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Regeneration of taste buds after reinnervation by peripheral or central sensory fibers of vagal ganglia
EXPERIMENTAL
NEUROLOGY
Regeneration Peripheral
25, 429437
of Taste
or Central
(1969)
Buds
Sensory
after
Reinnervation
Fibers
of Vagal
by Ganglia
~WDREW A. ZALEWSKI Laboratory of Neuropathology and Neuroanatomical Sciences, National Neurological Diseases and Stroke. National Institutes of Health, Public Service, U.S. Department of Health, Education and Welfare, Bethesda, Maryland 20014 Received
July
Institute
of
Health
7,1969
Denervation causes the degeneration and disappearance of taste buds in the rat’s vallate papilla. Buds will, however, reappear after reinnervation by the glossopharyngeal (self-reinnervation), vagus, or chorda tympani nerves. In all previous reinnervation studies, the anastomosis of the reinnervating nerve was performed distal to the sensory ganglia, that is, with peripheral fibers of the ganglia. The present experiment was performed to determine whether buds would reappear after reinnervation by central fibers of the gustatory ganglia. The vallate papilla of adult male rats was (studied after denervation and after reinnervation by peripheral (superior and nodose) or central (nodose) sensory fibers of the vagus nerve. All taste buds disappeared from the papilla within 2 weeks after denervation and, in the absence of innervation, none reappeared. Buds were found, however, 5 months after reinnervation by peripheral or central sensory fibers of the vague nerve. The regenerated buds were found only in their normal location in the trench walls, and not in the epithelium on the top of the papilla or in the epithelium surrounding it. Although more buds were present after peripheral than after central reinnervation, the results demonstrate that either peripheral or central sensory fibers of gustatory ganglia can cause taste bud regeneration. Despite the known morphological and physiological differences between peripheral and central fibers of gustatory sensory ganglia, each can exert a trophic influence sufficient to cause bud regeneration. Introduction
Taste buds are morphologically dependent on a trophic influence mediated by the intact gustatory nerve, for after transection of their nerve supply the buds degenerate and disappear. Studies have shown that buds will regenerate after reinnervation by gustatory nerves (glossopharyngeal, vagus, chorda tympani) , but not after reinnervation by motor (hypoglossal) or general sensory (auriculotemporal) nerves (2, 9, 23). Furthermore, a gustatory nerve can cause bud regeneration in any denervated gustatory epithelium
regardless
of whether
the nerve
normally
innervates
buds on the
front of the tongue (chorda tympani), back of the tongue (glossopharyngeal), or pharynx and larynx (vagus) (9, 23). In all the studies where 429
430
ZALEWSKI
gustatory nerves caused bud regeneration, the anastomosis was performed distal to the sensory ganglia; that is, reinnervation was by peripheral fibers of the ganglia. The present experiment was performed to determine whether taste buds would regenerate after reinnervation by central fibers of the ganglia. There are several well known differences between peripheral and central fibers of sensory ganglia that might affect the ability of central fibers to cause bud regeneration. First, the central fibers of sensory ganglia neurons are thinner than the peripheral fibers (17, 18). Consequently the thinner central fibers have less neuroplasm with which to exert a trophic influence. If bud regeneration, as in amphibian limb regeneration, was dependent on a threshold volume of neuroplasm (19), the central fibers because of their smaller volume could be trophically inadequate. Second, since the neurons of sensory ganglia are pseudounipolar cells, it is possible that a segregation of the trophic influence could occur at the locus at which the axon bifurcates into peripheral and central processes. If this were true the central fiber could not exert any trophic influence regardless of the amount of neuroplasm present. Third, the direction of impulse conduction is different in peripheral and central fibers. When central fibers reach the epithelium the presence of persistent impulse activity at the ends of central fibers could adversely affect the neuroepithelial interaction and preclude the development of buds. Finally, the ends of central fibers from synaptic contacts with neurons in the central nervous system. Ko such synapses have been described with peripheral nerve endings and the taste cells ( 16). This could indicate that a “special” type of contact (present in peripheral but not central fibers) might be needed to transmit the trophic influence. In the present experiment, the vallate papilla of rats was examined for taste buds after denervation, and after reinnervation by peripheral or central sensory fibers of the vagus nerve. Also since denervation causes a loss of alkaline phosphatase activity associated with the taste buds (20, 21), the effects of reinnervation on the enzyme were also studied. Materials
and
Methods
Osborne-Mendel male rats weighing 200-250 g were anesthetized by an intraperitoneal injection of chloral hydrate (400 mg/kg). The glossopharyngeal and vagus nerves were identified as they exited from the posterior lacerated foramen. Since the vallate papilla is innervated by right and left glossopharyngeal nerves (8)) both nerves were sectioned in order to denervate the papilla. After cutting these nerves, the proximal portions were implanted into adjacent muscle to prevent nerve regeneration. A small silk tie was also placed around each proximal segment so that at the time each animal was killed the fate of the glossopharyngeal nerves could be ascertained.
