Mark C. Whitehead

Forebrain projections to the rostral nucleus of the solitary tract in the hamster

The rostral nucleus of the solitary tract (NST) is the first central site of taste information processing. Specific anatomical subdivisions of the NST receive taste afferent input and contain interneurons and projection neurons that engage ascending or premotor taste pathways. The forebrain projects to the NST and can influence taste responses, but the anatomical relationship between forebrain inputs and the subdivisions of the NST and their cellular elements is not understood. To evaluate this, in this study, we used cholera toxin B (CTb) as a retrograde and anterograde marker. CTb was injected into the rostral NST to label, by retrograde transport, the sources of forebrain inputs. Cells were labeled bilaterally in the lateral and paraventricular hypothalamic nuclei, bed nucleus of the stria terminalis, central nuclei of the amygdala, and the agranular and dysgranular divisions of insular cortex. Within the medulla, labeled cells were located in the parvicellular reticular formation and spinal trigeminal nuclei. In addition, labeled cells and anterograde axonal labeling were present in the rostral NST contralateral to the injections. Injections of CTb centered in the dysgranular insular cortex, the site of most forebrain‐NST cells, labeled axon endings confined to the rostral NST. These endings were concentrated in the rostral central and ventral subdivisions. Corticofugal endings in the rostral central subdivision are positioned to influence microcircuits that include taste afferent synapses, presumed inhibitory interneurons, and neurons that project to the parabrachial nucleus. The many corticofugal endings in the ventral subdivision synapse among premotor neurons that ultimately influence salivatory and oromotor outflow. Intramedullary CTb labeling after NST injection indicates that the rostral central subdivision also receives projections from the contralateral rostral NST. J. Comp. Neurol. 422:429–447, 2000.

Organization of geniculate and trigeminal ganglion cells innervating single fungiform taste papillae: a study with tetramethylrhodamine dextran amine labeling

Single gustatory nerve fibers branch and innervate several taste buds. In turn, individual taste buds may receive innervation from numerous gustatory nerve fibers. To evaluate the pattern of sensory innervation of fungiform papilla-bearing taste buds, we used iontophoretic fluorescent injection to retrogradely label the fibers that innervate single taste papillae in the hamster. For each animal, a single taste papilla was injected through the gemmal pore with 3.3% tetramethylrhodamine dextran amine. Fungiform papillae either at the tongue tip (0.5-1.5 mm from the tip) or more posteriorly (1.5-3.0 mm from the tip) were injected. After one to seven days survival, the geniculate and trigeminal ganglia and the tongue were sectioned and examined for labeled cells and fibers, respectively. Analysis of the number and topographic distribution of geniculate cells innervating single taste papillae, shows that: (i) 15 +/- 4 (S.D.) ganglion cells converge to innervate a single fungiform taste bud; (ii) more ganglion cells innervate anterior- (range: 13-35 cells) than posterior-lying buds (range: five to 12 cells), which, in part, may be related to bud volume (microm3); and (iii) ganglion somata innervating a single taste bud are scattered widely within the geniculate ganglion. Analysis of labeled fibers in the tongue demonstrated that two to eight taste buds located within 2 mm of the injected taste bud share collateral innervation with the injected taste bud. Since all buds with labeled fibers were located in close proximity (within a 2-mm radius), widely dispersed geniculate ganglion cells converge to innervate closely spaced fungiform taste buds. Trigeminal ganglion (mandibular division) cells were also labeled in every case and, as with the geniculate ganglion, a dispersed cell body location and collateralization pattern among papillae were observed. This study shows that iontophoresis of tetramethylrhodamine dextran amine, selectively applied to individual peripheral receptor end-organs, effectively locates sensory ganglion cells in two different ganglia that project to these sites. Moreover, the marker demonstrates collateral branches of sensory afferents associated with the labeled fibers and the nearby receptor areas innervated by these collaterals. The labeling of single or clusters of receptor cells, as well as identified sensory afferents, affords future possibilities for combining this technique with immunocytochemistry to establish the relationships of innervation patterns with neurotransmitters and neurotropic substances within identified 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. J. Comp. Neurol. 455:11–24, 2003.

