Behavioural displays to gustatory stimuli in newborn rabbit pups

Motor displays in the face and head regions of 33 neonate rabbits(less than 24 hrs post partum) in response to taste stimulationwere examined. A droplet of taste solution was placed mediallyon the pup's lips and the ensuing behavioral repertoire wastallied over a 60 sec period in a double blind situation. Tastantsincluded 2 concentrations each of sucrose, saccharin, citricacid and quinine. Distilled water was used as a stimulant andfor intertrial rinses. Response characteristics to the varioustaste stimuli were differentiable, specific and reproduciblewithin and across animals. Certain response features were moreoften associated with one stimulus than with another. Quinineoften produced mouth opening (gaping) and head movements, whereassucrose was associated with a quiet animal licking and makingcharacteristic mouth movements. Sour reactions often resembledthose to sweet, but other features helped distinguish thoseresponses. Reactions proved to be concentration-dependent anddifferent from those to water. Quality and hedonic value wereusually accurately judged and corresponded to adult preferencebehaviors. It was inferred that rabbits at this early age arealready equipped with a functioning taste system up to the brainstemlevel. Cross-species comparisons of stereotyped reactions werediscussed.     

Development of sucrose preference in rabbit pups

Taste preference behavior in developing rabbit pups was examinedat 2–4 days, 6–8 days and 14–16 days postnatally.0.3 M sucrose and distilled water were presented alternatelyat 2 min. intervals, and volumes ingested were recorded. Sweetpreferences were not expressed until late in the second postnatalweek. Since it is known that rabbit pups exhibit distinctivefacial displays to different taste stimuli at birth (Ganchrowet al., 1979) the delay in the expression of sweet preferencewas postulated to be related to the postnatal maturation ofneural processes central to peripheral and brainstem gustatorycoordinating mechanisms.     

Ultrastructure of palatal taste buds in the perihatching chick.

Palatal taste buds of perihatching chicks were examined by electron microscopy. Four intragemmal cell types were characterized. 1) Light: with voluminous, electron-lucent cytoplasm containing scattered free ribosomes, rough and smooth endoplasmic reticulum, plump mitochondria, sparse perinuclear filaments, occasional Golgi bodies, and numerous clear and dense-cored vesicles. Clear vesicles sometimes aggregate in a presynaptic-like configuration apposed to an axonal profile. These cells contained large, spherical, uniformly granular nuclei with one nucleolus. 2) Dark: with dense cytoplasm containing filamentous bundles surrounding the nucleus, occasional clear vesicles, centrioles, rough endoplasmic reticulum, and compact mitochrondria. The apical cytoplasm noticeably lacks dense secretory granules. Irregular to lobulated nuclei are densely granular, and contain scattered clumps of chromatin, adhering especially to the inner leaflet of the nuclear membrane, and at least one nucleolus. Cytoplasmic extensions of dark cells envelop other intragemmal cell types and nerve fibers. Light and dark cells project microvilli into the taste pore. 3) Intermediate: contain gradations of features of light and dark cells. 4) Basal: darker than the other intragemmal cell types and confined to the ventral bud region. Putative afferent synapses in relation to light cells, and axo-axonal contacts are described. While the appearance of axo-axonal contacts may be a transient developmental event, other bud features are consonant with observations in adult chickens and suggest that the peripheral gustatory apparatus is mature at hatching in this precocial avian species.     

Taste Bud Cell Generation in the Perihatching Chick

Chick taste bud primordia initially appear in late gestationon embryonic day 17 (E17), 4 days before hatching. To trackDNA synthesis and subsequent taste bud cell proliferation betweenE17 and the second day post-hatching (H2), single 25 µCiinjections of tritiated thymidine (specific activity = 72.5Ci/mmol) were administered in ovo during E15, E16, E17 or E18.Anterior mandibular oral epithelium was processed for lightmicroscopic autoradiography. Sections through each taste bud'scenter were analysed for label (6 silver grains/gemmal cellnucleus), and bud diameter. Results indicated a major part ofgemmal cell DNA synthesis does not occur until after E19 irrespectiveof the day of thymidine injection, suggesting postmitotic orquiescent (decycled) cells assemble to form the early bud primordium(E17–19) based on local tissue interactions. All budsexamined from E20–H2 contained labelled cells. The dayof injection was important since 5-day survival cases afterE16 injection yielded about 25% the number of labelled cells/budas compared with equivalent survival cases following E17–18injections. These results are discussed with respect to parallelchanges in bud shape and increasing bud diameter, and cell proliferationin possible extra- and intragemmal sources of bud cells.     

The effect of β-bungarotoxin, or geniculate ganglion lesion on taste bud development in the chick embryo

Chick taste bud (gemmal) primordia normally appear on embryonic day (E) 16 and incipient immature, spherical-shaped buds at E17. In ovo injection of β-bungarotoxin at E12 resulted in a complete absence of taste buds in lower beak and palatal epithelium at developmental ages E17 and E21. However, putative gemmal primordia (solitary clear cells; small, cell groupings) remained, lying adjacent to salivary gland duct openings as seen in normal chick gemmal development. Oral epithelium was immunonegative to neural cell adhesion molecule (NCAM) suggesting gemmal primordia are nerve-independent. Some NCAM immunoreactivity was evident in autonomic ganglion-like cells and nerve fibers in connective tissue. After unilateral geniculate ganglion/otocyst excision on E2.5, at developmental ages E18 and posthatching day 1, ∼12% of surviving ipsilateral geniculate ganglion cells sustained ∼54% of the unoperated gemmal counts. After E18, proportional stages of differentiation in surviving developing buds probably reflect their degree of innervation, as well as rate of differentiation. Irrespective of the degree of geniculate ganglion damage, the proportion of surviving buds can be sustained at the same differentiated bud stage as on the unoperated side, or may differentiate to a later bud stage, consistent with the thesis that bud maturation, maintenance, and survival are nerve-dependent.     