VAGAL
NERVE
FIBERS
431
Reinnervation of the papilla was performed unilaterally and on the left side. Inasmuch as one glossopharyngeal can maintain more than 80% of the buds in the papilla (lo), unilateral reinnervation seemed an adequate test of cl nerve’s ability to cause taste bud regeneration. Either the peripheral portion of the vagus nerve or the central fibers of the nodose ganglion was joined to the distal stump of the transected glossopharyngeal nerve. To effect the anastomosis, the appropriate nerve ends were tucked into an arterial graft and held in place by topically added fibrinogen. Since the nodose ganglion is the larger of the two vagal sensory ganglia, it was felt enough neurons were utilized to determine whether central sensory fibers could initiate bud regeneration. The denervated papilla of three animals was studied 2 weeks postoperatively to insure that cutting the glossopharyngeal nerves caused the disappearance of the taste buds. Three denervated, three peripherally reinnervated, and six centrally reinnervated papillae were examined 5 months postoperatively. The site of the nerve anastomosis and the location of the glossopharyngeal nerves was identified each time an animal wav killed. In addition, the regenerated nerve from one peripherally and one centrally reinnervated papilla was cut distal to the anastomosis, and the effects of this denervation studied 3 days later. Frozen sections (6~) of the papillae were prepared and incubated to detect adenosine triphosphatase, cholinesterase, or alkaline phosphatase activity. Control slides were incubated without added substrate. The methods and incubation procedures have been reported (20). Results
Effects of Denemation (and Reinnervation on Taste Buds. Taste buds were readily identified by their adenosine triphosphatase activity. In the normal papilla (Fig. lA), buds were present only in the epithelium of the trench walls. The distribution and number of buds in the inner and outer trench walls was similiar, and each bud extended through the width of the epithelium. Control slides incubated without substrate did not reveal any buds. Within 2 weeks after denervation (Fig. lB), all buds had disappeared, and none was present in papillae examined after 5 months. The only additional effect of denervation was an atrophy of the epithelium where buds had been located (compare Fig. 1B with Fig. 1D). Buds were found, however, 5 months after either peripheral or central sensory vagal reinnervation (Fig. 1C, D) . The regenerated buds were in their normal position in the lower portions of the trench walls ; none was found in the epithelium on the top of the papilla or in the epithelium surrounding it. In both cases there was a tendency for buds to reappear in the inner rather than outer trench walls, but more buds were found after peripheral (Fig. 1C) than af-
432
ZALEWSKI
triphosphatase activity. A. Normal papilla. Taste buds are present FIG. 1. Adenosine in the epithelium of the lower portions of the trench walls ( x 72). E. Denervated pa pilla. Within 2 weeks after denervation, all taste buds had disappeared ( x 72). C. Peripheral sensory reinnervated papilla. Regenerated taste buds are present 5 months postoperatively ( x 72). D. Central sensory reinnervated papilla. Buds are present 5 months postoperatively. Fewer buds were found after central than after peripheral reinnervation. Note the marked atrophy of the epithelium (left trench of picture) which occurred when no buds or innervation was present.
