Neurotrophins BDNF and NT4 influence the development of the rodent gustatory system. Despite binding to the same receptor, TrkB, they have different roles. BDNF is chemo-attractive for gustatory neurons and regulates gustatory neuron targeting and number during development. NT4 regulates gustatory neuron number earlier in development than BDNF, but it is not chemo-attractive and does not regulate gustatory neuron targeting. To elucidate the mechanisms that regulate these processes we have examined which TrkB intracellular signalling pathways are required for specific aspects of gustatory development by studying the effect of specific point mutations in TrkB docking sites. We found that the TrkB/Shc docking site is involved in regulating the survival of geniculate ganglion neurons as a point mutation in this adaptor site (TrkbS/S) caused large losses of these neurons as early as E12.5. These losses were exacerbated throughout development until after birth. A point mutation in the TrkB/PLCγ (TrkbP/P) docking site did not cause loss of geniculate ganglion neurons at any point during development. Animals with a point mutation in both docking sites (TrkbD/D) caused a further decrease in neuron numbers compared to animals with a mutation in only one of the docking sites, similarly to what has previously been shown in Trkb null animals. We concluded that the TrkB/Shc docking site is crucial for determining the survival of geniculate ganglion neurons during mouse gustatory development, while the TrkB/PLCγ docking site does not affect the neuronal survival directly and likely plays a role in maintenance of these neurons. Examining the targeting of geniculate ganglion afferents into the tongue revealed large deficits in innervated neural bud and taste bud numbers in TrkbS/S animals both before and after birth. This was concluded to be reflecting the lack of neuronal survival in this ganglion, a result that was mirrored in TrkbD/D animals. TrkbP/P animals, on the other hand, exhibited a developmental delay in innervation. This was indicated by a low amount of innervated neural buds following the initial innervation period, which was compensated for by a large increase in the number of innervated taste buds by birth. By adulthood, the numbers of taste buds present on the tongues of TrkbP/P animals reached normal numbers compared to control animals. This suggested that the TrkB/PLCγ docking site is involved primarily in innervation. Finally, we examined the morphology of taste buds in newly born and adult animals. We found that the low amount of geniculate ganglion afferents innervating the tongue in TrkbS/S and TrkbD/D animals caused a decrease in size of taste buds. This effect was seen to be partially rescued by adulthood in TrkbS/S animals but not in TrkbD/D animals due to lack of viability. The morphology of taste buds was unaffected in TrkbP/P animals until adulthood, at which point the size of the taste buds was increased. These results are in agreement with previous findings showing dependency of taste bud morphology on the amount of innervation. Overall, our findings show a differential role of TrkB adaptor sites in gustatory development. Despite activated by the same ligands, the docking sites on this receptor are able to exert different influence on signalling pathways downstream of TrkB affecting neuronal survival, targeting and morphology of taste buds.

Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. Using the Gal4/UAS system, we have characterized the expression of Gr genes in detail, and find that gene expression falls into two distinct groups. The first group, which make up the majority of the genes analyzed, are co-expressed with Gr66a, and functional ablation of taste neurons expressing these genes demonstrates that these neurons mediate sensitivity to bitter substrates. A second, distinct group of taste neurons, express Gr5a and mediate sensitivity to trehalose. We find that these two sub-populations of taste neurons - attractive-type and avoidance-type - project afferents to discrete areas of the primary taste center in the CNS. These results demonstrate how bitter and sweet taste are coded for in the periphery and indicates that information about different taste modalities is initially segregated in the CNS. We have also used the Gal4/UAS system to thoroughly characterize the expression profile of a cluster of six Grs - Gr28a and Gr28b.a-b.e. We find that these receptors are expressed not only in taste neurons, but other sensory neurons, as well as neurons in the CNS. RNA in situ hybridization confirms this unusual expression pattern. In order to explore the function of these Grs, these genes have been deleted using ends-out homologous recombination, to produce Gr28 mutant flies. Initial behavioral experiments with Gr28 mutant flies suggest that these receptors may play a role in detecting aversive substrates and/or modulate avoidance responses to these substrates. / Dissertation