Fingerprinting taste buds: intermediate filaments and their implication for taste bud formation

Intermediate filaments in taste organs of terrestrial (human and chick) as well as aquatic (Xenopus laevis) species were detected using immunohistochemistry and electron microscopy. During development, the potential importance of the interface between the taste bud primordium and non-gustatory adjacent tissues is evidenced by the distinct immunoreactivity of a subpopulation of taste bud cells for cytokeratins and vimentin. In human foetuses, the selective molecular marker for taste bud primordia, cytokeratin 20, is not detectable prior to the ingrowth of nerve fibres into the epithelium, which supports the hypothesis that nerve fibres are necessary for initiating taste bud development. Another intermediate filament protein, vimentin, occurs in derivatives of mesoderm, but usually not in epithelium. In humans, vimentin immunoreactivity is expressed mainly in border (marginal) epithelial cells of taste bud primordia, while in chick, vimentin expression occurs in most taste bud cells, whereas non-gustatory epithelium is vimentin immunonegative. Our chick data suggest a relationship between the degree of vimentin expression and taste bud cell proliferation especially during the perihatching period. It is suggested that surrounding epithelial cells (human) and mesenchymal cells (chick) may be contributing sources of developing taste buds. The dense perinuclear network of intermediate filaments especially in dark (i.e. non-sensory) taste disc cells of Xenopus indicates that vimentin filaments also might be associated with cells of non-gustatory function. These results indicate that the mechanisms of taste bud differentiation from source tissues may differ among vertebrates of different taxa.     

Distribution of vimentin in the developing chick taste bud during the perihatching period.

The tissue environment within which taste bud cells develop has not been wholly elaborated. Previous studies of taste bud development in vertebrates, including the avian chick, have suggested that taste bud cells could arise from one, or several tissue sources (e.g. crest-mesenchyme, local ectoderm or endoderm). Thus, molecular markers which are present in gemmal as well as interfacing (peribud epithelium; mesenchyme-epithelium) regions, and their degree of expression during stages of taste bud development, are of special interest. The intermediate filament protein, vimentin, occurs in mesenchymal and mesodermally-derived (e.g. endothelial, fibroblast) cells as well as highly proliferating epithelium (e.g. tumors). The present study in chick gustatory tissue utilized antibodies against vimentin and the avidin-biotin-peroxidase technique to evaluate vimentin immunoreactivity (IR) within a timeframe which includes: 1) early stages of the taste bud primordium [embryonic days (E)17-E18)]; 2) the beginning of an accelerated bud cell proliferation at the time of initial, taste bud pore opening [around E19]; 3) attaining the adult complement of taste buds [around posthatch (H) day 1], and 4) completed organogenesis (H 17). During this time span, vimentin-IR was characterized in a region including and sometimes bridging taste bud and subepithelial connective tissue, whereas non-gustatory surrounding epithelium and salivary glands were vimentin-immuno-negative. Intragemmally, the proportion of vimentin-IR cells as related to total taste bud cells peaked at E19. These results indicate that vimentin expression, in part, is related to the onset of taste bud cell proliferation and suggest that mesenchyme could be one source of taste bud cells. Secondly, fibronectin, an extracellular matrix component of the epithelial basement membrane interface with mesenchyme, was expressed at or near the apical surfaces of taste bud cells projecting into the bud lumen, and in the basal gemmal region suggesting the possible role of fibronectin as a chemotactic anchor for differentiating and migrating taste bud receptor cells. Lastly, neuron-specific enolase-IR indicates that axonal varicosities are already present intragemmally at E17-E18, that is, during the incipient period of identifiable taste bud primordia.     

Scanning electron microscopy of developing papillae on the tongue of human embryos and fetuses

The appearance and differentiation of papillae on dorsal andlateral surfaces of human embryonic and fetal tongues, at variousdevelopmental ages, were studied by scanning electron microscopy.Formaldehyde and phosphate buffer fixation provided satisfactorypreservation. At 8–9 weeks, the anterior two-thirds ofthe tongue showed no obvious signs of papillae. In contrast,just anterior to the sulcus terminalis rounded elevations wereseen, suggesting initial signs of circumvallate papillae. At10–13 weeks, the distribution and shape of elevationson the anterior two-thirds of the tongue indicated the beginningof fungiform papillae. Openings located on the dorsal surfaceof many of these fungiforms contained an amorphous central structureprojecting out of the papilla. First signs of foliate papillaewere seen at 10 weeks. At 15–18 weeks, fungiform and filiformpapillae were recognized, although sometimes their borders wereobscured by scaling epithelial cells. At 23–26 weeks,all papillae exhibited their adult form. ^*Presented, in part, at the VIth International Symposium onOlfaction and Taste, Gif-sur-Yvette, Paris, France, 15–17thJuly, 1977.     

Identified taste bud cell proliferation in the perihatching chick [published erratum appears in Chem Senses 1998 Aug;23(4):459]

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.