Regeneration of taste buds by nongustatory nerve fibers

Previous cross-reinnervation studies in situ by other investigators have demonstrated that cutaneous sensory and motor axons are incapable of trophically supporting mammalian taste buds. The present experiments examined the gustatory trophic potency of chemosensory and barosensory axons of the carotid sinus nerve. We report here that morphologically normal taste buds appeared on cat circumvallate papillae at 2 to 19 months after cross-anastomosis of the carotid sinus and lingual nerves, branches of the IXth cranial (glossopharyngeal) nerve. However, neurophysiologic and histologic data also indicated that, despite microsurgical procedures designed to direct regenerating lingual nerve fibers toward the carotid body and carotid sinus, some lingual axons escaped the anastomosis and subsequently grew within their native distal stump. The principal objective of this study was thus to determine whether foreign innervation of taste buds did indeed occur, or regenerated lingual nerve fibers were instead responsible for the newly formed buds. Our results showed that stray lingual fibers were not responsible for the reappearance of taste buds because transection of the original proximal lingual nerve stump (cross-anastomosed to the distal carotid sinus nerve stump) did not reduce the incidence of taste buds or the accumulation of radiolabeled material axoplasmically transported from the petrosal (sensory) ganglion. Autoradiography of labeled tissue samples showed that more than 90% of the taste buds were labeled at 8 and 9 days after lingual nerve transection. These data support the hypothesis that sensory axons in the carotid sinus nerve share an important trophic chemistry with gustatory neurons.     

Regeneration of taste buds in tongue grafts after reinnervation by neurons in transplanted lumbar sensory ganglia

Taste buds disappear after denervation and reappear after nerve regeneration. Sensory neurons are responsible since reinnervation by motor or autonomic fibers of peripheral nerve fail to induce bud regeneration. However, we do not know whether some neurons in all sensory ganglia can support buds or whether gustatory (i.e., taste bud inducing) neurons are localized to specific cranial ganglia. The present study was therefore pefrormed to determine whether neurons in transplanted spinal ganglia could support taste buds similarly to those in transplanted cranial ganglia. Grafts of lumbar or vagal nodose ganglia were combined with grafts of tongue's vallate papillae in the anterior chamber of rats' eyes and the papillae examined for taste buds 35 days later. Neurons were present in all transplanted ganglia, and all papillae reinnervated by them contained regenerated taste buds. Nerve fibers could be traced from the transplanted ganglia to the epithelium of the tongue grafts which bore the regenerated taste buds. Papillae transplanted without ganglia lacked buds. These findings indicate that some neurons in all sensory ganglia can induce taste bud formation. The present results could occur if gustatory neurons are intrinsically present in all sensory ganglia, but an alternative interpretation is that the tongue grafts transformed some neurons into gustatory neurons and, hence, that neuronal plasticity is involved.     

Collateral reinnervation of taste buds after chronic sensory denervation: a morphological study

Peripheral transganglionic transport of horseradish peroxidase (HRP) was used to label afferent fibers in the taste buds and lingual epithelium 2-12 weeks after chronic chorda tympani or combined chorda tympani-lingual nerve lesions. From 4-12 weeks after a chronic chorda tympani lesion, taste buds could be found. These were innervated by fibers from the ipsilateral lingual nerve. From 8-12 weeks after a chronic chorda tympani-lingual nerve lesion, nerve fibers from the contralateral lingual nerve could be found in a few taste buds on the denervated side of the tongue. Thus, collateral sprouting took place over the midline in this instance. These findings indicate that intact gustatory axons do not sprout into denervated taste buds, but trigeminal fibers in the lingual nerve do have this ability.     

Persistence of taste buds in denervated fungiform papillae

Taste buds in hamster fungiform papillae persist in an atrophic state for as long as 330 days after chorda tympani denervation or 50 days after combined chorda tympani-lingual nerve resection. Although taste bud structure depends on innervation, there is no absolute neural requirement for taste bud survival.     

Shrinkage of ipsilateral taste buds and hyperplasia of contralateral taste buds following chorda tympani nerve transection

The morphological changes that occur in the taste buds after denervation are not well understood in rats, especially in the contralateral tongue epithelium. In this study, we investigated the time course of morphological changes in the taste buds following unilateral nerve transection. The role of the trigeminal component of the lingual nerve in maintaining the structural integrity of the taste buds was also examined. Twenty-four Sprague-Dawley rats were randomly divided into three groups: control, unilateral chorda tympani nerve transection and unilateral chorda tympani nerve transection + lingual nerve transection. Rats were allowed up to 42 days of recovery before being euthanized. The taste buds were visualized using a cytokeratin 8 antibody. Taste bud counts, volumes and taste receptor cell numbers were quantified and compared among groups. No significant difference was detected between the chorda tympani nerve transection and chorda tympani nerve transection + lingual nerve transection groups. Taste bud counts, volumes and taste receptor cell numbers on the ipsilateral side all decreased significantly compared with control. On the contralateral side, the number of taste buds remained unchanged over time, but they were larger, and taste receptor cells were more numerous postoperatively. There was no evidence for a role of the trigeminal branch of the lingual nerve in maintaining the structural integrity of the anterior taste buds.     