Philosophiae Doctor - PhD / Tsetse flies are the sole vectors of trypanosomes that cause nagana and sleeping sickness in animals and humans respectively in tropical Africa. Tsetse are unique: both sexes adults are exclusive blood-feeders, females are mated young and give birth to a single mature larva in sheltered habitats per pregnancy. Tsetse use chemoreception to detect and respond to chemical stimuli, helping them to locate hosts, mates, larviposition and resting sites. The detection is facilitated by chemoreceptors expressed on sensory neurons to cause specific responses. Specific molecular factors that mediate these responses are poorly understood in tsetse flies. This study aimed to identify and characterize genes that potentially mediate chemoreception in Glossina morsitans morsitans tsetse flies. These genes included sensory odorant (OR), gustatory (GR), ionotropic (IR), and related genes for odorant-binding (OBP), chemosensory (CSP) and sensory neuron membrane (SNMP) proteins. Synaptic transmission in higher brain sites may involve ionotropic glutamate-gated (iGluR) and metabotropic glutamate-gated (mGluR) receptors. The genes were annotated in G. m. morsitans genome scaffold assembly GMOY1.1 Yale strain using orthologs from D. melanogaster as query via TBLASTX algorithm at e-value below 1e-03. Positive blast hits were seeded as gene constructs in their respective scaffolds, and used as genomic reference onto which female fly-derived RNA sequence reads were mapped using CLC Genomics workbench suite. Seeded gene models were modified using RNA-Seq reads then viewed and re-edited using Artemis genome viewer tool. The genome was iteratively searched using the G. m. morsitans gene model sequences to recover additional similar hit sequences. The gene models were confirmed through comparisons against the NCBI conserved domains database (CDD) and non-redundant Swiss-Prot database. Trans-membrane domains and secretory peptides were predicted using TMHMM and SignalP tools respectively. Putative functions of the genes were confirmed via Blast2GO searches against gene ontology database. Evolutionary relationships amongst and between the genes were established using maximum likelihood estimates using best fitting amino acid model test in MEGA5 suite and PhyML tool. Expression profiles of genes were estimated using the RNA-seq data via CLCGenomics RNA-sequences analysis pipeline. Overall, 46 ORs, 14 GRs, and 19 IRs were identified, of which 21, 6 and 4 were manually identified for ORs, GRs, and IRs respectively. Additionally, 15 iGluRs, 6 mGluRs, 5 CSPs, 15 CD36-like, and 32 OBPs were identified. Six copies of OR genes (GmmOR41-46) were homologous to DmelOr67d, a single copy cis vacenyl acetate (cVA) receptor . Genes whose receptor homologs are associated with responses to CO2, GmmGR1-4, had higher expression profiles from amongst glossina GR genes. Known core-receptor homologs OR1, IR8a, IR25a and IR64a were conserved, and three species-specific divergent IRs (IR10a, IR56b and IR56d) were identified. Homologs of GluRIID, IR93a, and sweet taste receptors (Gr5a and Gr64a) were not identified in the genome. Homolog for LUSH protein, GmmOBP26, and sensory neuron membrane receptors SNMP1 and SNMP2 were conserved in the genome. Results indicate reduced repertoire of the chemosensory genes, and suggest reduced host range of the tsetse flies compared to other Diptera. Genes in multiple copies suggest their prioritization in chemoreception, which in turn may be tied to high specificity in host selection. Genes with high sequence conservation and expression profiles probably relate to their broad expression and utility within the fly nervous system. These results lay foundation for future comparative studies with other insects, provide opportunities for functional studies, and form the basis for re-examining new approaches for improving tsetse control tools and possible drug targets based on chemoreception.

The distribution of neurons in the rostral nucleus of the solitary tract (rNST) that respond to gustatory input from the anterior tongue was visualized by Fos protein immunohistochemistry following electrical stimulation of the chorda tympani (CT) nerve in rats. Maps of Fos-immunoreactive (Fos-ir) neurons were compared with the distribution of CT afferent terminal fields labeled by transganglionic transport of rhodamine-dextran in a separate group of animals. The primary concentration of Fos-ir neurons localized in register with the major terminal fields of CT afferent fibers, in the central third of the rostral 1.0 mm of the NST ipsilateral to the stimulated nerve. A similar correspondence in location and degree of labeling of Fos-ir neurons and afferent terminals was observed in the ipsilateral dorsal spinal trigeminal complex (Sp5) pars caudalis, near the obex, and the Sp5 pars oralis near the rostral pole of the rNST. Thus, the magnitude of Fos upregulation in brainstem targets of the CT nerve having chemosensory or nociceptive function, was proportional to the relative density of the CT afferent input. This correspondence, and the absence of labeling in neurons known to be one additional synapse away from the afferent input within gustatory or oral reflex pathways, suggests that the cell map obtained represents mainly neurons that are directly activated via primary afferent synapses from CT fibers. The availability of a method to histochemically identify a population of putative second-order taste neurons will facilitate analysis of the cellular/molecular properties of these neurons and of synaptic circuitry in the rNST.

