Distribution and role in regeneration of N-CAM in the basal laminae of muscle and Schwann cells

The neural cell adhesion molecule (N-CAM) is a membrane glycoprotein involved in neuron-neuron and neuron-muscle adhesion. It can be synthesized in various forms by both nerve and muscle and it becomes concentrated at the motor endplate. Biochemical analysis of a frog muscle extract enriched in basal lamina revealed the presence of a polydisperse, polysialylated form of N-CAM with an average Mr of approximately 160,000 as determined by SDS-PAGE, which was converted to a form of 125,000 Mr by treatment with neuraminidase. To define further the role of N-CAM in neuromuscular junction organization, we studied the distribution of N-CAM in an in vivo preparation of frog basal lamina sheaths obtained by inducing the degeneration of both nerve and muscle fibers. Immunoreactive material could be readily detected by anti-N-CAM antibodies in such basal lamina sheaths. Ultrastructural analysis using immunogold techniques revealed N-CAM in close association with the basal lamina sheaths, present in dense accumulation at places that presumably correspond to synaptic regions. N-CAM epitopes were also associated with collagen fibrils in the extracellular matrix. The ability of anti-N-CAM antibodies to perturb nerve regeneration and reinnervation of the remaining basal lamina sheaths was then examined. In control animals, myelinating Schwann cells wrapped around the regenerated axon and reinnervation occurred only at the old synaptic areas; new contacts between nerve and basal lamina had a terminal Schwann cell capping the nerve terminal. In the presence of anti-N-CAM antibodies, three major abnormalities were observed in the regeneration and reinnervation processes: (a) regenerated axons in nerve trunks that had grown back into the old Schwann cell basal lamina were rarely associated with myelinating Schwann cell processes, (b) ectopic synapses were often present, and (c) many of the axon terminals lacked a terminal Schwann cell capping the nerve-basal lamina contact area. These results suggest that N-CAM may play an important role not only in the determination of synaptic areas but also in Schwann cell-axon interactions during nerve regeneration.     

Substance P-like immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies

Substance P-immunoreactive (SP-1) structures in the carotid bodies of rats and cats were examined with the light and electron microscopes. In both species SP-I varicose nerve fibers were located singly in the interstitial connective tissue in close association with blood vessels. They were small unmyelinated fibers enveloped in a common Schwann cell sheath with other SP-negative fibers. Some of SP-I fibers contained large dense-cored granules and small clear vesicles in addition to microtubules and mitochondria and probably represented nerve fiber varicosities. The latter often were found incompletely invested by Schwann cell sheaths. SP-fibers were found occasionally in the envelopes of supporting cells at the periphery of parenchymal cell groups. However, none of the nerve terminals making synaptic contacts with glomus cells exhibited SP-like immunoreactivity. In cat carotid bodies some glomus cells showed moderate to intense SP-like immunoreactivity. The intense SP-I glomus cells displayed numerous dense-cored vesicles of 85 to 140 nm in diameter and frequently showed synaptic contacts with SP-negative nerve terminals. In rat carotid bodies we were unable to detect consistent SP-immunoreactivity in glomus cells. Our results do not favor the hypothesis that SP is a neurotransmitter/modulator in the chemoreceptor afferents synapsing on glomus cells in either the cat or rat carotid body. However our results support the hypothesis that SP in cat glomus cells may play a role in the modulation of chemoreceptor activity.     

Substance-P-containing fibers in the incisive papillae of the rat hard palate. Light- and electron-microscopic immunohistochemical study

Substance P (SP)-containing fibers in the incisive papillae of rat hard palates, which include various components of sensory receptors, i.e. mechanoreceptors, free nerve endings and chemosensory corpuscles (taste buds), were examined using immunoperoxidase techniques and light and electron microscopes. Immunolabeled fibers were consistently distributed in the medial part of the orifice of the incisive canals, i.e. in the taste-bud-enriched region. Dense immunolabeled fibers were found in subgemmal regions and in the lamina propria papillae. Some fine fibers entered and ascended the taste buds or occasionally the epithelium outside the taste buds. In addition, a rich innervation by SP-containing fibers close to blood capillaries was clearly identified. Electron microscopy revealed no specialized synaptic contact between the immunolabeled fibers and taste bud cells. Synaptic-like images could be found only between nonimmunolabeled nerve endings and the underlying taste bud cells. In the lamina propria papillae, mechanoreceptors observed in the present study contained no immunoperoxidase end products, whereas free nerve endings with an immunolabeled small-diameter axon (630-730 nm in diameter) were frequent. Similar axons were located at the adventitia of the blood capillaries. The possible functional role of SP-containing fibers in the incisive papillae was given attention.     

