Charlotte M. Mistretta

Alterations in Size, Number, and Morphology of Gustatory Papillae and Taste Buds in BDNF Null Mutant Mice Demonstrate Neural Dependence of Developing Taste Organs

Sensory ganglia that innervate taste buds and gustatory papillae (geniculate and petrosal) are reduced in volume by about 40% in mice with a targeted deletion of the gene for brain‐derived neurotrophic factor (BDNF). In contrast, the trigeminal ganglion, which innervates papillae but not taste buds on the anterior tongue, is reduced by only about 18%. These specific alterations in ganglia that innervate taste organs make possible a test for roles of lingual innervation in the development of appropriate number, morphology, and spatial pattern of fungiform and circumvallate papillae and associated taste buds. We studied tongues of BDNF null mutant and wild‐type littermates and made quantitative analyses of all fungiform papillae on the anterior tongue, the single circumvallate papilla on the posterior tongue, and all taste buds in both papilla types. Fungiform papillae and taste buds were reduced in number by about 60% and were substantially smaller in diameter in mutant mice 15–25 days postnatal. Remaining fungiform papillae were selectively concentrated in the tongue tip region. The circumvallate papilla was reduced in diameter and length by about 40%, and papilla morphology was disrupted. Taste bud number in the circumvallate was reduced by about 70% in mutant tongues, and the remaining taste buds were smaller than those on wild‐type tongues. Our results demonstrate a selective dependence of taste organs on a full complement of appropriate innervation for normal growth and morphogenesis. Effects on papillae are not random but are more pronounced in specific lingual regions. Although the geniculate and petrosal ganglia sustain at least half of their normal complement of cell number in BDNF −/− mice, remaining ganglion cells do not substitute for lost neurons to rescue taste organs at control numbers. Whereas gustatory ganglia and the taste papillae initially form independently, our results suggest interdependence in later development because ganglia derive BDNF support from target organs and papillae require sensory innervation for morphogenesis. J. Comp. Neurol. 409:13–24, 1999. 

Organ Cultures of Embryonic Rat Tongue Support Tongue and Gustatory Papilla Morphogenesis In Vitro Without Intact Sensory Ganglia

Taste buds on the mammalian tongue are confined to the epithelium of three types of gustatory papillae: the fungiform, circumvallate, and foliate. The gustatory papillae are composed of an epithelium that covers a broad connective tissue core, with extensive innervation to taste bud and nongustatory epithelial locations. Although the temporal sequence of gustatory papilla development is known for several species, factors that regulate initiation, growth, and maintenance of the papillae are not understood. We tested the hypothesis that sensory innervation is required for the initial formation and early morphogenesis of fungiform papillae in a patterned array. An organ culture of the embryonic rat tongue was developed to provide an in vitro system for studying mechanisms involved in fungiform papilla morphogenesis in patterns on the anterior tongue. Tongues were dissected from embryos at 13 days of gestation (E13), a time when the tongue has not yet fully formed and gustatory papillae have not yet appeared, and at 14 days of gestation (E14), when the tongue is well formed and papillae make their initial morphological appearance. Dissected tongues were maintained at the gas/liquid interface in standard organ culture dishes, fed with DMEM/F12 plus 2% B‐27 supplement and 1% fetal bovine serum. After 1, 2, 3, or 6 days in culture, tongues were processed for scanning electron or light microscopy, or immunocytochemistry. Tongues cultured from E13 or E14 underwent extensive morphogenesis and growth in vitro. Furthermore, fungiform papillae developed on these tongues on a culture day equivalent to E15 in vivo; that is, after 2 days for cultures begun at E13 and 1 day for those begun at E14. Because E15 is the characteristic time for gustatory papilla formation in the intact embryo, results demonstrate that the cultured tongues retain important temporal information related to papilla development. In addition, fungiform papillae formed in the tongue cultures in the stereotypic pattern of rows. The papillae were large structures with epithelial and mesenchymal cell integrity, and an intact epithelial basement membrane was indicated with laminin immunoreactivity. The cultures demonstrate that gustatory papilla morphogenesis can progress in the absence of an intact sensory innervation. To exclude a potential developmental role for autonomic ganglion cells that are located in the posterior rat tongue, cultures consisting of only the anterior half of E14 tongues were established. Fungiform papilla development progressed in half tongues in a manner directly comparable to whole tongue cultures. Therefore, robust, reproducible development of fungiform papillae in patterns is supported in rat tongue cultures from E13 or E14, without inclusion of intact sensory or major, posterior tongue autonomic ganglia. This is direct evidence that papillae will form and develop further in vitro without sensory ganglion support. The data also provide the first detailed account of in vitro development of the entire embryonic tongue. J. Comp. Neurol. 377:324–340, 1997.