While equine palatability research has been well documented, the majority of research has been conducted in mature, non-lactating horses (Betz & Lanter, 1980; Van den berg et al., 2016; Goodwin et al., 2005; Francis et al., 2020). While palatability research primarily aims to detect flavor preferences to mask medications, utilize in treats, etc., other aspects focus on acceptance and behavior (Senturklu & Landblom, 2015; Goodwin et al., 2005; Van den berg et al., 2016; Franics et al., 2020). Although a substantial amount of data is conducted on canine olfaction, data pertaining to equine olfaction is quite limited (Francis et al., 2020; Jenkins et al., 2018; Kokocinska-Kusiak et al., 2021). The objective of this study was to investigate the flavor and aroma preferences of lactating equine mare and foal pairings. Stock-type mare and foal pairings (n = 4), aged 6.5 ± 3.9 weeks, were utilized in paired preference testing using apple, anise, peppermint and control palatants. Testing was split into flavor testing (0730-0830 hours) and aroma testing (1630-1730 hours). Mares and foals were loaded into test stalls and allotted 30 minutes to freely interact with treatments. Data was recorded via live stream cameras and evaluated for categorical and numerical data. Variables of interest included: first treatments investigated, consumed, and finished; maternal effects of treatment locations, time spent consuming each treatment, time spent rest recumbent, percentage of treatments consumed, and total voluntary consumption. Data were analyzed using SAS for academics with significance set at 5%. Flavor analysis revealed no preference of first flavor investigated (P = 0.4918), consumed (P = 0.4018) or finished (P = 0.5424) of mares. Foals were also found to exhibit no flavor preferences for first investigated (P = 0.5996), consumed (P = 0.4278), or finished (P = 0.9914). A location effect was exhibited by mares and found that flavor treatments in location 2 and 3 were more likely to be first consumed (P < 0.0001). Foals were more likely to first finish those flavor treatments in location 1 (P = 0.0003). No period effect was exhibited for flavors first investigated (P =0.7552), consumed (P = 0.5404) or finished (P = 0.7903). A period effect was exhibited for first finished flavor treatments of mares (P = 0.0170). Further analysis found that mares were more likely to finish the peppermint flavor for periods 1 and 2 compared to periods 3 and 4 (P = 0.0518). The mean time spent foals spent consuming flavors was not significant (P = 0.2120) and no period effects were exhibited (P = 0.1713). However, time spent resting decreased significantly from period 1 to 4 (P = 0.0476). Likewise, there was no flavor treatment effect on the mean percentage of treatments consumed (P = 0.8431), nor was there a period effect (P = 0.4125). Aroma analysis also found no preference for first investigated (P = 0.8472), consumed (P = 0.8472), or finished (P = 0.8166) of mares. Similarly, foals exhibited no aroma preferences for first investigated (P = 0.5206), consumed (P = 0.5206), or finished (P = 0.1650). A period effect was exhibited for mares for first aroma treatments investigated (P = 0.0101) and consumed (P = 0.01) across periods. However, further analysis found that there was no difference for treatments across periods. A location effect was also exhibited by mares, with location 1 being selected at a lower frequency than locations 2, 3 and 4 (P = 0.0003). Interestingly, locations 3 and 4 had a higher frequency of being the first aromatic treatments finished (P = 0.0008). Foals were found to first consume (P = 0.0315), and finish (P = 0.0013), those treatments located in location 1. Comparative analysis of relationships between mare and foal selective actions found that pairings made similar flavor selections for first investigated treatments (P = 0.1865) but selected different aromatic treatments (P = 0.0415). Likewise, pairings selected similar flavor treatments (P = 0.1062) but selected different aromatic treatments for first consumption (P = 0.0415). Findings suggest that while flavor and aromatic preferences are not strong for mare and foal pairings. However, comparative analysis of selections between the two groups found selections to be similar for flavor testing and different for aromatic testing.