Neural Organization of the Median Ocellus of the Dragonfly : II. Synaptic structure

Two types of presumed synaptic contacts have been recognized by electron microscopy in the synaptic plexus of the median ocellus of the dragonfly. The first type is characterized by an electron-opaque, button-like organelle in the presynaptic cytoplasm, surrounded by a cluster of synaptic vesicles. Two postsynaptic elements are associated with these junctions, which we have termed button synapses. The second synaptic type is characterized by a dense cluster of synaptic vesicles adjacent to the presumed presynaptic membrane. One postsynaptic element is observed at these junctions. The overwhelming majority of synapses seen in the plexus are button synapses. They are found most commonly in the receptor cell axons where they synaptically contact ocellar nerve dendrites and adjacent receptor cell axons. Button synapses are also seen in the ocellar nerve dendrites where they appear to make synapses back onto receptor axon terminals as well as onto adjacent ocellar nerve dendrites. Reciprocal and serial synaptic arrangements between receptor cell axon terminals, and between receptor cell axon terminals and ocellar nerve dendrites are occasionally seen. It is suggested that the lateral and feedback synapses in the median ocellus of the dragonfly play a role in enhancing transients in the postsynaptic responses.     

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.     

Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites

Axons regenerate to reinnervate denervated skeletal muscle fibers precisely at original synaptic sites, and they differentiate into nerve terminals where they contact muscle fibers. The aim of this study was to determine the location of factors that influence the growth and differentiation of the regenerating axons. We damaged and denervated frog muscles, causing myofibers and nerve terminals to degenerate, and then irradiated the animals to prevent regeneration of myofibers. The sheath of basal lamina (BL) that surrounds each myofiber survives these treatments, and original synaptic sites on BL can be recognized by several histological criteria after nerve terminals and muscle cells have been completely removed. Axons regenerate into the region of damage within 2 wk. They contact surviving BL almost exclusively at original synaptic sites; thus, factors that guide the axon's growth are present at synaptic sites and stably maintained outside of the myofiber. Portions of axons that contact the BL acquire active zones and accumulations of synaptic vesicles; thus by morphological criteria they differentiate into nerve terminals even though their postsynaptic targets, the myofibers, are absent. Within the terminals, the synaptic organelles line up opposite periodic specializations in the myofiber's BL, demonstrating that components associated with the BL play a role in organizing the differentiation of the nerve terminal.     

Characterization of orexin A immunoreactivity in the rat area postrema

The distribution of orexin A immunoreactivity and the synaptic relationships of orexin A-positive neurons in the rat area postrema were studied using both light and electron microscopy techniques. At the light microscope level, numerous orexin A-like immunoreactive fibers were found within the area postrema. Using electron microscopy, immunoreactivity within fibers was confined primarily to the axon terminals, most of which contained dense-cored vesicles. Both axo-somatic and axo-dendritic synapses made by orexin A-like immunoreactive axon terminals were found, with these synapses being both symmetric and asymmetric in form. Orexin A-like immunoreactive axon terminals could be found presynaptic to two different immunonegative profiles including the perikarya and dendrites. Occasionally, some orexin A-like immunoreactive profiles, most likely to be dendrites, could be seen receiving synaptic inputs from immunonegative or immunopositive axon terminals. The present results suggest that the physiological function of orexin A in the area postrema depends on synaptic relationships with other immunopositive and immunonegative neurons, with the action of orexin A mediated via a self-modulation feedback mechanism.     

