"Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin.

Taste buds contain a variety of morphological and histochemical types of elongate cells. Serotonin, neuron-specific enolase (NSE), ubiquitin carboxyl terminal hydrolase (PGP 9.5), and neural cell adhesion molecule (N-CAM) all have been described as being present in the morphologically defined Type III taste cells in rats. In order to determine whether these substances coexist in a single cell, we undertook immunohistochemical and ultrastructural analysis of taste buds in rats. Double-label studies show that PGP 9.5 and NSE always colocalize. In contrast, PGP 9.5 and serotonin seldom colocalize. Further, whereas the serotonin-immunoreactive cells are always slender and elongate, the PGP 9.5/NSE population comprise two morphological types--one slender, the other broader and pyriform. Although gustducin-immunoreactive taste cells appear similar in overall shape to the pyriform PGP 9.5/NSE population, gustducin never colocalizes with PGP 9.5 or NSE. The serotonin-immunoreactive taste cells have an invaginated nucleus, synaptic contacts with nerve fibers, and taper apically to a single, large microvillus. These are all characteristics of Type III taste cells described previously in rabbits (Murray [1973] Ultrastructure of Sensory Organs I. Amsterdam: North Holland. p 1-81). PGP 9.5-immunoreactive taste cells exhibit two morphological varieties. One type is similar to the serotonin-immunoreactive population, containing an invaginated nucleus, synapses with nerve fibers, and a single large microvillus. The other type of PGP 9.5-immunoreactive taste cell has a large round nucleus and the apical end of the cell tapers to a tuft of short microvilli, which are characteristics of Type II taste cells. Thus, in rats, some Type III cells accumulate serotonin but do not express PGP 9.5, whereas others express PGP 9.5 but do not accumulate amines. Similarly, Type II taste cells come in at least two varieties: those immunoreactive for gustducin and those immunoreactive for PGP 9.5.

Ultrastructural localization of gustducin immunoreactivity in microvilli of type II taste cells in the rat.

Gustducin is a transducin-like G protein (guanine nucleotide-binding protein) that is expressed in taste bud cells. Gustducin is believed to be involved in bitter and possibly sweet taste transduction. In the present study, we demonstrate that a subset of type II cells displays immunoreactivity to antisera directed against gustducin in taste buds of rat circumvallate papilla. Immunogold particles are present both in the microvilli and cytoplasm of the immunoreactive cells. Quantitative analysis of the data suggests that the number of colloidal gold particles (P<0.001) and nanogold particles (P<0.01) in the immunoreactive type II cells are much greater than in type I cells. There are also approximately 2.5 times (P<0.05) as many colloidal gold particles associated with the microvilli versus the cytoplasm in the immunoreactive type II cells. The ultrastructural distribution of gustducin immunoreactivity is consistent with its proposed role in the initial events of sensory transduction by gustatory receptor cells.

Taste cells with synapses in rat circumvallate papillae display SNAP-25-like immunoreactivity.

SNAP-25 is a 25 kDa protein believed to be involved in the processes of membrane fusion and exocytosis associated with neurotransmitter release. In the present study we present evidence that SNAP-25-like immunoreactivity can be used as a marker for taste cells with synapses in rat circumvallate papillae. SNAP-25 immunoreactivity is present in most intragemmal nerve processes and a small subset of taste cells. Intense immunoreactivity is associated with the nerve plexus located below the base of the taste bud. Of a total of 87 taste cells with synapses onto nerve processes, 80 of the presynaptic taste cells had SNAP-25 immunoreactivity. The association of SNAP-25 immunoreactivity with taste cells possessing synapses suggests that these cells may be gustatory receptor cells. Because this SNAP-25 antibody can label taste cells with synapses, it may also serve as a useful tool for future studies correlating structure with function in the taste bud.

Identified taste bud cell proliferation in the perihatching chick.