Cyclopamine and jervine in embryonic rat tongue cultures demonstrate a role for Shh signaling in taste papilla development and patterning : fungiform papillae double in number and form in novel locations in dorsal lingual epithelium

From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior tongue, the fungiform papillae are patterned in rows. Among the many molecules that have potential roles in regulating papilla location and pattern, Sonic hedgehog (Shh) has been localized within early tongue and developing papillae. We used an embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla morphogenesis and patterning to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium. Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla, regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue cultures with standard medium or cyclopamine, and was conspicuously localized in the basement membrane region of the papillae. Ptc protein had a similar distribution to Shh, although the immunoproduct was more diffuse. Fungiform papillae did not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline furrow, nor was development of the single circumvallate papilla altered. The results demonstrate a prominent role for Shh in fungiform papilla induction and patterning and indicate differences in morphogenetic control of fungiform and circumvallate papilla development and numbers. Furthermore, a previously unknown, broad competence of dorsal lingual epithelium to form fungiform papillae on both anterior and posterior oral tongue is revealed.

Swallowing in fetal sheep.

Swallowing was measured in fetal sheep by using electromagnetic flowmeter heads chronically implanted in the fetal esophagus. The fetus swallows 20 to 200 milliliters of amniotic fluid in two to seven discrete episodes per day. The episodes are 1 to 9 minutes in duration and occur at seemingly random intervals. Swallowing is influenced by the condition of the fetus and may be the first manifestation of eating and drinking behavior.

Developmental neurobiology of salt taste sensation

A principal process in the homeostatic control of sodium levels is salt intake, and the sense of taste has a primary role in regulating ingestion. Because ingestion of sodium chloride (NaCl) is essential for life, the taste system for salt sensation might be expected to exhibit mature functional characteristics from very early development. However, major changes in gustatory nerve responses to NaCl take place during development. In sheep and rat, the peripheral nerve responses to NaCl are of low magnitude during early development. Progressively, the taste system acquires an increasing proportion of fibers that respond maximally to NaCl. The sodium responsiveness emerges in the context of shifting peripheral innervation patterns and the apparent addition of functional receptor membrane channels sensitive to the sodium transport blocker, amiloride. These developmental processes can be altered by early manipulation of sodium in the diet.

Superior Laryngeal Nerve Response Patterns to Chemical Stimulation of Sheep Epiglottis

Responses were recorded from single fibers of the sheep superior laryngeal nerve during stimulation of the epiglottis with 0.5 M KCl, NH4Cl, NaCl and LiCl, distilled water, 0.005 M citric acid, and 0.01 N HCl. Recordings were made from both lambs and ewes. KCl elicited a response from 99% of fibers followed in order of effective stimulation by NH4Cl, HCl, distilled water, citric acid, NaCl and LiCl. Analysis of the variation in response frequency with time demonstrated differences in the response patterns for these stimuli. The pattern of frequency over time is sufficient to discriminate among the salts, between some of the salts and acids, and between some of the salts and water. Therefore the response pattern may be significant in initiating the various reflex activities that occur during chemical stimulation of the larynx.

Transganglionic transport of HRP from the circumvallate papilla of the rat

To learn whether horseradish peroxidase (HRP) injections in gustatory papillae on the tongue can be used to study central topographical projections of taste buds and papillae, injections were made into the circumvallate papilla in rats. Labeled central projections after papilla injections were compared to projections after applying HRP to the cut glossopharyngeal nerve. Papilla injections result in HRP transport by afferent and efferent fibers of the glossopharyngeal nerve, and the pattern of central projections is similar to that after labeling the cut nerve. Projections include a separation in the brainstem of afferent, dorsally located fibers and efferent, ventrally located fibers. Afferent fibers project to the solitary nucleus and the trigeminal system. Efferent projections label muscle motorneurons in the nucleus ambiguus and the cells of origin of parasympathetic preganglionic fibers, which from the inferior salivatory nucleus. The parasympathetic neurons labeled after papilla projections are preganglionic fibers to Remaks ganglia in the tongue; post-ganglionic fibers of these ganglia are the secretomotor supply to the von Ebners glands. In summary, injections of HRP into gustatory papillae reliably label central projections of the papilla and can be used for studies to discern topography in central projections of the taste system. Injections into the circumvallate papilla also have demonstrated that the parasympathetic neurons innervating von Ebners glands are located in the inferior salivatory nucleus.