Fine Structure of Subepithelial “Free” and Corpuscular Trigeminal Nerve Endings in Anterior Hard Palate of the Rat

Axonally transported protein labeled many trigeminal nerve endings in subepithelial regions of the anterior hard palate of the rat. Sensory endings were most numerous in the lamina propria near the tips of the palatal rugae where large connective tissue and epithelial papillae interdigitated. Two kinds of sensory ending were found there: “free” endings, and a variety of corpuscular endings. The “free” sensory endings consisted of bundles of unmyelinated axons separated from the connective tissue by relatively unspecialized Schwann cells covering part or all of their surface and a completely continuous basal lamina; they were commonly found running parallel to the epithelium or near corpuscular endings. The corpuscular sensory endings all had a specialized nerve form, specialized Schwann cells, and axonal fingers projecting into the corpuscular basal lamina or connective tissue. There were at least four distinct types of corpuscular ending: Ruffini-like endings were found among dense collagen bundles, and they had a flattened nerve ending with a flattened Schwann lamella on either side. Meissner endings had an ordered stack of flattened nerve terminals with flattened Schwann cells and much basal lamina within and around the corpuscle. Simple corpuscles were single nerve endings surrounded by several layers of concentric lamellar Schwann processes. Glomerular endings were found in lamina propria papillae or encircling epithelial papillae; they were a tangle of varied neural forms each of which had apposed flattened Schwann cells, and a layer of basal lamina of varied thickness. Fibroblasts often formed incomplete partitions around Meissner and simple corpuscles.

The axoplasm of all kinds of subepithelial sensory endings contained numerous mitochondria and vesicles, as well as occasional multivesicular bodies and lysosomes; the axoplasm of all endings was pale with few microtubules and neurofilaments. The specialized lamellar Schwann cells had much pinocytotic activity. Four kinds of junctions were found between the corpuscular sensory endings and the lamellar Schwann cells: (1) symmetric densities that resemble desmosomes; (2) asymmetric densities with either the neuronal or glial membrane more dense; (3) neural membrane densities adjacent to Schwann parallel inner and outer membrane densities; and (4) sites of apparent Schwann endocytosis associated with neural blebs. The “free” sensory endings only made occasional desmosome-like junctions with their Schwann cells.

These observations are discussed in relation to possible mechanosensory transduction mechanisms, with particular attention to axoplasmic structure, axonal fingers, and neural and nonneural cell associations.     

Substance P-like immunoreactivity in adrenal chromaffin cells and intra-adrenal nerve fibers of rats

Summary The present peroxidase-antiperoxidase immunohistochemical study demonstrated a relatively small number of cells with substance P(SP)-like immunoreactivity in the adrenal medulla of rats. These cells were found alone or in small groups, were polygonal in shape and lacked long cytoplasmic processes. At immunoelectron microscopy, the immunoreactive cells were characterized by abundant granular vesicles, and the immunoreactive material was confined to the round core of the vesicles. Thus, it is suggested that SP co-exists with catecholamines in a population of chromaffin cells of the rat adrenal medulla. In addition a few SP-immunoreactive nerve fibers with varicosities were found in the adrenal medulla of rats. They extended between small clusters of chromaffin cells and had their dotlike terminals around and within the cell clusters. The SP-immunoreactive nerve fibers were characterized by the presence of abundant small clear vesicles mixed with a few large granular vesicles; the immunoreactivity appeared in the latter, but was also perfused throughout the entire axoplasm. The nerve fibers formed synapses on nonimmunoreactive chromaffin cells. Judging from the presence of bundles of SP-immunoreactive nerve fibers penetrating the adrenal capsule and cortex as well as the absence of SP-immunoreactive ganglion cells in the medulla, the intramedullary SP-immunoreactive nerve fibers seem to be extrinsic in origin.     

Immunocytochemical observations of corticotropin-releasing-factor-containing neurons in the rat hypothalamus with special reference to neuronal communication

Synapses between neurons with corticotropin-releasing-factor-(CRF)-like immunoreactivities and other immunonegative neurons in the hypothalamus of colchicine-treated rats, especially in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) were observed by immunocytochemistry using CRF antiserum. The immunoreactive nerve cell bodies and fibers were numerous in both the PVN and the SON. The CRF-containing neurons had synaptic contacts with immunonegative axon terminals containing a large number of clear synaptic vesicles alone or combined with a few dense-cored vesicles. We also found CRF-like immunoreactive axon terminals making synaptic contacts with other immunonegative neuronal cell bodies and fibers. And since some postsynaptic immunonegative neurons contained many large neurosecretory granules, they are considered to be magnocellular neurosecretory cells. These findings suggest that CRF functions as a neurotransmitter and/or modulator in addition to its function as a hormone.     