Developing taste buds in the anterior mandibular floor of perihatching chicks were studied by high voltage electron microscopic autoradiography in order to identify proliferating gemmal cell types. Montaged profiles of 29 taste buds in five cases euthanized between embryonic day 21 and posthatching day 2 were analyzed after a single [3H]thymidine injection administered on embryonic day 16, 17 or 18. Results showed that dark cells comprised 55% of identified (n = 900 cells) and 62% of labeled (n = 568 cells) gemmal cells as compared with light, intermediate, basal or perigemmal bud cells. Dark cells had both a greater (P < 0.05) number of labeled cells and a greater amount of label (grains/nucleus) than the other four bud cell types, irrespective of injection day. The nuclear area (micron 2) of dark cells was not significantly larger (P > 0.05) than that of the other gemmal cell types and therefore cannot account for the greater amount for label in the dark cells. Interestingly, only dark cells showed a positive correlation (P < 0.003) between amount of label and nuclear area. Results suggest that, during the perihatching period of robust cell proliferation, dividing dark cells may give rise primarily, but not exclusively, to dark cell progeny.

High-voltage electron microscopy and 3-D reconstruction of solitary chemosensory cells in the anterior dorsal fin of the Gadid fish Ciliata mustela (Teleostei).

Solitary chemosensory cells (SCCs) are secondary sensory cells present in the epidermis of most primary aquatic vertebrates. In rocklings, the epidermis of the anterior dorsal fin (ADF) contains approximately 5 million SCCs. High-voltage electron microscopy and three-dimensional reconstructions from serial sections were used to examine the ultrastructure, arrangement, and synaptic contacts of the SCCs in the rockling ADF. Approximately 15% of all cells in the fin ray epidermis are SCCs, which occupy roughly 30% of the epidermal volume. These spindle-shaped cells are 25-30 microm long and up to 10 microm wide and terminate apically in a microvillus protruding 2-5 microm above the epidermal surface. SCCs contain abundant endoplasmic reticulum and a large Golgi apparatus in their proximal regions. The distal parts of SCCs contain characteristic vesicles, elongate mitochondria, and longitudinal strands of intermediate filaments. Synapses between SCCs and nerves resemble those found in teleost taste buds. One to four synaptic contacts per SCC were found. We hypothesize that the apparent secretory activity of the SCCs serves to replenish the apical membrane and mucus. Furthermore, parallel sampling of several hundred SCCs by single nerve fibers may serve low-threshold detection rather than stimulus localization.

Comparison of high-pressure freezing/freeze substitution and chemical fixation of catfish barbel taste buds.

The barbel taste buds of catfish are widely used as a model system for investigating the structure and function of vertebrate taste buds. We have examined the ultrastructure of the taste buds of the channel catfish, Ictalurus punctatus, as part of a comparative study of the morphology of taste buds in various mammalian and non-mammalian vertebrates. Since conventional chemical fixation methods have limited usefulness for certain kinds of ultrastructural studies (i.e., localization of diffusible substances or labeling techniques requiring retention of biological activity), we have developed methods for fixing catfish barbel taste buds by high-pressure freezing followed by freeze-substitution (HPF/FS) and have compared the ultrastructure of taste buds fixed by this technique and by chemical fixation procedures. The morphological details of the cells within taste buds are significantly affected by the method of fixation employed. In general, membrane contours are smoother and intracellular organelles more regular in shape in HPF/FS samples as compared with the chemically fixed specimens. Absolute and relative electron-densities of various tissue components are also affected by the fixation method employed. Certain ultrastructural features are more clearly visualized by one or the other of the fixation procedures. Fixation of barbel taste buds by HPF/FS not only provides an alternative view of the ultrastructure of taste bud cells but also offers a method of tissue preparation that may prove to be preferable to chemical methods for ultrastructural investigations involving procedures such as immunohistochemical labeling.

Taste bud cell generation in the perihatching chick.