VAGAL
NERVE
FIBERS
433
ter central (Fig. 1D) reinnervation. Although quantitative counts were not made, it was evident that neither type of reinnervation restored the normal number of taste buds. In the one peripherally and one centrally reinnervated papilla in which the nerve was subsequently cut, degenerating buds were found. These buds were smaller than normal, and they appeared to be moving away from the basement membrane towards the epithelial surface. That they were indeed denervated was suggested by the lack of cholinesterase staining beneath the regions of epithelium containing the degenerating buds. An intense nerve fiber cholinesterase reaction was always present beneath the buds of the normal or reinnervated papillae. Effects of Deneruation and Reinnervation on Alkaline Phosphatase Activity. In the normal papilla, alkaline phosphatase (Fig. 2A) was detected only in that region of the trench wall epithelium in which the taste buds were located. No enzyme was demonstrated in control slides incubated without added substrate. Coincident with taste buds loss, most enzyme activity disappeared 2 weeks after denervation (Fig. ZB). At 5 months some activity was still detected, but this was due to enzyme present in leucocytes which had invaded the epithelium. The enzyme was restored on the epithelial surface only in those regions where regenerated taste buds appeared (Fig. 2C, D) . Enzyme activity seemed to be localized around the taste bud pore (Fig. 2D), and not in the stratified squamous epithelium between the buds. Although the distribution of the enzyme depended on the number and position of the regenerated buds, the localization was similiar after peripheral or central reinnervation. Discussion
Regenerate taste buds appeared in the vallate papilla after reinnervation by peripheral or central sensory fibers of the vagus nerve. Despite the known morphological (fiber diameter, synaptic ending) and physiological (direction of impulse conduction) differences between these fibers, each can exert a trophic influence sufficient to cause bud regeneration. The utilization of nodose ganglion neurons that are isolated from the central nervous system (i.e., central sensory reinnervation) clearly demonstrates that the development and maintenance of buds is not dependent on reflex connections, and that the trophic influence does not emanate from the central nervous system. A similar conclusion has been reached from results of an experiment in which only central transection (i.e., proximal to the sensory ganglia) of the gustatory nerve has been performed (13). Morphological studies of sensory ganglia, however, have as yet to reveal differences between those neurons which are tropically effective and those which are not, even though only two or three types have been described (3, 4, 14). In a recent comparative study of spinal and cranial ganglia, similiar cell types
434
FIG. 2. Alkaline phosphatase activity. ity is present in the lower portions of ( X72). B. Denervated trench wall of alkaline phosphatase activity is lost 2 sensory reinnervated papilla (C) and
ZhLEWSIiI
A. Normal papilla. Alkaline phosphatase activthe trench walls where the taste buds are located papilla. Coincident with taste bud degeneration, weeks after denervation ( X300). C. D. Central trench wall (D). -1lkalinc phosphatase activity
VAGAL
NERVE
FIBERS
435
were found (3)) nevertheless no buds appeared when the lingual epithelium was reinnervated by spinal ganglion cells (6). The cells of cranial ganglia must, therefore, differ metabolically from those of spinal ganglia cells. Furthermore, the evidence to date suggests that only gustatory neurons can produce the trophic effect on taste buds. The possibility that general sensoneurons might be quantitatively inadequate in this regard has been ruled out experimentally. When the vallate papilla was bilaterally reinnervated by general sensory neurons, thereby increasing the number of fibers and volume of neuroplasm acting on the epithelium ( 11) , no buds were found (23). Although bud regeneration is dependent on a trophic influence of certain cranial ganglia, it cannot be concluded that only one of the cell types present is responsible. Although nerve fibers are readily demonstrated beneath the regenerated buds, their source remains unknown ; they could issue from any of the neurons present in the ganglia. No attempt has been made to relate either the normal or regenerated fibers to their cells of origin. On the other hand, the fact that smaller numbers of taste buds reappear after nerve regeneration (either original or other gustatory nerve) (23) could mean that specific neurons do indeed exert the trophic influence. Regions of cholinesterase activity were at times found beneath the epithelium of the trench walls where no buds appeared (Zalewski, unpublished), perhaps these enzymatic sites represent regenerated general sensory nerve fibers. To date, only a few drugs have been administered in hope of replacing the trophic influence of the gustatory nerve. Testosterone has been shown to cause the appearance of buds on the top of the vallate papilla (1, 22, 23) and in the epithelium surrounding it (23). This effect occurs, however, only if the gustatory nerve is intact, for administration of testosterone fails to prevent the degeneration of denervated taste buds (22). Testosterone acts with but not in place of the gustatory nerve. Adrenocorticotropin has also been injected, but it also did not support buds after denervation (Zalewski. unpublished). Alkaline phosphatase was restored only in those regions of the epithelium where regenerated buds appeared. The localization of the enzyme seemed to correspond to the region of the taste bud pore (Fig. 2D). Most likely, the enzyme is present on the membranes of the microvilli of the taste bud cells since it has been localized electronmicroscopically in the microvilli of epithelial cells of the intestine (12) and in the proximal convoluted tubules of associated with the taste buds is restored after 5 months. No enzyme is present in the trench walls lacking taste buds (C). Note that the enzyme is present over the free surface of the buds (D), and not in the stratified squamous epithelium between the buds ( X 72, X300). Similar findings were observed after peripheral sensory reinnervation.