Developmental changes in neurophysiological taste response from the medulla in sheep

To determine whether functional characteristics of the taste system change during development, electrophysiological taste responses were recorded from neurons in the solitary complex (nucleus and tractus solitarius) in the medulla of fetal, newborn and adult sheep. Taste stimuli included NH4Cl, KCl, NaCl, LiCl, citric acid, and HCl, applied to the anterior tongue. Fetal neurons at all ages (84-137 days of gestation) responded to stimulation of the tongue with NH4Cl and KCl, but responses to NaCl and LiCl were only obtained in older fetuses (after 114 days of gestation), lambs and adults. Responses to citric aicd were obtained at all ages; however, HCl responses were only infrequently obtained in young fetuses. Other developmental changes included a progressive decrease in latency of the responses to NH4Cl, KCl, citric acid and HCl, and an increase in the duration of the neural response discharge as a function of gestational age. Since taste buds do not acquire the structural characteristics of the adult until the last third of gestation (approximately 100-147 days), these functional changes in taste response characteristics take place concurrently with structural development. Mammalian fetuses swallow amniotic fluid in utero, and, therefore, the fetal taste system is stimulated during structural and functional development. Thus, there is an opportunity for fetal gustatory experience to influence the developing taste system.

Distinctive spatiotemporal expression patterns for neurotrophins develop in gustatory papillae and lingual tissues in embryonic tongue organ cultures.

Abstract. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) mRNAs are expressed in the developing rat tongue and taste organs in specific spatiotemporal patterns. BDNF mRNA is present in the early lingual gustatory papilla epithelium, from which taste buds eventually arise, prior to the arrival of gustatory nerve fibers at the epithelium, whereas NT-3 initially distributes in the mesenchyme. However, a direct test for neural dependence of neurotrophin expression on the presence of innervation in tongue has not been made, nor is it known whether the patterns of neurotrophin expression can be replicated in an in vitro system. Therefore, we used a tongue organ culture model that supports taste papilla formation while eliminating the influence from sensory nerve fibers, to study neurotrophin mRNAs in lingual tissues. Rat tongue cultures were begun at embryonic day 13 or 14 (E13, E14), and BDNF, NT-3, nerve growth factor (NGF) and neurotrophin-4 (NT-4) mRNAs were studied at 0, 2, 3 and 6 days in culture. BDNF transcripts were localized in the gustatory epithelium of both developing fungiform and circumvallate papillae after 2 or 3 days in culture, and NT-3 transcripts were in the subepithelial mesenchyme. The neurotrophin distributions were comparable to those in vivo at E13–E16. In 6-day tongue cultures, however, BDNF transcripts in anterior tongue were not restricted to fungiform papillae but were more widespread in the lingual epithelium, while the circumvallate trench epithelium exhibited restricted BDNF labeling. The NT-3 expression pattern shifted in 6-day organ cultures in a manner comparable to that in the embryo in vivo, and was expressed in the lingual epithelium as well as mesenchyme. NGF mRNA expression was subepithelial throughout 6 days in cultures. NT-4 mRNA was not detected. The neurotrophin mRNA distributions demonstrate that temporospatial localization of neurotrophins observed during development in vivo is retained in the embryonic tongue organ culture system. Furthermore, initial neurotrophin expression in the developing lingual epithelium, mesenchyme, and/or taste papillae is not dependent on intact sensory innervation. We suggest that patterns of lingual neurotrophin mRNA expression are controlled by the influence of local tissue interactions within the tongue at early developmental stages. However, the eventual loss of restricted BDNF mRNA localization from fungiform papillae in anterior tongue suggests that sensory innervation may be important for restricting the localized expression of neurotrophins at later developmental stages, and for maintaining the unique phenotypes of gustatory papillae.

Effects of Dietary NaCl Deprivation During Early Development on Behavioral and Neurophysiological Taste Responses

In order to determine whether the gustatory system can be modified by restricting dietary NaCl during early development, neurophysiological taste responses were recorded in rats at various times after deprivation, and behavioral taste preferences were measured in adults. Rats deprived of dietary NaCl from the third day of gestation to 12 days postnatally and then placed on a NaCl-replete diet had chorda tympani nerve responses similar to those of nondeprived rats when recordings were made at 28 days of age and older; however, preferences for NaCl solutions over water were significantly less than those of controls when tested at adulthood. NaCl deprivation in pups from the third day of gestation to approximately 35 days postnatally resulted in altered chorda tympani nerve responses to NaCl but not to other stimuli such as NH4Cl and KCl. Therefore, restriction of dietary NaCl at a period in the rats development when peripheral and central taste responses are changing results in short-term alterations in peripheral neural responses and in long-term changes in preference behaviors.