Ultrastructure of orexin-1 receptor immunoreactivities in the spinal cord dorsal horn

The ultrastructural properties of orexin 1-receptor-like immunoreactive (OX1R-LI) neurons in the dorsal horn of the rat spinal cord were examined using light and electron microscopy techniques. At the light microscopy level, the most heavily immunostained OX1R-LI neurons were found in the ventral horn of the spinal cord, while some immunostained profiles, including nerve fibers and small neurons, were also found in the dorsal horn. At the electron microscopy level, OX1R-LI perikarya were identified containing numerous dense-cored vesicles which were more heavily immunostained than any other organelles. Similar vesicles were also found within the axon terminals of the OX1R-LI neurons. The perikarya and dendrites of some of the OX1R-LI neurons could be seen receiving synapses from immunonegative axon terminals. These synapses were found mostly asymmetric in shape. Occasionally, some OX1R-LI axon terminals were found making synapses on dendrites that were OX1R-LI in some cases and immunonegative in others. The synapses made by OX1R-LI axon terminals were found both asymmetric and symmetric in appearance. The results provide solid morphological evidence that OX1R is transported in the dense-cored vesicles from the perikarya to axon terminals and that OX1R-LI neurons in the dorsal horn of the spinal cord have complex synaptic relationships both with other OX1R-LI neurons as well as other neuron types.     

Substance P-like immunoreactivity in adrenal chromaffin cells and intra-adrenal nerve fibers of rats

The present peroxidase-antiperoxidase immunohistochemical study demonstrated a relatively small number of cells with substance P(SP)-like immunoreactivity in the adrenal medulla of rats. These cells were found alone or in small groups, were polygonal in shape and lacked long cytoplasmic processes. At immunoelectron microscopy, the immunoreactive cells were characterized by abundant granular vesicles, and the immunoreactive material was confined to the round core of the vesicles. Thus, it is suggested that SP co-exists with catecholamines in a population of chromaffin cells of the rat adrenal medulla. In addition a few SP-immunoreactive nerve fibers with varicosities were found in the adrenal medulla of rats. They extended between small clusters of chromaffin cells and had their dot-like terminals around and within the cell clusters. The SP-immunoreactive nerve fibers were characterized by the presence of abundant small clear vesicles mixed with a few large granular vesicles; the immunoreactivity appeared in the latter, but was also perfused throughout the entire axoplasm. The nerve fibers formed synapses on nonimmunoreactive chromaffin cells. Judging from the presence of bundles of SP-immunoreactive nerve fibers penetrating the adrenal capsule and cortex as well as the absence of SP-immunoreactive ganglion cells in the medulla, the intramedullary SP-immunoreactive nerve fibers seem to be extrinsic in origin.     

Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.     

Evidence for receptor nerve endings in tendons and related tissues of an electric teleost, Gnathonemus petersii

Tendons and muscle aponeurosis associated with muscle lodged in the mental appendage of a weakly electric mormyrid fish, Gnathonemus petersii, have been examined for nerve terminations in them, using methods of silver impregnation, histology and electron microscopy. Large myelinated afferent nerve fibres innervate the tendinous tissues and form nerve terminals in the perimysium, the muscle-tendinous junctions and the tendons, the last recalling the tendon organs of higher vertebrates. Nerve terminals are characterized by loss of myelin, Schwann sheath and basal lamina, the neurolemma being freely exposed to either fibroblasts and their laminar processes or the collagen fibres and the tendon intercellular matrix. Nerve terminals lack synaptic specialisation of their membrane. This study is the first demonstration of sensory nerve terminals (tension receptors) associated with tendons and related tissue in a teleost fish.     

Neuron-specific enolase,neurofilament protein and S-100 protein in the olfactory mucosa of human fetuses

Summary Immunohistochemical examination for neuronspecific enolase (NSE), neurofilament protein (NFP), and S-100 protein was performed in the olfactory mucosa of human fetuses. NSE and NFP immunoreactivities were found in the olfactory receptor cells, while no S-100 immunoreactive cells were recognized within the olfactory epithelium. The anti-NSE serum stained various types of nerve bundles in the lamina propria mucosae; a population of the NSE-positive nerve bundles was also immunoreactive for NFP. The anti-S-100 serum clearly demonstrated Schwann cells associated with the nerve fibers in the lamina propria mucosae. These findings 1) suggest a possibility of NSE and NFP as new marker substances for olfactory cells and 2) indicate that immunohistochemistry is a useful tool to analyse the cellular components of the olfactory organs in normal and pathological conditions.     