436
ZALEWSKI
the kidney (15). Localization studies at the ultrastructural level might help explain why the enzyme is present in the taste bud cells of the rat’s vallate and foliate but not fungiform papillae (20) in spite of the fact that comparable types of cells are found (5, 7). Furthermore, this ezymatic difference is not due to the differences in innervation (21) . All gustatory nerves cause the appearance in buds of the vallate papilla (23), but none induce it in buds of the fungiform papillae (Zalewski, unpublished). From the preceding discussion, it is evident that continued studies of the neuron, epithelium, and extra neuroepithelial factors (hormones) are needed before the trophic mechanism(s) regulating taste buds is understood (11). References 1. ALLARA, E. 1952. Sull’ influenza esercitata dagli ormon-sulla stattura dell formazioni gustative di mus rattus albinus. Rizr. Biol. 44 : 209-229. 2. AREY, L. B., and F. L. MONZINGO. 1942. Can hypoglossal nerve fibers induce the formation of taste buds. Quart. Bull. A~orthzuesfrr~r Uirriv. Med. School. 16: 170-178. 3. CARMEL, P. W., and B. M. STEIN. 1969. Cell changes in sensory ganglia following proximal and distal nerve section in the monkey. /. Cor~b. Neural. 1%: 145-166. 4. DE CASTRO, F. 1932. Sensory ganglia of the cranial and spinal nerves, normal and pathological, vol. 1, pp 93-143. 112 “Cytology and Cellular Pathology of the Nervous System.” W. Penfield, [ed.]. Hoeber, New York. 5. FARBMAN, A. I. 1965. Fine structure of the taste bud. J. Ultrastruct. Rcs. 12: 328-350. 6. FARBMAN, A. I., and M. ZIEGNER. 1968. Differentiation of fetal at tongue grafts in the anterior chamber of the eye. dnat. Record 160 : 347. GRAY, E. G., and K. C. WATKINS. 1965. Electron microscopy of taste buds of the rat. Z. Zellforsch. Mikroskop. .dnat., ADt. Histochrm. 66 : 583-595. 8. GUTH, L. 1957. The effects of glossopharyngeal nerve transection n the circutnvallate papilla of the rat. Anat. Record 126: 715-731. 9. GUTH, L. 1958. Taste buds on the cat’s circumvallate papilla after reimlervation by glossopharyngeal, vagus, and hypoglossal nerves. *-~JuI~. Record 130 : 25-37. 10. GUTH, L. 1963. Histological changes following partial denervation of the circumvallate papilla of the rat. E.~fitl. Nczcrol. 8 : 336-349. 11. GUTH, L. 1969. Trophic effects of vertebrate neurons. Ncrwoscienccs Rcs. Program Bull. 7 : l-73. 12. HUGON, J., and M. BORGERS. 1968. Fine structural localization of acid and alkaline phosphatase activities in the absorbing cells of the duodenum of rodents. Histockemie 12: 42-66. 13. KAMRIN, R. P., and M. SINGER. 1953. Influences of sensory neurons isolated from the central nervous system on maintenance of taste buds and regeneration of barbels in the catfish, Ameriurus Nebulosus. A?n. J. Physiol. 174 : 146-148. 14. LIEBERMAN, A. R. 1969. Light and electron-microscope observations on the Golgi apparatus of normal and axotomized primary sensory neurons. .I. .-l~taf. 104: 309-32s. 7.
VAGAL
NERVE
FIBERS
437
15. MAYAHARA, H., and K. OGAWA. 1968. The effect of thickness of specimen on the ultrastructural localization of alkaline phosphatase activity in the rat proximal convoluted tubule. J. Histochem. Cytochem. 16 : 721-724. 16. MURRAY, R. G., and A. MURRAY. 1967. Fine structure of taste buds of rabbit foliate papillae. J. Ultrastruct. Res. 19 : 327-353. 17. CAJAL, RAT&NY S. 1909. “Histologie due Systeme nerveux de 1 1’Homme et des VertebrPs.” Tome 1: “Generalites, Moelle, Ganglions rachidiens, Bulbe et Protuberance.” Maloine, Paris. 18. RANSON, S. W., and H. K. DAVENPORT. 1931. Sensory unmyelinated fibers in the spinal nerves. Am. J. Anat. 48: 331-353. 19. SINGER, M., B. RZEHAB, and C. S. MAIER. 1967. The relation between the caliber of the axon and the trophic activity of nerves in limb regeneration. J. Exptl. 2’001. 166: 89-97. 20. ZALEWSKI, A. A. 1968. Changes in phosphatase enzymes following denervation of the vallate papilla of the rat. Exptl. Newol. 22 : 40-51. 21. ZALEWSKI, A. A. 1969a. Role of nerve and epithelium in the regulation of alkaline phosphatase activity in gustatory papillae. Exptl. Neurol. 23 : 18-28. 22. ZALEWSKI, A. A. 1%9b. Neurotrophic-hormonal interaction in the regulation of taste buds in the rat’s vallate papilla. J. Newobiol. 1: 123-132. 23. ZALEWSKI, A. A. 1969~. Combined effects of testosterone and motor, sensory, or gustatory nerve reinnervation on the regeneration of taste buds in the rat. Exptl.