Chick taste bud primordia initially appear in late gestation on embryonic day 17 (E17), 4 days before hatching. To track DNA synthesis and subsequent taste bud cell proliferation between E17 and the second day post-hatching (H2), single 25 muCi injections of tritiated thymidine (specific activity = 72.5 Ci/mmol) were administered in ovo during E15, E16, E17 or E18. Anterior mandibular oral epithelium was processed for light microscopic autoradiography. Sections through each taste bud's center were analysed for label (> or = 6 silver grains/gemmal cell nucleus), and bud diameter. Results indicated a major part of gemmal cell DNA synthesis does not occur until after E19 irrespective of the day of thymidine injection, suggesting postmitotic or quiescent (decycled) cells assemble to form the early bud primordium (E17-19) based on local tissue interactions. All buds examined from E20-H2 contained labelled cells. The day of injection was important since 5-day survival cases after E16 injection yielded about 25% the number of labelled cells/bud as compared with equivalent survival cases following E17-18 injections. These results are discussed with respect to parallel changes in bud shape and increasing bud diameter, and cell proliferation in possible extra- and intragemmal sources of bud cells.

Application of serial sectioning and three-dimensional reconstruction to the study of taste bud ultrastructure and organization.

The lingual taste buds of mammals are complex organs containing dozens of cells of varying morphology and numerous nerve fibers that are intermingled among the cellular processes. Some of the taste bud cells form synaptic contacts with these nerve fibers. Important questions remain to be answered regarding the structure and function of the cells of various types within taste buds and the means by which responses to gustatory stimuli are transmitted to the nerve fibers that communicate with the brain. Using both conventional and high voltage electron microscopy, we have examined serially sectioned taste buds from the tongues of mice and rabbits in order to address these issues and to obtain more complete information than that available from sampling of sections. The technique of computer-assisted 3-D reconstruction was used to generate models of whole taste buds and individual cellular and neural elements within taste buds from the serial sections. Analysis of serially sectioned taste buds from mice and rabbits has revealed that in both of these species relatively few (30% or less) of the cells within the taste buds form synaptic contacts with nerve fibers. In the foliate taste buds of rabbits, all of the cells that are presynaptic to nerve fibers are of a single morphological type (type III). The cells that are presynaptic to nerve fibers within the taste buds of mice are morphologically diverse. A pattern of synaptic connectivity exists within murine taste buds such that a given nerve fiber receives synaptic input only from taste cells that are ultrastructurally similar. In the taste buds of both mice and rabbits, we have observed both divergence and convergence of synaptic input from the putative taste receptor cells onto nerve fibers, suggesting that at the level of the taste bud there is some integration of the information generated by individual receptor cells. In addition to typical chemical synapses, other cytoplasmic specializations (such as subsurface cisternae and atypical mitochondria) may be involved in interactions between taste bud cells and nerve fibers.

Electrophysiological and morphological properties of light and dark cells isolated from mudpuppy taste buds.

Isolated Necturus taste receptor cells were studied by giga-seal whole-cell recording and electron microscopy to correlate electrophysiological properties with taste cell structural features. Dark (type I) cells were identified by the presence of dense granular packets in the supranuclear and apical regions of the cytoplasm. In response to a series of depolarizing voltage commands from a holding potential of -80 mV, these cells exhibited a transient, TTX-sensitive inward Na+ current, a sustained outward K+ current, and a slowly inactivating inward Ca++ current. Light (type II) cells were identified by a lack of granular packets and by an abundance of smooth endoplasmic reticulum distributed throughout the cell. In addition, isolated light cells had clear vesicular inclusions in the cytoplasm and blebs on the plasma membrane. Light cells were divided into two functional populations based upon electrophysiological criteria: cells with inward and outward currents, and cells with outward currents only. Light cells with inward and outward currents had voltage-activated Na+, K+, and Ca++ currents with properties similar to those of dark cells. In contrast, the second group of light cells had only voltage-activated outward K+ currents in response to depolarizing voltage commands. These data suggest that dark cells and light cells with inward and outward currents are capable of generating action potentials and releasing neurotransmitters onto gustatory afferent neurons in response to taste stimulation. In contrast, light cells with outward currents only likely serve a different function in the taste bud.

Ontogenesis and taste bud cell turnover in the chicken. I. Gemmal cell renewal in the hatchling.

