Innervation of taste buds revealed with Brainbow-labeling in mouse.

Nerve fibers that surround and innervate the taste bud were visualized with inherent fluorescence using Brainbow transgenic mice that were generated by mating the founder line L with nestin-cre mice. Multicolor fluorescence revealed perigemmal fibers as branched within the non-taste epithelium and ending in clusters of multiple rounded swellings surrounding the taste pore. Brainbow-labeling also revealed the morphology and branching pattern of single intragemmal fibers. These taste bud fibers frequently innervated both the peripheral bud, where immature gemmal cells are located, and the central bud, where mature, differentiated cells are located. The fibers typically bore preterminal and terminal swellings, growth cones with filopodia, swellings, and rounded retraction bulbs. These results establish an anatomical substrate for taste nerve fibers to contact and remodel among receptor cells at all stages of their differentiation, an interpretation that was supported by staining with GAP-43, a marker for growing fibers and growth cones.

Effects of targeted activation of tongue muscles on oropharyngeal patency in the rat.

Laboratory rats were acutely implanted with an electrode array composed of eight independently controllable contacts applied to ventral and dorsal aspects of the left and right hypoglossal nerves (HGNs) and their branches. Bipolar intramuscular electromyographic (EMG) electrodes were implanted into the left and right genioglossus, hyoglossus and styloglossus muscles to identify which muscles were activated during stimulation via the contacts. Elicited movements, including changes in the position of the tongue and in the size and the shape of the airway, were documented video-graphically through a surgery microscope and an endoscope. Constant current electrical stimulation activated various combinations of electrode contacts and the stimulation patterns were correlated with corresponding oral movements, airway sizes, and EMG activities. Results demonstrate that graded responses and differential activation of the various tongue muscles are achievable by stimulation of specific contacts in the electrode array. These effects are interpreted to result from the targeted activation of regions of the nerve lying under and between the electrodes. Further testing established that the muscle responses elicited by unilateral electrical stimulation with the present approach can be smoothly graded, that the muscle responses resulted in opening of the airway and could be reliably maintained for long durations.

Nucleus of the solitary tract in the C57BL/6J mouse: Subnuclear parcellation, chorda tympani nerve projections, and brainstem connections.

The nucleus of the solitary tract (NST) processes gustatory and related somatosensory information rostrally and general viscerosensory information caudally. To compare its connections with those of other rodents, this study in the C57BL/6J mouse provides a subnuclear cytoarchitectonic parcellation (Nissl stain) of the NST into rostral, intermediate, and caudal divisions. Subnuclei are further characterized by NADPH staining and P2X2 immunoreactivity (IR). Cholera toxin subunit B (CTb) labeling revealed those NST subnuclei receiving chorda tympani nerve (CT) afferents, those connecting with the parabrachial nucleus (PBN) and reticular formation (RF), and those interconnecting NST subnuclei. CT terminals are densest in the rostral central (RC) and medial (M) subnuclei; less dense in the rostral lateral (RL) subnucleus; and sparse in the ventral (V), ventral lateral (VL), and central lateral (CL) subnuclei. CTb injection into the PBN retrogradely labels cells in the aforementioned subnuclei; RC and M providing the largest source of PBN projection neurons. Pontine efferent axons terminate mainly in V and rostral medial (RM) subnuclei. CTb injection into the medullary RF labels cells and axonal endings predominantly in V at rostral and intermediate NST levels. Small CTb injections within the NST label extensive projections from the rostral division to caudal subnuclei. Projections from the caudal division primarily interconnect subnuclei confined to the caudal division of the NST; they also connect with the area postrema. P2X2 -IR identifies probable vagal nerve terminals in the central (Ce) subnucleus in the intermediate/caudal NST. Ce also shows intense NADPH staining and does not project to the PBN.

Types of taste circuits synaptically linked to a few geniculate ganglion neurons.

The present study evaluates the central circuits that are synaptically engaged by very small subsets of the total population of geniculate ganglion cells to test the hypothesis that taste ganglion cells are heterogeneous in terms of their central connections. We used transsynaptic anterograde pseudorabies virus labeling of fungiform taste papillae to infect single or small numbers of geniculate ganglion cells, together with the central neurons with which they connect, to define differential patterns of synaptically linked neurons in the taste pathway. Labeled brain cells were localized within known gustatory regions, including the rostral central subdivision (RC) of the nucleus of the solitary tract (NST), the principal site where geniculate axons synapse, and the site containing most of the cells that project to the parabrachial nucleus (PBN) of the pons. Cells were also located in the rostral lateral NST subdivision (RL), a site of trigeminal and sparse geniculate input, and the ventral NST (V) and medullary reticular formation (RF), a caudal brainstem pathway leading to reflexive oromotor functions. Comparisons among cases, each with a random, very small subset of labeled geniculate neurons, revealed "types" of central neural circuits consistent with a differential engagement of either the ascending or the local, intramedullary pathway by different classes of ganglion cells. We conclude that taste ganglion cells are heterogeneous in terms of their central connectivity, some engaging, predominantly, the ascending "lemniscal," taste pathway, a circuit associated with higher order discriminative and homeostatic functions, others engaging the "local," intramedullary "reflex" circuit that mediates ingestion and rejection oromotor behaviors.

