated for 1 h at 20~ in medium containing 2 • 10-4M iso-OMPA (tetraisopropylpyrophosphoramide)as an inhibitor of the non-specific cholinesterase. After washing with 50 % ethanol, they were stretched on glass slides, dried in air, dehydrated, and finally mounted in balsam. For sectioning, small blocks (3-4 mm 3) of the brain were frozen rapidly in dry-ice iso-pentane, and dried in vacuo for six days at -45~ then two days at -20~ and one day at room temperature. They were then treated initially with paraformaldehyde gas (RH = 47 %) for 1 h at 80~ and infiltrated in vacuo with paraffin for 10 rain at 60~C. The blocks were sectioned (8-10 ~tmthick), and examined with a fluorescence microscope. For the acetylchofinesterase (ACHE)reaction, frozen sections (15-30 Inn thick) were made from other specimens of brain, fixed with 4 % formaldehyde, and then treated as described above.
Results A s c h e m a t i c illustration o f the arterial s u p p l y to the fowl b r a i n is given in Fig. 1. A l t h o u g h the stem vessels are the cerebral c a r o t i d a n d the v e r t e b r o - b a s i l a r systems, the l a t t e r is e x t r e m e l y u n d e r d e v e l o p e d , a n d the arterial b l o o d s u p p l y to the b r a i n seems m o s t l y to be p r o v i d e d b y the former. T h e cerebral c a r o t i d a r t e r y is divided into a n t e r i o r a n d p o s t e r i o r rami, a n d t h o u g h the p o s t e r i o r r a m u s connects with the b a s i l a r artery, an a n t e r i o r c o m m u n i c a t i n g a r t e r y is lacking, so t h a t the Willis ring is incomplete.
Adrenergic lnnervation. The a d r e n e r g i c nerve plexuses in the cerebral c a r o t i d artery are c o m p o s e d o f thin fibres, o r g a n i z e d into e l o n g a t e d m e s h w o r k s a l o n g the arterial axis. A s the size o f the a r t e r y decreases, the plexus fibres b e c o m e thinner a n d the m e s h w o r k s m o r e elongated. Plexuses in the a n t e r i o r ramus, the m i d d l e cerebral a n d the c e r e b r o - e t h m o i d a l arteries are m o r p h o l o g i c a l l y similar to those o f the cerebral c a r o t i d a r t e r y (Figs. 2, 4, 5, 6), b u t in the p o s t e r i o r r a m u s they are less dense a n d their m e s h w o r k o f fibres is m o r e e l o n g a t e d (Fig. 3). The nerve fibres in the b a s i l a r a n d the v e r t e b r a l arteries are finer t h a n those in the cerebral c a r o t i d system, a n d are inclined to r u n p a r a l l e l with the arterial axis (Figs. 8, 9). In the small pial arteries, the
Cerebral Blood Vessel Innervation in the Domestic Fowl
47
Figs. 14-19. Cholinergic nerve plexuses in cerebral blood vessels demonstrated by the thiocholine technique Fig. 14. The cholinergic innervation in a whole stretched preparation of the cerebral carotid artery (a), the anterior (b) and the posterior (g) rami. Arrow indicates a stem nerve. • 64 Fig. 15. The cholinergic plexuses in the anterior ramus. Arrow indicates cholinergic stem nerve, x 64 Fig. 16. The posterior cerebral artery. Arrow indicates a stem nerve, x 64 Fig. 17. The middle cerebral artery, x 64 Fig. 18. The cerebro-ethmoidal artery, x 64 Fig. 19. Small branches of the middle cerebral artery. • 64
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Figs. 20--25. Cholinergic nerve plexuses in the cerebral blood vessels Fig. 20. The basilar artery. Arrowhead indicates a cholinergic nerve cell. • 64 Fig. 21. The confluenceof the vertebral arteries (k) to the basilar artery (0. Arrow indicatesa stem nerve. • 64 Fig. 22. Cross section of the internal carotid artery in the carotic canal. Arrow indicates the adrenergic plexuses in the adventitia-media border, and the arrowhead indicates a large stem nerve which contains cholinergic fibres, x 64 Fig. 23. Cross section of the anterior ramus. Arrows indicate the cholinergic fibres, x 64 Fig. 24. Cross section of the vertebral arteries. Arrows indicate the cholinergic nerve bundles. • 64 Fig. 25. A cholinergicnerve fibre (arrow) whichoriginates from a cholinergicnervecell(arrowhead) in the brain and terminates in a capillary wall (asterisk). x 320
fibres m a y be straight or spiral (Fig. 7). I n the carotic canal, the d i s t r i b u t i o n of nerve fibres in the i n t e r n a l carotid artery is less dense t h a n in the cerebral carotid artery, a n d a large fluorescent nerve b u n d l e runs parallel with the artery (Fig. 10). I n cross sections, these adrenergic fibres are located in the vascular wall in the t u n i c a adventitia, adjacent to the tunica media, a n d their density of distribution closely c o r r e s p o n d s with the degree of density shown by the plexus m e s h w o r k in the whole m o u n t p r e p a r a t i o n s (Figs. 11, 12). O n l y a few nerve fibres o f peripheral origin are observed in the p a r e n c h y m a l arterial wall, a n d in some p a r e n c h y m a l capillaries a n d arterioles, fibres c a n n o t be detected. Instead, nerve fibres originating from the intracerebral nerve cells are seen, a n d such fibres are c o m m o n in the d i e n c e p h a l o n (Fig. 13).