Taste bud cell turnover rate was examined in oral epithelium of the precocial chick, which at hatching contains the adult complement of taste buds. Forty newly hatched chicks received single or double pulse injections of tritiated thymidine (specific activity was 6.7 Curies/millimole; dosage was 0.5 microCuries/g body weight, intraperitoneally). Anterior mandibular epithelium was processed for light microscopic autoradiography at 2 and 16 hours, as well as 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, and 20 days after the initial pulse. In a coded and randomized procedure, the section (7 microns) through the bud's center was selected for counting > or = 6 silver grains over round-clear and gracile-dense gemmal cell nuclei. The mean number of labelled cells/bud varied significantly (P < or = 0.01) during the first four posthatch days, yielding the fastest gemmal cell turnover rates (3.4-4.4 days) yet reported in vertebrates. Average bud diameter also significantly changed during the first four posthatch days, and was reflected in shifts of the distribution of 40-69 microns and > or = 70 microns diameter buds. Both an increase in labelled bud cells and bud diameter during the first two posthatch days may reflect high proliferation rates in initially maturing buds. Subsequent decrease in bud diameter between 2 and 3 days postinjection may indicate splitting of large-diameter (> or = 70 microns) buds and/or normal bud cell death due to failure of sensory afferentation. Bud-splitting alone, however, cannot account for significant decreases in bud cell label which did not occur before 4-6 days postinjection.

Aspects of vertebrate gustatory phylogeny: morphology and turnover of chick taste bud cells.

The taste bud is a receptor form observed across vertebrates. The present report compares chick taste buds to those of other vertebrates using light and electron microscopy. Unlike mammals, but common to many modern avians, the dorsal surface of chick anterior tongue lacks taste papillae and taste buds. Ultrastructurally, chick buds located in the anterior floor of the mouth (as in some reptiles and amphibians) and palate contain dark, intermediate, light, and basal cell types. Dark, intermediate, and light cells extend microvilli into intragemmal lumina and pores communicating with the oral cavity. As specialized features, dark cell apices lack dense granules and exhibit short microvilli relative to light and intermediate cells. Dark cell cytoplasmic fingers envelop intragemmal nerve fibers and cells as in other species, and sometimes contain abundant clear vesicles. Nerve profile expansions often are located adjacent to dark, intermediate, and light cell nuclei. Classical afferent synaptic contacts are rarely observed. Taste cell turnover is suggested by mitotic and degenerating figures in chick buds. In addition, tritiated thymidine injected into hatchlings, whose anterior mandibular oral taste bud population approximates that in adults, reveals a turnover rate of about 4.5 days. This is about half that observed in altricial mammals, reflecting a species difference or developmental factor in precocial avians. It is concluded that chick taste buds exhibit morphologic features common to other vertebrate buds with specializations reflecting the influences of niche, glandular relations, and/or age.

HVEM ultrastructural analysis of mouse fungiform taste buds, cell types, and associated synapses.

We have used high voltage electron microscopy and computer-generated three-dimensional reconstructions from serial sections to elucidate the structure of taste bud cells and their associated synapses in fungiform taste buds of the mouse. Five fungiform taste buds (two of which were serially sectioned) were examined with the high-voltage electron microscope (HVEM). We identified the synaptic connections from taste cells onto sensory nerve fibers and classified the presynaptic taste cells based on previously established ultrastructural criteria. From those data we have distinguished dark, intermediate, and light cells in murine fungiform taste buds. Synapses in murine fungiform taste buds are fewer in number, but contain many more vesicles than synapses in either foliate or circumvallate taste buds. Synapses in mouse circumvallate and foliate taste buds typically contain a few to several synaptic vesicles per section, whereas fungiform synapses may have in excess of 100 vesicles per profile. The significance of these differences in the numbers of synapses and synaptic structure between fungiform and circumvallate/foliate synapses is not known. Based on the small number of synapses observed in fungiform taste buds, we speculate that fungiform taste buds have only a few cells transducing sensory stimuli at any given time. Alternatively, communication of sensory information from the taste receptor cells to the afferent nerve fibers may be mediated by some other mechanism(s) in addition to classical chemical synapses.

Multifunctional features of a gastrodermal sensory cell in Hydra: three-dimensional study.