Evaluation of methods to reduce formaldehyde levels of cadavers in the dissection laboratory.

Dissection of conventionally embalmed cadavers exposes students, staff, and faculty to formaldehyde, a probable carcinogen. Therefore, prudent practices should seek to minimize formaldehyde exposure. In this study, we evaluated two commercially available chemicals, InfuTrace and Perfect Solution, for their effectiveness in reducing ambient formaldehyde levels. Four cadavers embalmed conventionally with formaldehyde and/or with the above agents were compared for their formaldehyde levels under conditions that strictly controlled for air circulation and for locations and methods of testing, and during activities that simulated student dissecting. For InfuTrace, one cadaver was reinfused with InfuTrace after initial standard perfusion with formaldehyde; a second cadaver had InfuTrace injected into the thoracic and abdominal body cavities after formaldehyde perfusion. For Perfect Solution, the product was used for embalming a third cadaver in lieu of formaldehyde. For a control, a fourth cadaver was embalmed with the standard formaldehyde solution. Testing of personal and ambient room air samples and of fluid obtained from the cadavers was performed and analyzed in a blinded fashion. Results indicated that both Perfect Solution, substituted for standard formaldehyde embalming, and InfuTrace infused through the vasculature after formaldehyde embalming, resulted in lower concentrations of formaldehyde than embalming with formaldehyde solution alone or in combination with body cavity injection of InfuTrace. These differences in formaldehyde concentrations are consistent across measuring methods, for example, of room air, of breathing zone air during cadaver handling and dissection, and of liquid samples obtained from the cadavers. Perfect Solution yielded suboptimum fixation and a different texture, color, and smell than the formaldehyde treatments.

Exuberant neuronal convergence onto reduced taste bud targets with preservation of neural specificity in mice overexpressing neurotrophin in the tongue epithelium.

A mouse fungiform taste bud is innervated by only four to five geniculate ganglion neurons; their peripheral fibers do not branch to other buds. We examined whether the degree or specificity of this exclusive innervation pattern is influenced by brain-derived neurotrophic factor (BDNF), a prominent lingual neurotrophin implicated in taste receptoneural development. Labeled ganglion cells were counted after injecting single buds with different color markers in BDNF-lingual-overexpressing (OE) mice. To evaluate the end-organs, taste buds and a class of putative taste receptor cells were counted from progeny of BDNF-OE mice crossbred with green fluorescent protein (GFP) (gustducin) transgenic mice. Fungiform bud numbers in BDNF-OE mice are 35%, yet geniculate neuron numbers are 195%, of wild-type mice. Neurons labeled by single-bud injections in BDNF-OE animals were increased fourfold versus controls. Injecting three buds, each with different color markers, resulted in predominantly single-labeled ganglion cells, a discrete innervation pattern similar to controls. Thus, hyper-innervation of BDNF-OE buds involves many neurons innervating single buds, not increased fiber branching. Therefore, both wild-type and BDNF-OE mice exhibit, in fungiform buds, the same, "discrete" receptoneural pattern, this despite dramatic neurotrophin overexpression-related decreases in bud numbers and increases in innervation density. Hyperinnervation did not affect GFP positive cell numbers; proportions of GFP cells in BDNF-OE buds were the same as in wild-type mice. Total numbers of ganglion cells innervating buds in transgenic mice are similar to controls; the density of taste input to the brain appears maintained despite dramatically reduced receptor organs and increased ganglion cells.

Discrete innervation of murine taste buds by peripheral taste neurons.