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Cholinergic Innervation. In general, the AChE-positive nerve fibres in the cerebral arteries are considerably less numerous than adrenergic ones, and the ACHEactivity is generally weak. The cerebral carotid artery plexuses are composed of fine fibres, in elongated meshworks along the arterial axis (Fig. 14). Their density and AChE-activity rapidly diminishes toward the distal portion of the anterior and posterior rami and their peripheral branches (Figs. 15, 16, 17, 19). However, in the cerebro-ethmoidal artery, which is the end branch of the anterior ramus and anastomoses with the ethmoidal artery, the density and AChE-activity increases again (Fig. 18). In the distal portion of the basilar artery, which connects with the posterior ramus, the density and the AChE-activity of the nerve plexuses are approximately similar to the distal portion of the anterior ramus (Fig. 20). In the proximal portion of the small basilar artery arising from the vertebral artery, one or two large nerve bundles with a high AChE-activity are constantly observed (Fig. 21), but in the small pial and parenchymal arteries they are not commonly seen. In the carotic canal, on the other hand, a large AChE-positive nerve bundle runs parallel with the internal carotid artery and is identical with that observed by fluorescence microscopy (Figs. 10, 22). It is of interest to note that isolated cholinergic nerve cells occur scattered throughout the nerve plexuses of the large cerebral arteries (Fig. 20). Like the adrenergic nerves, in the cross sections of the blood vessels, the cholinergic fibres are similarly located in the tunica adventitia (Figs. 23, 24), though in the small pial arteries the AChE-positive fibres can scarcely be seen. Cholinergic nerves of peripheral origin are not evident in the small parenchymal artery, arterioles and capillaries, and these vessels appear to be directly innervated by nerve fibres arising from cholinergic nerve cells in the brain, especially in the diencephalon (Fig. 25). It is interesting to note that the parenchymal capillaries and arterioles show a strongly positive AChE-activity in their walls. Discussion
It has been established that the mammalian cerebral arteries have a dual innervation, and both adrenergic and cholinergic nerve plexuses coexist in the vascular walls (Schenk and Badawi, 1968; Edvinsson, 1975; Wasano et al., 1975). A dual innervation is also found in reptiles, and in the cerebral arteries of a snake, the innervation is essentially similar to that of mammals, except that adrenergic plexuses are more prominent than cholinergic ones (Iijima et al., 1977). In the turtle, however, the cholinergic plexuses are significantly less well developed, and more closely resemble the amphibian pattern where, in the bullfrog at least, the adrenergic fibres are well developed as in reptiles, but cholinergic nerves cannot be detected (Tagawa et al., 1979). In the present study, the adrenergic nerve plexuses of the cerebral blood vessels of the fowl are denser than those in mammals and the other vertebrates that have so far been studied, but the cholinergic plexuses are less dense and show weaker AChE-activity than in mammals and snakes. It has previously been reported that the cerebrovascular nerves of mammals (Edvinsson, 1975) and turtles (Iijima, 1977) enter the cranial cavity along the internal carotid and the vertebral arteries, and in the snake along the cerebral
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c a r o t i d a n d o p h t h a l m i c arteries (Iijima et al., 1977). In the present study, however, the cholinergic nerves enter the cranial cavity chiefly a l o n g the internal c a r o t i d a n d p a r t l y a l o n g the v e r t e b r a l a n d e t h m o i d a l arteries. In the case o f the adrenergic nerves, they m o s t likely enter the cranial cavity along the internal c a r o t i d a n d p a r t l y a l o n g the v e r t e b r a l arteries. I n rats, p a r e n c h y m a l arteries are i n n e r v a t e d directly by n o n - s y m p a t h e t i c a d r e n e r g i c fibres o r i g i n a t i n g f r o m the i n t r a c e r e b r a l cell bodies ( F a l c k et al., 1965, 1968; H a r t m a n et al., 1972; E d v i n s s o n et al., 1973a, b), a n d in their electron m i c r o s c o p i c study, Rennels a n d N e l s o n (1975) have p r o v i d e d conclusive evidence o f the existence o f a x o n terminals in capillaries o f the cat h y p o t h a l a m u s . In o u r p r e v i o u s h i s t o c h e m i c a l studies on the b r a i n s o f the snake, turtle a n d bullfrog, we have similarly n o t e d t h a t the i n t r a c e r e b r a l capillaries a n d arterioles are dually i n n e r v a t e d b y p a r e n c h y m a l nerve cells (Iijima, 1977; Iijima et al., 1977; T a g a w a et al., 1979), a n d in the present s t u d y this central i n n e r v a t i o n is also evident in the c e r e b r a l vessels o f the fowl. T h e vessel walls o f the i n t r a c e r e b r a l arterioles a n d capillaries have an ACHEpositive activity in the fowl, as has also been r e c o r d e d in m a m m a l i a n ( F l u m e r f e l t et al., 1973), reptilian (Iijima, 1977; Iijima et al., 1977) a n d a m p h i b i a n species (Shen et al., 1955; B r i g h t m a n a n d Albers, 1959; Contestabile, 1976; T a g a w a et al., 1979).