Most studies of gustatory coding have been performed in either anesthetized or awake, passively stimulated rats. In this dissertation the influences of behavioral state on gustatory processing in awake rats are described. In the first set of experiments, the effects of non-contingent tastant delivery on the chemical tuning of single neurons were explored. Tastants were delivered non-contingently through intra-oral cannulas to restrained, non water-deprived rats while single unit responses were recorded from the gustatory cortex (GC). As the subjects' behavior progressed from acceptance to rejection of the tastants, the chemical tuning of the neurons changed as well. This suggests that the subjects' behavioral state powerfully influences gustatory processing. In the second set of experiments, rats were trained to lick for fluid reinforcement on an FR5 schedule while single unit activity was recorded from GC. In this case, the chemical tuning was much more stable. Under this paradigm, chemosensory responses were rapid (~ 150 ms) and broadly tuned. In the third study, it was found that ensembles of GC neurons could discriminate between tastants and their concentrations on a single trial basis, and such discrimination was accomplished with a combination of rate and temporal coding. Ensembles of GC neurons also anticipated the identity of the upcoming stimulus when the tastant delivery was predictable. Finally, it was found that ensembles of GC neurons could discriminate between the bitter stimuli nicotine and quinine. Nicotine is both a bitter tastant and a trigeminal stimulant, and when the acetylcholine receptors in the lingual epithelium were blocked with mecamylamine, the ensembles failed to discriminate nicotine from quinine.

<p>Gustation, also known as taste perception, is critical for the survival of most animal species. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of sugars, bitter or toxic compounds, and pheromones. However, with a few notable exceptions, the functions of most GRs involved in feeding are unknown. Our research has focused on a cluster of highly-related Drosophila Grs, known as the Gr64 family, that have been shown to be critical for the perception of multiple sugars. Furthermore, we have demonstrated that another gene related to the Gr64 genes, Gr61a, is a sugar receptor that is narrowly tuned to a subset of pyranose sugars and may (along with the Gr64 genes) be indispensable for early fly development. </p><p>As a complementary approach to our behavioral analysis, we have examined the expression pattern of the Drosophila sugar receptors using knock-in driver alleles created by homologous recombination. As expected, most of these drivers have shown strong expression in various taste tissues. Intriguingly, some of these knock-in alleles also show expression in the maxillary palp and antenna, tissues previously thought to be involved only in olfaction. These expression patterns raise interesting questions about the true range of function of these chemosensory receptors and whether or not they might be involved in olfaction as well as gustation.</p> / Dissertation

Among humans, the density of lingual fungiform papillae (DFP) is correlated with taste sensitivity. The purpose of this dissertation was to investigate the evolution of the primate gustatory system through a comparative analysis of DFP. This investigation was conducted in three separate studies. The first study took a broad perspective incorporating data from 37 primate species to assess the relationships among DFP, body mass, taste sensitivity, and diet. Among the major findings of this first study: (1) Sucrose sensitivity was negatively correlated with DFP and positively correlated with papilla area. (2) Sucrose sensitivity was not correlated with the percent of leaves or fruit in the diet. (3) DFP and papilla area were correlated with diet. (4) The relationships between fungiform papillae and diet differed among different taxonomic groups. The second study of DFP investigated whether there are sex differences in the DFP of non-human primates, as there are in humans. In all five primate species investigated, females had higher mean DFPs than males. These sex differences were significant in Pan troglodytes and Cebus apella, and not significant in Alouatta palliata, Cercopithecus aethiops, or Varecia variegata. Pan, Cebus, and Homo share large relative brain sizes with associated life history parameters making each offspring very costly. Accordingly it was suggested that sex differences in DFP may be due to the particularly high risk of lacking nutrients or ingesting toxins for females of these three species. The third study was a comparison of phenylthiocarbamide (PTC) taste ability and DFP in humans and chimpanzees. The major questions addressed in this study were (1) Is DFP correlated with PTC phenotype in chimpanzees as it is in humans? (2) Are there sex differences in PTC genotype and phenotype as there are in DFP? Although females had greater DFPs than males, and significantly more females had the genotype for higher PTC taste sensitivity, there was no correlation between DFP and PTC phenotype. Several explanations for the differences between human and chimpanzee results were offered, including small sample sizes for chimpanzees and greater accuracy in determining PTC sensitivity among humans. / text

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