The reinnervation of the tongue and salivary glands after lingual nerve injuries in cats

The recovery of fibres in the chorda tympani and lingual nerves has been investigated in cats following nerve injury by recording the receptor properties of gustatory, thermosensitive and mechanosensitive units and the return of vasomotor and secretomotor responses. The combined trunk of the chorda tympani and lingual nerves was either crushed (4 animals) or sectioned (3 animals) unilaterally and recovery allowed for 12 weeks. After nerve crush, integrated whole nerve activity recorded from the chorda tympani during stimulation of the tongue with gustatory or thermal stimuli revealed a response profile which was similar to controls. After nerve section little or no activity could be recorded. Recordings made from 52 single units in the chorda tympani after nerve crush revealed that the proportions of gustatory, thermosensitive and mechanosensitive units were similar to those of controls. The units had slower conduction velocities, responded less vigorously and to a narrower range of stimuli. Recordings made from 46 units in the chorda tympani after nerve section revealed very few gustatory or thermosensitive units, the majority were purely mechanosensitive and the decrease in conduction velocity was greater than after nerve crush. Electrical stimulation of efferent vasodilator fibres in both the chorda tympani and lingual nerves, evoked a temperature rise on the dorsal surface of the tongue. This effect was completely restored after nerve crush but was significantly smaller after nerve section. The flow rate of saliva from the submandibular salivary gland was not significantly changed by nerve crush but was significantly smaller after nerve section. There was no evidence for functional reinnervation of gustatory or secretomotor terminals by inappropriate fibre types.     

Restoration of denervated skeletal muscle transplants after reinnervation in rats

The present study describes reinnervation and restoration of rat skeletal muscle denervated for the duration of 3, 6 or 12 months. Denervation of extensor digitorum longus (EDL) muscle was achieved by cutting and ligating the donor rat sciatic nerve in situ. At 3, 6 and 12 months, the denervated EDL muscles were removed and transplanted into an innervated normal leg of another rat. In addition, normal (i.e., no prior denervation) muscles were transplanted as controls for comparison. The muscles were analyzed at 4 and 12 weeks after transplantation. The EDL muscle weight and myofiber size decreased with extended denervation times. After transplantation, the muscles underwent regeneration and reinnervation, and recovered as determined by an increase in muscle mass and myofiber size. The 3-month denervated muscle regenerates recovered completely, and were similar to the non-denervated normal muscle regenerates. Reinnervation, and partial recovery of muscle weight and myofiber size was observed in 6- and 12-month denervated muscle transplants. These results document that while regeneration and reinnervation does occur in denervated muscles after transplantation, the extent of recovery is related to the duration of denervation.     

Collateral reinnervation by the superior laryngeal nerve after recurrent laryngeal nerve injury

This study investigates the role of the intact superior laryngeal nerve (SLN) in the reinnervation process of one of the laryngeal muscles, the posterior cricoarytenoid muscle (PCA), following recurrent laryngeal nerve (RLN) injury. Using a chronic RLN injury model in the adult rat, PCA reinnervation was assessed by retrograde double-tracing techniques in combination with electrophysiology and immunohistochemistry of muscle sections. The results demonstrate that the PCA receives dual innervation from both laryngeal nerves even in the uninjured system. Functionally significant collateral reinnervation originates from intact SLN fibers following RLN injury, mainly due to intramuscular sprouting rather than by recruitment of more motor neurons. This may be important when choosing surgical and/or medical treatment for patients with RLN injury.     

Ultrastructure of mouse foliate taste buds: synaptic and nonsynaptic interactions between taste cells and nerve fibers