Newel.
24:
285-297.
NEUROLOGY
Regeneration Peripheral
25, 429437
of Taste
or Central
(1969)
Buds
Sensory
after
Reinnervation
Fibers
of Vagal
by Ganglia
~WDREW A. ZALEWSKI Laboratory of Neuropathology and Neuroanatomical Sciences, National Neurological Diseases and Stroke. National Institutes of Health, Public Service, U.S. Department of Health, Education and Welfare, Bethesda, Maryland 20014 Received
July
Institute
of
Health
7,1969
Denervation causes the degeneration and disappearance of taste buds in the rat’s vallate papilla. Buds will, however, reappear after reinnervation by the glossopharyngeal (self-reinnervation), vagus, or chorda tympani nerves. In all previous reinnervation studies, the anastomosis of the reinnervating nerve was performed distal to the sensory ganglia, that is, with peripheral fibers of the ganglia. The present experiment was performed to determine whether buds would reappear after reinnervation by central fibers of the gustatory ganglia. The vallate papilla of adult male rats was (studied after denervation and after reinnervation by peripheral (superior and nodose) or central (nodose) sensory fibers of the vagus nerve. All taste buds disappeared from the papilla within 2 weeks after denervation and, in the absence of innervation, none reappeared. Buds were found, however, 5 months after reinnervation by peripheral or central sensory fibers of the vague nerve. The regenerated buds were found only in their normal location in the trench walls, and not in the epithelium on the top of the papilla or in the epithelium surrounding it. Although more buds were present after peripheral than after central reinnervation, the results demonstrate that either peripheral or central sensory fibers of gustatory ganglia can cause taste bud regeneration. Despite the known morphological and physiological differences between peripheral and central fibers of gustatory sensory ganglia, each can exert a trophic influence sufficient to cause bud regeneration. Introduction
Taste buds are morphologically dependent on a trophic influence mediated by the intact gustatory nerve, for after transection of their nerve supply the buds degenerate and disappear. Studies have shown that buds will regenerate after reinnervation by gustatory nerves (glossopharyngeal, vagus, chorda tympani) , but not after reinnervation by motor (hypoglossal) or general sensory (auriculotemporal) nerves (2, 9, 23). Furthermore, a gustatory nerve can cause bud regeneration in any denervated gustatory epithelium
regardless
of whether
the nerve
normally
innervates
buds on the
front of the tongue (chorda tympani), back of the tongue (glossopharyngeal), or pharynx and larynx (vagus) (9, 23). In all the studies where 429
430
ZALEWSKI
gustatory nerves caused bud regeneration, the anastomosis was performed distal to the sensory ganglia; that is, reinnervation was by peripheral fibers of the ganglia. The present experiment was performed to determine whether taste buds would regenerate after reinnervation by central fibers of the ganglia. There are several well known differences between peripheral and central fibers of sensory ganglia that might affect the ability of central fibers to cause bud regeneration. First, the central fibers of sensory ganglia neurons are thinner than the peripheral fibers (17, 18). Consequently the thinner central fibers have less neuroplasm with which to exert a trophic influence. If bud regeneration, as in amphibian limb regeneration, was dependent on a threshold volume of neuroplasm (19), the central fibers because of their smaller volume could be trophically inadequate. Second, since the neurons of sensory ganglia are pseudounipolar cells, it is possible that a segregation of the trophic influence could occur at the locus at which the axon bifurcates into peripheral and central processes. If this were true the central fiber could not exert any trophic influence regardless of the amount of neuroplasm present. Third, the direction of impulse conduction is different in peripheral and central fibers. When central fibers reach the epithelium the presence of persistent impulse activity at the ends of central fibers could adversely affect the neuroepithelial interaction and preclude the development of buds. Finally, the ends of central fibers from synaptic contacts with neurons in the central nervous system. Ko such synapses have been described with peripheral nerve endings and the taste cells ( 16). This could indicate that a “special” type of contact (present in peripheral but not central fibers) might be needed to transmit the trophic influence. In the present experiment, the vallate papilla of rats was examined for taste buds after denervation, and after reinnervation by peripheral or central sensory fibers of the vagus nerve. Also since denervation causes a loss of alkaline phosphatase activity associated with the taste buds (20, 21), the effects of reinnervation on the enzyme were also studied. Materials
and
Methods
Osborne-Mendel male rats weighing 200-250 g were anesthetized by an intraperitoneal injection of chloral hydrate (400 mg/kg). The glossopharyngeal and vagus nerves were identified as they exited from the posterior lacerated foramen. Since the vallate papilla is innervated by right and left glossopharyngeal nerves (8)) both nerves were sectioned in order to denervate the papilla. After cutting these nerves, the proximal portions were implanted into adjacent muscle to prevent nerve regeneration. A small silk tie was also placed around each proximal segment so that at the time each animal was killed the fate of the glossopharyngeal nerves could be ascertained.