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. J. Comp. Neurol. 511:753–772, 2008.

SCANNING ELECTRON MICROSCOPY OF DENERVATED TASTE BUDS IN HAMSTER : MORPHOLOGY OF FUNGIFORM TASTE PORES

Taste pores of fungiform papillae are critical for taste function. Taste nerve injury affects the pore, rendering it refractory to staining with vital dyes. Whether pores of denervated fungiform papillae disappear or undergo more modest structural changes to account for diminished staining was the subject of the present study.

Transganglionic degeneration in the gustatory system consequent to chorda tympani damage

The chorda tympani taste nerve is prone to damage in humans. Chorda tympani damage results in taste loss accompanied by altered taste sensations, e.g., phantom tastes. To understand taste alterations this study explores the central and peripheral anatomical consequences of taste nerve injury in an animal model. The chorda tympani was severed in the middle ear of hamsters and the animals were allowed to survive for 2-161 days when sections of the brain were stained for degenerating axons with the Fink-Heimer method. Degenerating axons were present in the chorda tympani termination zone in the nucleus of the solitary tract of every case. Thus, peripheral nerve damage in the taste system results in degeneration of central axonal endings as in other sensory systems (e.g., trigeminal, vestibular). To evaluate whether the central degeneration results from ganglion cell death, geniculate ganglion cells were labeled with Fast blue by tongue injections before neurotomy, and the cells were counted 13-48 days after neurotomy. Numbers of labeled cells from experimental ganglia did not differ significantly from those in control ganglia. Moreover, the experimental cells could be double-labeled by tongue injections with a second marker, diamidino yellow or nuclear yellow, after 40 days postneurotomy. We conclude that degeneration of central axons after taste nerve section represents a long-lasting transganglionic process that likely disrupts the synaptology of the central taste system. The altered synaptology could relate to taste phenomena of central origin reported for nerve-injured patients. Geniculate ganglion cells generally survive neurotomy and can regenerate axons to the tongue.

Neural cell adhesion molecule, neuron-specific enolase and calcitonin gene-related peptide immunoreactivity in hamster taste buds after chorda tympani/lingual nerve denervation

Hamster fungiform papilla taste buds persist in an atrophic form following sensory denervation. While atrophic and innervated taste buds are morphologically similar, it is not known whether their gemmal cells have similar molecular characteristics. Three neurochemicals, neural cell adhesion molecule, neuron-specific enolase, and calcitonin gene-related peptide have been implicated in trophic phenomena, synaptogenesis and cell recognition in neurons and sensory neuroepithelia. The present study uses immunocytochemical localization of these molecular markers to characterize normal and denervated fungiform taste buds following unilateral chorda tympani/lingual nerve denervation in hamsters. In normal taste buds, immunoreactivity to neural cell adhesion molecule, neuron-specific enolase, and calcitonin gene-related peptide was present in a group of cells located centrally in the bud as well as in fungiform nerve fibres and endings. After denervation, gemmal cell immunoreactivity to all three markers was reduced and often confined to a single or a few bud cell(s). Also, fibre staining was absent except for sparse calcitonin gene-related peptide-immunoreactive fibres associated with blood vessels and within the fungiform papillae. These remaining fibres may be autonomic or somatomotor in origin. These results indicate that sensory denervation of hamster taste buds reduces, but does not wholly eliminate the immunoreactivity of surviving gemmal cells to neural cell adhesion molecule, neuron-specific enolase, and calcitonin gene-related peptide. While the number of taste bud cells expressing the markers appears to be nerve-dependent, immunoreactivity in sensory-denervated bud cells of hamster may reflect the influence of local tissue factors.

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. J. Comp. Neurol. 455:25–39, 2003.

Nucleus of the solitary tract in the C57BL/6J mouse: Subnuclear parcellation, chorda tympani nerve projections, and brainstem connections: Mouse NST atlas and 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. J. Comp. Neurol. 522:1565–1596, 2014.