Innervation of the guinea pig spleen studied by electron microscopy

The innervation of the guinea pig spleen was investigated by electron microscopy. Unmyelinated nerve fibers in the capsulotrabecular and arterial systems were found to contain large and small granular and small agranular synaptic vesicles in their terminals and are thought to be sympathetic adrenergic in nature. They influence the contraction of the smooth muscle cells by diffusion innervation in these systems. These nerve terminals were also scattered in both the red and the white pulp. Pulp nerves wrapped by Schwann cells were further enclosed by myofibroblastic reticular cells. This condition revealed that the pulp nerves pass through the connective-tissue spaces of the reticular fibers, which contain elastic fibers, collagenous fibrils, and lamina densa-like materials of the usual basement laminae. One of the target cells for the pulp nerves is considered to be the myofibroblastic reticular cell in the reticular meshwork. Neurotransmitter substances released from the naked adrenergic nerve terminals travel through the reticular fibers and may play a role, by both close association innervation and diffusion innervation, in the contraction of reticular cells to expose the reticular fibers. At the exposed sides, connective-tissue elements of the reticular fibers are bathed with blood plasma, and the included naked nerve terminals, devoid of Schwann cells but with basement laminae of these cells, face free cells at some distance or are in close association with free cells, especially lymphocytes, macrophages, and plasma cells. The close ultrastructural relationship between the naked adrenergic nerve terminals and immunocytes strongly suggests that there is an intimate relationship between the immune system and the sympathetic nervous system through both close association innervation and diffusion innervation. Thus splenic adrenergic nerves of the guinea pig may play a triple role in 1) contraction of smooth muscle cells to regulate blood flow in the organ, 2) induction of the exposure of reticular fibers by contraction of the reticular cells in order to form a close relationship of the nerve terminals with the immunocytes, and 3) subsequent neuroimmunomodulation of the immunocytes.     

Light- and electron-microscopic study of substance P-immunoreactive neurons in the guinea pig retina

Substance P (SP) immunoreactivity in the guinea pig retina was studied by light and electron microscopy. The morphology and distribution of SP-immunoreactive neurons was defined by light microscopy. The SP-immunoreactive neurons formed one population of amacrine cells whose cell bodies were located in the proximal row of the inner nuclear layer. A single dendrite emerged from each soma and descended through the inner plexiform layer toward the ganglion cell layer. SP-immunoreactive processes ramified mainly in strata 4 and 5 of the inner plexiform layer. SP-immunoreactive amacrine cells were present at a higher density in the central region around the optic nerve head and at a lower density in the peripheral region of the retina. The synaptic connectivity of SP-immunoreactive amacrine cells was identified by electron microscopy. SP-labeled amacrine cell processes received synaptic inputs from other amacrine cell processes in all strata of the inner plexiform layer and from bipolar cell axon terminals in sublamina b of the same layer. The most frequent postsynaptic targets of SP-immunoreactive amacrine cells were the somata of ganglion cells and their dendrites in sublamina b of the inner plexiform layer. Amacrine cell processes were also postsynaptic to SP-immunoreactive neurons in this sublamina. No synaptic outputs onto the bipolar cells were observed.     

Ultrastructural changes in the nerves innervating the cerebral artery after sympathectomy

Summary The ultrastructure of the innervation of the anterior cerebral artery of the rat was studied in control animals and in animals after superior cervical ganglionectomy.Fluorescence histochemistry shows a periarterial network of intensely fluorescent fibers which are divided into two groups, adventitial and periadventitial. The fluorescence begins to decrease 26 hours after, and completely disappears about 32 hours after, ganglionectomy.Fine structural changes are first observed 18 hours after ganglionectomy, when the axoplasm of degenerating axons becomes electron dense. This density gradually increases up to about 32 hours. By 32 hours most axons with disintegrating axolemmas become inclusion bodies of the Schwann cells. At this stage, synaptic vesicles can still be distinguished as less dense areas, but the membrane structures of synaptic vesicles and mitochondria are difficult to recognize. The degenerating axons are gradually absorbed and by 38 hours dense, residual bodies are observed in the Schwann cells. Generally speaking, the degeneration occurs first in the adventitial fibers and then in the periadventitial fibers. The transient appearance of small, granular vesicles is noticed in axon terminals about 18 hours after denervation, although very few small, granular vesicles are seen in control tissue or at later stages of degeneration.     