References Brightman, M.W., Albers, R.W.: Species differences in the distribution of extraneuronal cholinesterase within the vertebrate central nervous system. J. Neurochem. 4, 244-250 (1959) Contestabile, A.: Histochemical characterization of cholinesterase activity in the frog brain with special reference to its localization on the wall of blood vessels. Histochem. J. 8, 513-521 (1976) Edvinsson, L.: Neurogenic mechanisms in the cerebrovascular bed. Autonomic nerves, amine receptors and their effects on cerebral blood flow. Acta Physiol. Scand. (Suppl.) 427, 1-35 (1975) Edvinsson, L., Lindvall, M., Nielsen, K.C., Owman, Ch.: Are brain vessels innervated also by central (non-sympathetic) adrenergic neurones? Brain Res. 63, 496-499 (1973a) Edvinsson, L., Nielsen, K.C., Owrnan, Ch., West, K.A.: Evidence of vasoconstrictor sympathetic nerves in brain vessels of mice. Neurol. 23, 73-77 (1973b) Falck, B.: Observations on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta Physiol. Scand. (Suppl. 197) 56, 1-25 (1962) Falck, B., Mchedlishvili, G.I., Owman, Ch.: Histochemical demonstration of adrenergic nerves in cortex-pia of rabbit. Acta Pharmacol. Toxicol. 23, 133-142 (1965) Falck, B., Nielsen, K.C., Owman, Ch.: Adrenergic innervation of the pial circulation. Scand. J. Cli. Lab. Invest. (Suppl.) 102, 96-98 (1968) Flumerfelt, B.A., Lewis, P.R., Gwyn, D.G.: Cholinesterase activity of capillaries in the rat brain. A light and electron microscopic study. Histochem. J. 5, 67-77 (1973) Hartman, B.K., Zide, D., Udenfriend, S.: The use of dopamine fl-hydroxylase as a marker for the central noradrenergic nervous system in rat brain. Proc. Natl. Acad. Sci. (U.S.A.) 69, 2722-2726 (1972) Iijima, T.: A histochemical study of the innervation of cerebral blood vessels in the turtle. J. Comp. Neur. 176, 307-314 (1977) Iijima, T., Wasano, T., Tagawa, T., AndS, K.: A histochemical study of the innervation of cerebral blood vessels in the snake. Cell Tissue Res. 179, 143-155 (1977) Karnovsky, M.I., Roots, L.: A "direct-coloring" thiocholine method for cholinesterases. J. Histochem. Cytochem. 12, 219-221 (1964) Motavkin, P.A., Dovbish, T.V.: Histochemical characteristics of acetylcholinesterase of the nerves innervating the brain vessels. Acta Morphol. Acad. Sci. Hung. 19, 159-173 (1971) Nelson, E., Rennels, M.: Innervation of intracranial arteries. Brain 93, 475-490 (1970)
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Owman, Ch., Edvinsson, L., Nielsen, K.C.: Autonomic neuroreceptor mechanisms in brain vessels. Blood Vessels 11, 2-31 (1974) Peerless, S_I., Yasargil, M.G.: Adrenergic innervation of the cerebral blood vessels in the rabbit. J. Neurosurg. Psychiatry 35, 148-154 (1971) Rennels, M.L., Nelson, E.: Capillary innervation in the mammalian central nervous system: An electron microscopic demonstration (1). Am. J. Anat. 144, 233-241 (1975) Schenk, E.A., Badawi, A.EI.: Dual innervation of arteries and arterioles. A histochemical study. Z. 'Zellforsch. 91, 170-177 (1968) Shen, S.C., Greenfield, P., Boe11, E.J.: The distribution of cholinesterase in the frog brain. J. Comp. Neurol. 102, 717-743 (1955) Tagawa, T., AndS, K., Wasano, T., Iijima, T.: A histochemical study of the innervation of cerebral blood vessels in the bullfrog. J. Comp. Neurol. 183, 25-32 (1979) Wasano, T., Tagawa, T., Iijima, T., AndS, K.: Histochemical studies on the adrenergic and cholinergic nerves innervating the cerebral arteries of vertebrates. Proc. 10th Int. Cong. Anat. Tokyo p. 144 (1975) Accepted January 22, 1979