Computer-assisted, three-dimensional reconstructions of two gastrodermal sensory cells from transmission electron micrographs of serial sections of Hydra revealed a unipolar morphology with the nucleus near an apical cilium and a simple unbranched axon with a widened terminal. The sensory cells were similar in size and shape to a unipolar sensory cell isolated from macerated gastrodermis and examined with scanning electron microscopy. In thin sections, the cells were characterized by the presence of numerous dense-cored vesicles in the axon and its terminal. A few dense-cored vesicles were aligned at electron-dense synaptic foci in the axon terminal of the sensory cell, which formed an axo-axonal synapse with a nearby centrally located ganglion cell and a neuromuscular synapse with the basal myoneme of a digestive cell. The ganglion cell possessed a perikaryal cilium and a slender axon that extended adjacent to the sensory cell terminal, where it formed an en passant axo-axonal synapse in reciprocal arrangement with that of the sensory cell. In addition, the ganglion cell axon formed a neuromuscular synapse in sequence with the sensory cell axo-axonal synapse. The presence of a large number of neurosecretory-like granules, apical cilium and reciprocal interneuronal and neuromuscular synaptic loci suggests that this gastrodermal sensory cell, characterized ultrastructurally for the first time, represents a third type of multifunctional neuron in Hydra. Thus, Hydra may contain primitive stem-like neurons, which are sensory-motor and also function in both neurosecretion and neurotransmission.

HVEM serial-section analysis of rabbit foliate taste buds: I. Type III cells and their synapses.

Serially sectioned rabbit foliate taste buds were examined with high voltage electron microscopy (HVEM) and computer-assisted, three-dimensional reconstruction. This report focuses on the ultrastructure of the type III cells and their synapses with sensory nerve fibers. Type III cells have previously been proposed to be the primary gustatory receptor cells in taste buds of rabbits and other mammals. Within rabbit foliate taste buds, type III cells constitute a well-defined, easily recognizable class and are the only taste bud cells observed to form synapses with intragemmal nerve fibers. Among 18 type III cells reconstructed from serial sections, 11 formed from 1 to 6 synapses each with nerve fibers; 7 reconstructed type III cells formed no synapses. Examples of both convergence and divergence of synaptic input from type III cells onto nerve fibers were observed. The sizes of the active zones of the synapses and numbers of vesicles associated with the presynaptic membrane specializations were highly variable. Dense-cored vesicles 80-140 nm in diameter were often found among the 40-60 nm clear vesicles clustered at presynaptic sites. At some synapses, these large dense-cored vesicles appeared to be the predominant vesicle type. This observation suggests that there may be functionally different types of synapses in taste buds, distinguished by the prevalence of either clear or dense-cored vesicles. Previous investigations have indicated that the dense-cored vesicles in type III cells may be storage sites for biogenic amines.

Ultrastructure of substance P- and CGRP-immunoreactive nerve fibers in the nasal epithelium of rodents.

The respiratory and olfactory mucosae of rats and mice were examined at ultrastructural levels for the presence of intraepithelial nerve endings. Immunocytochemical studies utilizing antisera directed against substance P and calcitonin gene-related peptide (CGRP) revealed numerous intraepithelial peptide-immunoreactive fibers near the basal region of the epithelium. Occasional transepithelial fibers were observed to extend outward to nearly reach the epithelial surface. In no cases, however, did the transepithelial fibers reach the surface, but instead, stopped at the line of tight junctions approximately 1 micron from the surface. No specialized contacts between the nerve fibers and the epithelial cells were observed. The transepithelial fibers provide a possible anatomical substrate for the sensitivity of the trigeminal nerve to many air-borne chemical stimuli. That potential chemical stimuli must traverse the tight-junctional barrier may explain why lipid solubility is related to effectiveness for trigeminal stimuli.

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.

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.

Ultrastructure of mouse vallate taste buds: III. Patterns of synaptic connectivity.