VAGAL
NERVE
FIBERS
431
Reinnervation of the papilla was performed unilaterally and on the left side. Inasmuch as one glossopharyngeal can maintain more than 80% of the buds in the papilla (lo), unilateral reinnervation seemed an adequate test of cl nerve’s ability to cause taste bud regeneration. Either the peripheral portion of the vagus nerve or the central fibers of the nodose ganglion was joined to the distal stump of the transected glossopharyngeal nerve. To effect the anastomosis, the appropriate nerve ends were tucked into an arterial graft and held in place by topically added fibrinogen. Since the nodose ganglion is the larger of the two vagal sensory ganglia, it was felt enough neurons were utilized to determine whether central sensory fibers could initiate bud regeneration. The denervated papilla of three animals was studied 2 weeks postoperatively to insure that cutting the glossopharyngeal nerves caused the disappearance of the taste buds. Three denervated, three peripherally reinnervated, and six centrally reinnervated papillae were examined 5 months postoperatively. The site of the nerve anastomosis and the location of the glossopharyngeal nerves was identified each time an animal wav killed. In addition, the regenerated nerve from one peripherally and one centrally reinnervated papilla was cut distal to the anastomosis, and the effects of this denervation studied 3 days later. Frozen sections (6~) of the papillae were prepared and incubated to detect adenosine triphosphatase, cholinesterase, or alkaline phosphatase activity. Control slides were incubated without added substrate. The methods and incubation procedures have been reported (20). Results
Effects of Denemation (and Reinnervation on Taste Buds. Taste buds were readily identified by their adenosine triphosphatase activity. In the normal papilla (Fig. lA), buds were present only in the epithelium of the trench walls. The distribution and number of buds in the inner and outer trench walls was similiar, and each bud extended through the width of the epithelium. Control slides incubated without substrate did not reveal any buds. Within 2 weeks after denervation (Fig. lB), all buds had disappeared, and none was present in papillae examined after 5 months. The only additional effect of denervation was an atrophy of the epithelium where buds had been located (compare Fig. 1B with Fig. 1D). Buds were found, however, 5 months after either peripheral or central sensory vagal reinnervation (Fig. 1C, D) . The regenerated buds were in their normal position in the lower portions of the trench walls ; none was found in the epithelium on the top of the papilla or in the epithelium surrounding it. In both cases there was a tendency for buds to reappear in the inner rather than outer trench walls, but more buds were found after peripheral (Fig. 1C) than af-
432
ZALEWSKI
triphosphatase activity. A. Normal papilla. Taste buds are present FIG. 1. Adenosine in the epithelium of the lower portions of the trench walls ( x 72). E. Denervated pa pilla. Within 2 weeks after denervation, all taste buds had disappeared ( x 72). C. Peripheral sensory reinnervated papilla. Regenerated taste buds are present 5 months postoperatively ( x 72). D. Central sensory reinnervated papilla. Buds are present 5 months postoperatively. Fewer buds were found after central than after peripheral reinnervation. Note the marked atrophy of the epithelium (left trench of picture) which occurred when no buds or innervation was present.