Heterogeneous distribution of neurocalcin-immunoreactive nerve terminals in the mouse adrenal medulla

Neurocalcin is a novel calcium-binding protein found in bovine brain tissue. We investigated immunoreactivity for neurocalcin in the mouse adrenal medulla using light and electron microscopy. The immunoreactivity was present in nerve fibers, nerve terminals, and ganglion cells in the adrenal medulla, but chromaffin cells, sustentacular cells, and Schwann cells were negative in reaction. Nerve bundles containing neurocalcin-immunoreactive fibers passed through the adrenal cortex and extended into the medulla. Immunopositive nerve fibers branched off and projected varicose terminals around the chromaffin cells. These varicose terminals contained small and large-cored vesicles and made synapses with the chromaffin cells. We performed paraformaldehyde-induced fluorescence-histochemical studies for catecholamine combined with immunohistochemical studies for neurocalcin. Neurocalcin-immunoreactive nerve terminals were more abundant at noradrenaline (fluorescent) cell-rich regions than at adrenaline (non-fluorescent) cell-rich regions. These results show that neurocalcin-immunoreactive nerves mainly innervate noradrenaline-containing chromaffin cells in the mouse adrenal medulla and that neurocalcin may regulate synaptic function in the nerve terminals. Received: 21 October 1996 / Accepted: 12 February 1997     

Calbindin D28k-like immunoreactivity in the developing and regenerating circumvallate papilla of the rat

The distribution of calbindin D28k (CB)-like immunoreactivity (-LI) in the circumvallate papilla (CVP) was examined during development and regeneration following bilateral crush injury to the glossopharyngeal nerve in the rat. In the adult CVP, CB-like immunoreactive (-IR) nerve fibers were observed in the subgemmal region and some penetrated into the taste buds. CB-LI was also detected in the cytoplasm of the spindle-shaped gustatory cells in the lower half of the trench epithelium, which contained numerous synaptic vesicles and bundles of intermediate filaments. These CB-IR gustatory cells made synapse-like contacts with CB-IR nerve terminals. Some CB-IR nerve terminals made contacts with the gustatory cells negative for CB-LI. At least three developmental stages were defined with regard to the developmental changes in the distribution of CB-LI: (1) Stage I (embryonic day (E) 18–postnatal day (P)5): CB-IR nerve fibers appeared in the lamina propria just beneath the newly-formed CVP at E18, but the gustatory epithelium of the CVP contained no CB-IR structures. Taste buds with taste pores appeared at P1. (2) Stage II (P5–10): thin CB-IR nerve fibers began entering the trench epithelium, but no CB-IR cells were observed. (3) Stage III (P10–adult): in addition to the intragemmal and perigemmal CB-IR nerve fibers, very few CB-IR cells appeared in the taste buds around P10, and their numbers increased progressively. The changes in the distribution of taste buds and CB-LI following glossopharyngeal nerve injury were similar to those observed during development. On post-operative day (PO) 4, the taste buds and CB-IR cells decreased markedly in number. These CB-IR cells became round in shape, and the number of CB-IR nerve fibers decreased markedly. On PO8, both taste buds and CB-IR cells disappeared completely. The regenerated taste buds were first observed on PO12, increased rapidly in number by PO20, and increased slowly thereafter. CB-IR nerve fibers accumulated at the subgemmal region and began penetrating into the trench wall epithelium around PO16. CB-IR cells appeared between PO20 and PO24, and their numbers increased progressively and reached the normal level on PO40. The topographical localizations of the taste buds and CB-IR cells during development and regeneration were comparable to those of normal animals. The delay of the time courses for appearance of CB-IR nerve fibers and CB-IR cells compared to the appearance of taste buds during development and regeneration suggests that CB in the gustatory epithelium may participate in the survival of the taste bud cells rather than in the induction of the taste buds.