We have used serial high voltage electron micrographs and computer-generated, three-dimensional reconstructions to study morphological relationships and patterns of synaptic connectivity in taste buds from the circumvallate papillae of the mouse. The intragemmal arborizations of 40 sensory nerve fibers were examined from 7 taste buds that were sectioned serially. We identified the synaptic connections from taste cells onto the reconstructed nerve fibers and classified the presynaptic taste cells based on previously established ultrastructural criteria. From these data we were able to extract the following information for the reconstructed nerve fibers: (1) the morphology of intragemmal nerve fibers and their arborizations within the taste bud, (2) the total number of synaptic connections from taste bud cells onto the nerve fibers, and (3) the taste cell types associated with each of the synapses. Fifty-six synapses were studied. Synapses were often found to be located at either the branch points or terminations of nerve fiber processes. The maximum number of taste cells observed to synapse onto a single nerve fiber was 5. Several nerve fibers had no apparent synapses. Dark cells (type I), intermediate cells, and light cells (type II) all formed synaptic connections with sensory nerve fibers. In no cases did dark cells and light cells synapse onto the same sensory nerve fiber. Our observation that any given nerve fiber receives its synaptic input from morphologically similar taste cells provides evidence for specificity in taste bud synaptic connections. We speculate that the observed pattern of synaptic connections is related to taste bud function. Since all of the synapses onto a given nerve fiber are from morphologically similar taste cells, we postulate that there is a correlation between taste cell morphology and sensory responsiveness. Intracellular electrophysiological studies on taste cells, in which responses to focally applied chemical stimuli are followed by characterization of the ultrastructural features of the same taste cells, will prove or disprove this hypothesis.

Ultrastructure of mouse vallate taste buds: II. Cell types and cell lineage.

The lifespan of cells in the mouse taste bud was examined with high-voltage electron microscopic (HVEM) autoradiography (ARG) after giving a single injection of 3H-thymidine. Animals were killed at 1 hour, 6 hours, 12 hours, 24 hours, and then daily up through 10 days postinjection. Lingual tissues were prepared for HVEM ARG so that we could identify and characterize labeled cells. Four categories of taste cells were identified: basal, dark, intermediate, and light cells. Basal cells were polygonal cells located near the basolateral sides of the taste buds and were characterized primarily by the presence of filaments attached to the nuclear envelope. Dark and light cells had the typical features described by previous authors. Intermediate cells had features in between those of dark and light cells. Over 90% of the cells labeled in the first 2 days following injection of 3H-thymidine were basal cells. Labeled dark cells appeared 6 hours after injection, reached their peak incidence at the fourth day postinjection, and then gradually decreased. Labeled intermediate cells were identified after the appearance of dark cells (12 hours) and reached a peak incidence at the fifth day after injection of 3H-thymidine. Lastly, labeled light cells were first observed on the fourth day postinjection and continued to increase until the tenth day, when they constituted 45% of the labeled cells. These data support the hypothesis that there is one cell line in the mouse vallate taste bud that undergoes morphological changes in its lifespan.

Ultrastructure of mouse vallate taste buds. I. Taste cells and their associated synapses.

The ultrastructural features of murine vallate taste bud cells and their associated synapses have been examined in thin and thick sections with conventional transmission electron microscopy and high-voltage electron microscopy. Computer-assisted reconstructions from serial sections were utilized to aid in visualization of taste bud cell-nerve fiber synapses. We have classified taste bud cells on the basis of previously established criteria-namely, size of the nucleus, shape and density of chromatin, density of cytoplasm, and presence or absence of dense-cored or clear vesicles, other cytoplasmic organelles, and synaptic foci. Both dark cells and light cells are present, as well as cells with intermediate morphological characteristics. Synapses were observed from taste bud cells onto nerve fiber processes. In virtually all instances, synapses are associated with the nuclear region of the taste cell. These synapses are characterized by the presence of 40-70 nm clear vesicles embedded in a thickened presynaptic membrane separated from the postsynaptic membrane by a 16-30 nm cleft. Synapses are not unique to any particular cell type. Dark, intermediate, and light cells all synapse onto nerve fibers. Two general types of synapses exist: spot (or macular) and fingerlike. In the latter, the postsynaptic region of the neuronal process protrudes into an invagination of the taste cell membrane. Differences in synaptic morphology are not correlated with taste cell type. In some cases a single taste cell was observed to possess both macular and fingerlike synapses adjacent to one another, forming a synaptic complex onto a single neuronal process. On the basis of the presence of synaptic contacts, we conclude that both "dark" and "light" cells are gustatory receptors.