VAGAL
NERVE
FIBERS
433
ter central (Fig. 1D) reinnervation. Although quantitative counts were not made, it was evident that neither type of reinnervation restored the normal number of taste buds. In the one peripherally and one centrally reinnervated papilla in which the nerve was subsequently cut, degenerating buds were found. These buds were smaller than normal, and they appeared to be moving away from the basement membrane towards the epithelial surface. That they were indeed denervated was suggested by the lack of cholinesterase staining beneath the regions of epithelium containing the degenerating buds. An intense nerve fiber cholinesterase reaction was always present beneath the buds of the normal or reinnervated papillae. Effects of Deneruation and Reinnervation on Alkaline Phosphatase Activity. In the normal papilla, alkaline phosphatase (Fig. 2A) was detected only in that region of the trench wall epithelium in which the taste buds were located. No enzyme was demonstrated in control slides incubated without added substrate. Coincident with taste buds loss, most enzyme activity disappeared 2 weeks after denervation (Fig. ZB). At 5 months some activity was still detected, but this was due to enzyme present in leucocytes which had invaded the epithelium. The enzyme was restored on the epithelial surface only in those regions where regenerated taste buds appeared (Fig. 2C, D) . Enzyme activity seemed to be localized around the taste bud pore (Fig. 2D), and not in the stratified squamous epithelium between the buds. Although the distribution of the enzyme depended on the number and position of the regenerated buds, the localization was similiar after peripheral or central reinnervation. Discussion
Regenerate taste buds appeared in the vallate papilla after reinnervation by peripheral or central sensory fibers of the vagus nerve. Despite the known morphological (fiber diameter, synaptic ending) and physiological (direction of impulse conduction) differences between these fibers, each can exert a trophic influence sufficient to cause bud regeneration. The utilization of nodose ganglion neurons that are isolated from the central nervous system (i.e., central sensory reinnervation) clearly demonstrates that the development and maintenance of buds is not dependent on reflex connections, and that the trophic influence does not emanate from the central nervous system. A similar conclusion has been reached from results of an experiment in which only central transection (i.e., proximal to the sensory ganglia) of the gustatory nerve has been performed (13). Morphological studies of sensory ganglia, however, have as yet to reveal differences between those neurons which are tropically effective and those which are not, even though only two or three types have been described (3, 4, 14). In a recent comparative study of spinal and cranial ganglia, similiar cell types
434
FIG. 2. Alkaline phosphatase activity. ity is present in the lower portions of ( X72). B. Denervated trench wall of alkaline phosphatase activity is lost 2 sensory reinnervated papilla (C) and
ZhLEWSIiI
A. Normal papilla. Alkaline phosphatase activthe trench walls where the taste buds are located papilla. Coincident with taste bud degeneration, weeks after denervation ( X300). C. D. Central trench wall (D). -1lkalinc phosphatase activity
VAGAL
NERVE
FIBERS
435
were found (3)) nevertheless no buds appeared when the lingual epithelium was reinnervated by spinal ganglion cells (6). The cells of cranial ganglia must, therefore, differ metabolically from those of spinal ganglia cells. Furthermore, the evidence to date suggests that only gustatory neurons can produce the trophic effect on taste buds. The possibility that general sensoneurons might be quantitatively inadequate in this regard has been ruled out experimentally. When the vallate papilla was bilaterally reinnervated by general sensory neurons, thereby increasing the number of fibers and volume of neuroplasm acting on the epithelium ( 11) , no buds were found (23). Although bud regeneration is dependent on a trophic influence of certain cranial ganglia, it cannot be concluded that only one of the cell types present is responsible. Although nerve fibers are readily demonstrated beneath the regenerated buds, their source remains unknown ; they could issue from any of the neurons present in the ganglia. No attempt has been made to relate either the normal or regenerated fibers to their cells of origin. On the other hand, the fact that smaller numbers of taste buds reappear after nerve regeneration (either original or other gustatory nerve) (23) could mean that specific neurons do indeed exert the trophic influence. Regions of cholinesterase activity were at times found beneath the epithelium of the trench walls where no buds appeared (Zalewski, unpublished), perhaps these enzymatic sites represent regenerated general sensory nerve fibers. To date, only a few drugs have been administered in hope of replacing the trophic influence of the gustatory nerve. Testosterone has been shown to cause the appearance of buds on the top of the vallate papilla (1, 22, 23) and in the epithelium surrounding it (23). This effect occurs, however, only if the gustatory nerve is intact, for administration of testosterone fails to prevent the degeneration of denervated taste buds (22). Testosterone acts with but not in place of the gustatory nerve. Adrenocorticotropin has also been injected, but it also did not support buds after denervation (Zalewski. unpublished). Alkaline phosphatase was restored only in those regions of the epithelium where regenerated buds appeared. The localization of the enzyme seemed to correspond to the region of the taste bud pore (Fig. 2D). Most likely, the enzyme is present on the membranes of the microvilli of the taste bud cells since it has been localized electronmicroscopically in the microvilli of epithelial cells of the intestine (12) and in the proximal convoluted tubules of associated with the taste buds is restored after 5 months. No enzyme is present in the trench walls lacking taste buds (C). Note that the enzyme is present over the free surface of the buds (D), and not in the stratified squamous epithelium between the buds ( X 72, X300). Similar findings were observed after peripheral sensory reinnervation.