The peripheral taste system likely maintains a specific relationship between ganglion cells that signal a particular taste quality and taste bud cells responsive to that quality. We have explored a measure of the receptoneural relationship in the mouse. By injecting single fungiform taste buds with lipophilic retrograde neuroanatomical markers, the number of labeled geniculate ganglion cells innervating single buds on the tongue were identified. We found that three to five ganglion cells innervate a single bud. Injecting neighboring buds with different color markers showed that the buds are primarily innervated by separate populations of geniculate cells (i.e., multiply labeled ganglion cells are rare). In other words, each taste bud is innervated by a population of neurons that only connects with that bud. Palate bud injections revealed a similar, relatively exclusive receptoneural relationship. Injecting buds in different regions of the tongue did not reveal a topographic representation of buds in the geniculate ganglion, despite a stereotyped patterned arrangement of fungiform buds as rows and columns on the tongue. However, ganglion cells innervating the tongue and palate were differentially concentrated in lateral and rostral regions of the ganglion, respectively. The principal finding that small groups of ganglion cells send sensory fibers that converge selectively on a single bud is a new-found measure of specific matching between the two principal cellular elements of the mouse peripheral taste system. Repetition of the experiments in the hamster showed a more divergent innervation of buds in this species. The results indicate that whatever taste quality is signaled by a murine geniculate ganglion neuron, that signal reflects the activity of cells in a single taste bud.

Brain-derived neurotrophic factor-, neurotrophin-3-, and tyrosine kinase receptor-like immunoreactivity in lingual taste bud fields of mature hamster.

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), as well as their respective tyrosine kinase (Trk) receptors, TrkB and TrkC, influence peripheral target cell innervation, survival, and proliferation. In the mature taste system the role of neurotrophins and their receptors is not known. The mature hamster is an intriguing model because anterior lingual fungiform, unlike posterior lingual foliate and circumvallate, taste buds survive denervation. In light of this difference, we examined whether the degree of neurotrophin- or neurotrophin receptor-like immunoreactivity (IR) normally differs among lingual gemmal fields. In single- and double-labeled immunofluorescent experiments, 3,209 taste bud sections (profiles) from 13 hamsters were examined for immunopositive gemmal cells or nerve fibers using antibodies to BDNF and NT-3, their respective receptors TrkB and TrkC, and the neural marker ubiquitin c-terminal hydrolase L-1 [protein gene product (PGP) 9.5]. In each gemmal field, more than 75% of taste bud profiles showed immunopositivity to BDNF, NT-3, and TrkB. Across bud fields, BDNF-, TrkB-, and BDNF/TrkB-like IR, as well as PGP 9.5 and PGP 9.5/BDNF-like IR in centrally located, fungiform bud cells was greater (P < 0.0001 to P < 0.002) than in circumvallate or foliate buds. Within bud fields, the number of BDNF-like, labeled bud cells/bud profile was greater than that for NT-3-like IR in fungiform (P < 0.0002) and foliate (P < 0.0001) buds. TrkC was immunonegative in gemmal cells. The average density of TrkB- and TrkC-like fiber IR was more pronounced in fungiform than posterior gemmal-bearing papillae. Thus, fungiform papillae, whose taste buds are least affected by denervation, exhibit specific neurotrophin and receptor enrichment.

Brain-derived neurotrophic factor-, neurotrophin-3-, and tyrosine kinase receptor-like immunoreactivity in lingual taste bud fields of mature hamster after sensory denervation.

Unlike lingual taste buds in most mammals, fungiform buds on the anterior tongue of mature hamster survive sensory denervation. The role of the neurotrophin ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), and their respective tyrosine kinase (Trk) receptors, TrkB and TrkC, in denervated taste buds is not known. The present report investigates changes in the degree of gemmal cell immunoreactivity (IR) (i.e., number of immunoreactive cells/bud profile) and density of nerve fiber-IR of these markers in unilaterally denervated mature hamsters. The fungiform bud field after chorda tympani/lingual nerve resection is compared with the nerve-dependent, posterior tongue foliate and circumvallate bud fields after glossopharyngeal nerve resection. Four weeks post lesion, the number of denervated fungiform buds matched that on the unoperated side, whereas denervated foliate and circumvallate bud counts decreased by 72% and 38%, respectively. In taste buds that survived on the posterior tongue, the degree of foliate bud cell BDNF-, NT-3-, and TrkB-like IR, and circumvallate bud cell BDNF- and NT-3-like IR, significantly decreased compared with the unoperated side. In contrast, for anterior tongue fungiform bud cells, the degree of neurotrophin- and receptor-like IR was relatively less affected: NT-3- and TrkB-like IR were unchanged; BDNF-like IR, although significantly decreased, was also maintained. Moreover, TrkB-like fiber IR was essentially eliminated within and surrounding fungiform buds. Hence, NT-3-, BDNF-, and TrkB-like IR in fungiform gemmal cells may reflect an autocrine capacity promoting survival. Because TrkC-like IR in bud cells is absent (i.e., immunonegative), and sparse in fibers intragemmally and perigemmally, NT-3 may also bind to bud cell TrkB so as to sustain fungiform gemmal cell viability post denervation.