High voltage electron microscopy and conventional transmission electron microscopy were used to examine the ultrastructure of foliate taste buds of mice. Computer-assisted, three-dimensional reconstructions from serial sections were used to visualize regions of interaction between taste cells and nerve fibers. Based on criteria previously established for murine vallate taste buds (Kinnamon et al., '85), foliate taste cells were classified as dark, light, or intermediate depending on their cytoplasmic content and the characteristics of their nuclei. Cells of foliate taste buds display a continuous range of morphologies, from "typical" dark cells to "typical" light cells. Cells of dark, intermediate, and light morphologies all make afferent synapses onto nerve processes, suggesting that cells of all 3 types are sensory in function. Synapses between taste cells and nerve processes may be either macular or fingerlike in shape. No efferent synapses were found. In addition to conventional synapses, taste cells exhibit 2 other types of specializations at sites of apposition with nerve fibers: subsurface cisternae and atypical mitochondria. Subsurface cisternae are narrow sacs of endoplasmic reticulum that are closely apposed to the inner leaflet of the taste cell membrane. Possible functions of subsurface cisternae include synthesis of synaptic membrane components, modification of the electrical or adhesive properties of the taste cell membrane, and exchange of trophic factors with nerve processes. Atypical mitochondria are usually much larger than typical taste cell mitochondria, and their cristae often display a swollen, twisted configuration. These mitochondria are closely apposed to the inside of the taste cell membrane adjacent to nerve fibers. Atypical mitochondria may be providing unusual amounts of energy for metabolic reactions in their vicinities or participating in calcium buffering in the taste cell. Within taste cells, presynaptic specializations, subsurface cisternae, and mitochondria are often clustered together to form "synaptic ensembles." We hypothesize that the functions served by the subsurface cisternae and mitochondria, as well as synaptic transmission, may be important in interactions between taste cells and nerve fibers.     

Dystonin deficiency reduces taste buds and fungiform papillae in the anterior part of the tongue

The anterior part of the tongue was examined in wild type and dystonia musculorum mice to assess the effect of dystonin loss on fungiform papillae. In the mutant mouse, the density of fungiform papillae and their taste buds was severely decreased when compared to wild type littermates (papilla, 67% reduction; taste bud, 77% reduction). The mutation also reduced the size of these papillae (17% reduction) and taste buds (29% reduction). In addition, immunohistochemical analysis demonstrated that the dystonin mutation reduced the number of PGP 9.5 and calbindin D28k-containing nerve fibers in fungiform papillae. These data together suggest that dystonin is required for the innervation and development of fungiform papillae and taste buds.     

Neural induction of taste buds

Bilateral innervation allows more than 80% of the 610 vallate taste buds to survive removal of one IXth nerve in adult rats. Removal of both IXth nerves in neonatal or adult rats results in the absence of taste buds. In studying development, we found that removing or crushing one IXth nerve in three-day-old neonates profoundly decreased the number of vallate taste buds that subsequently developed. Specifically, after removal of one IXth nerve at 3 days, only 228 taste buds formed, compared with 496 taste buds that one nerve would maintain in adults. Thus, during normal development, the right and left IXth nerves interact synergistically, as at least 150 more taste buds develop than predicted by the sum of the independent action of each IXth nerve. This suggests that vallate taste buds are induced by the IXth nerve. A second example of synergism, representing evidence for the neural induction of taste buds, came from experiments in which we crushed the left IXth nerve 3 days after birth and found that these regenerated IXth nerve axons induced 4 times as many taste buds in the presence of the normal right IXth nerve (118 taste buds) as in its early absence (30 taste buds). We conclude that taste buds are neurally induced and that axons of the IXth nerve interact synergistically in inducing them, rather than competing for targets. We propose that in development innervated progenitor cells form stem cells which lead to taste bud cells.     

Failure of reinnervation of Pacinian corpuscle after nerve crush

Summary Denervated Pacinian corpuscles from the slow loris (Nycticebus coucang) obtained on the 49th and 75th post-operative days were subjected to electron microscopic investigation. Unremoved myelin debris occurred within the endoneurial tube during the early stages of regeneration and there was extensive fibrosis in the later stages. These factors may prevent the reinnervating nerve fibre from entering the corpuscle. The changes in the blood vessels may be due to disuse atrophy.     

Synapsin I-like immunoreactivity in nerve fibers associated with lingual taste buds of the rat

Immunoreactivity to synapsin I, a neuronal phosphoprotein, was localized in free-floating tissue sections prepared from lingual tissue of rats. Many nerve fibers within the tissue exhibited clear immunoreactivity including motor endplates on striated muscle, autonomic fibers innervating blood vessels or glands, and sensory fibers innervating muscles or the lingual epithelium including taste buds. Numerous immunoreactive fibers occurred within each taste bud, with fewer, fine fibers being dispersed in the epithelium between taste buds. The majority of the intragemmal immunoreactive fibers extended throughout the taste buds most of the distance outward from the basal lamina toward the surface of the epithelium. Fine, perigemmal fibers reached nearly to the epithelial surface. Ultrastructural analysis of the immunoreactive sensory fibers revealed that synapsin I-immunoreactivity occurred diffusely throughout the cytoplasm, and heavily in association with microvesicles. The synaptic vesicles at the taste receptor cell-to-afferent fiber synapse were, however, not immunoreactive for synapsin I, although these vesicles fall into the size class shown to be immunoreactive in other systems. This absence of synapsin I may be a common property of vesicles in axonless short receptor cells.