436
ZALEWSKI
the kidney (15). Localization studies at the ultrastructural level might help explain why the enzyme is present in the taste bud cells of the rat’s vallate and foliate but not fungiform papillae (20) in spite of the fact that comparable types of cells are found (5, 7). Furthermore, this ezymatic difference is not due to the differences in innervation (21) . All gustatory nerves cause the appearance in buds of the vallate papilla (23), but none induce it in buds of the fungiform papillae (Zalewski, unpublished). From the preceding discussion, it is evident that continued studies of the neuron, epithelium, and extra neuroepithelial factors (hormones) are needed before the trophic mechanism(s) regulating taste buds is understood (11). References 1. ALLARA, E. 1952. Sull’ influenza esercitata dagli ormon-sulla stattura dell formazioni gustative di mus rattus albinus. Rizr. Biol. 44 : 209-229. 2. AREY, L. B., and F. L. MONZINGO. 1942. Can hypoglossal nerve fibers induce the formation of taste buds. Quart. Bull. A~orthzuesfrr~r Uirriv. Med. School. 16: 170-178. 3. CARMEL, P. W., and B. M. STEIN. 1969. Cell changes in sensory ganglia following proximal and distal nerve section in the monkey. /. Cor~b. Neural. 1%: 145-166. 4. DE CASTRO, F. 1932. Sensory ganglia of the cranial and spinal nerves, normal and pathological, vol. 1, pp 93-143. 112 “Cytology and Cellular Pathology of the Nervous System.” W. Penfield, [ed.]. Hoeber, New York. 5. FARBMAN, A. I. 1965. Fine structure of the taste bud. J. Ultrastruct. Rcs. 12: 328-350. 6. FARBMAN, A. I., and M. ZIEGNER. 1968. Differentiation of fetal at tongue grafts in the anterior chamber of the eye. dnat. Record 160 : 347. GRAY, E. G., and K. C. WATKINS. 1965. Electron microscopy of taste buds of the rat. Z. Zellforsch. Mikroskop. .dnat., ADt. Histochrm. 66 : 583-595. 8. GUTH, L. 1957. The effects of glossopharyngeal nerve transection n the circutnvallate papilla of the rat. Anat. Record 126: 715-731. 9. GUTH, L. 1958. Taste buds on the cat’s circumvallate papilla after reimlervation by glossopharyngeal, vagus, and hypoglossal nerves. *-~JuI~. Record 130 : 25-37. 10. GUTH, L. 1963. Histological changes following partial denervation of the circumvallate papilla of the rat. E.~fitl. Nczcrol. 8 : 336-349. 11. GUTH, L. 1969. Trophic effects of vertebrate neurons. Ncrwoscienccs Rcs. Program Bull. 7 : l-73. 12. HUGON, J., and M. BORGERS. 1968. Fine structural localization of acid and alkaline phosphatase activities in the absorbing cells of the duodenum of rodents. Histockemie 12: 42-66. 13. KAMRIN, R. P., and M. SINGER. 1953. Influences of sensory neurons isolated from the central nervous system on maintenance of taste buds and regeneration of barbels in the catfish, Ameriurus Nebulosus. A?n. J. Physiol. 174 : 146-148. 14. LIEBERMAN, A. R. 1969. Light and electron-microscope observations on the Golgi apparatus of normal and axotomized primary sensory neurons. .I. .-l~taf. 104: 309-32s. 7.
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15. MAYAHARA, H., and K. OGAWA. 1968. The effect of thickness of specimen on the ultrastructural localization of alkaline phosphatase activity in the rat proximal convoluted tubule. J. Histochem. Cytochem. 16 : 721-724. 16. MURRAY, R. G., and A. MURRAY. 1967. Fine structure of taste buds of rabbit foliate papillae. J. Ultrastruct. Res. 19 : 327-353. 17. CAJAL, RAT&NY S. 1909. “Histologie due Systeme nerveux de 1 1’Homme et des VertebrPs.” Tome 1: “Generalites, Moelle, Ganglions rachidiens, Bulbe et Protuberance.” Maloine, Paris. 18. RANSON, S. W., and H. K. DAVENPORT. 1931. Sensory unmyelinated fibers in the spinal nerves. Am. J. Anat. 48: 331-353. 19. SINGER, M., B. RZEHAB, and C. S. MAIER. 1967. The relation between the caliber of the axon and the trophic activity of nerves in limb regeneration. J. Exptl. 2’001. 166: 89-97. 20. ZALEWSKI, A. A. 1968. Changes in phosphatase enzymes following denervation of the vallate papilla of the rat. Exptl. Newol. 22 : 40-51. 21. ZALEWSKI, A. A. 1969a. Role of nerve and epithelium in the regulation of alkaline phosphatase activity in gustatory papillae. Exptl. Neurol. 23 : 18-28. 22. ZALEWSKI, A. A. 1%9b. Neurotrophic-hormonal interaction in the regulation of taste buds in the rat’s vallate papilla. J. Newobiol. 1: 123-132. 23. ZALEWSKI, A. A. 1969~. Combined effects of testosterone and motor, sensory, or gustatory nerve reinnervation on the regeneration of taste buds in the rat. Exptl.
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285-297.