Doctor of Philosophy in Zoology (PhD)
Taste processing in Drosophila melanogaster
Most projects centre on the neural circuits underlying taste processing and feeding in the fly brain. Potential projects include looking at circuit mechanisms underlying taste integration, hunger, feeding, taste memories, chemosensory integration, and taste circuit development.
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To survive animals must find and consume nutritive foods. The chemical composition of food sources is evaluated using gustation. Because of its importance to survival, feeding behaviors are tightly regulated. Changes in feeding occur in response to the external environment and the animal’s internal physiological state. Expendable behaviours can be suppressed during starvation to prioritize feeding. This thesis examines the role of two factors in feeding decisions: the location of taste input and starvation. Pharyngeal sense organs in Drosophila are the last evaluation point before food is ingested. It was previously unclear whether they served a unique function in feeding. To investigate this, we focused on appetitive gustatory pharyngeal neurons and showed they express nine gustatory receptors which respond to sweet compounds. Mutants lacking peripheral taste have functional pharyngeal sense organs. In the absence of peripheral taste cues, the pharyngeal sense organs can drive the choice and ingestion of sweet compounds by sustaining consumption. Knocking down pharyngeal neurons in these mutants allowed us to examine a sweet-blind fly in a short term feeding assay. Putatively sweet-blind flies do not show a preference for sweet compounds in a short-term feeding assay, suggesting that nutrient sensing is not operating in this context. Starved flies have an increased tolerance for bitter foods, which is mediated by sensitization and desensitization of sweet and bitter taste, respectively. Mechanisms that cause the sensitization of sweet taste have been studied, but those that underlie desensitization of bitter taste were unknown. We identify a pair of octopaminergic/tyraminergic modulatory neurons called the ventrolateral cluster of octopaminergic neurons (OA-VLs). Because OA-VLs exist in close physical proximity to bitter neuron axon terminals but are not postsynaptic to them, we examined their function as modulators of bitter neuron output. Tonic firing rate of OA-VLs decreases as a function of starvation. Octopamine and tyramine are sufficient to potentiate bitter neuron response in starved flies, suggesting that reduction in OA-VL activity during starvation depotentiates bitter neuron output. Silencing OA-VLs causes a reduction in bitter neuron output and increased acceptance of bitter compounds. OA-VLs may act directly on bitter sensory neurons through the Oct-Tyr receptor.
The ability to detect various tastes greatly enhances survivorship. The detection of nutritious or toxic foods leads to a promotion or inhibition of feeding. Although the characterization of taste-detection at the periphery is extensive, their connection to second-order taste neurons is only beginning to be elucidated. In Drosophila melanogaster, we can harness powerful genetic tools to help map out the neuronal circuitry that translates the sensory inputs of taste to the motor commands of feeding. Our objective is to identify higher-order neurons that bridge this sensorimotor circuitry. Understanding how taste information is processed in fruit flies may shed light on how its mammalian counterparts do so, as well. To accomplish this, we exploit the Sip-Triggered Optogenetic Behaviour Enclosure (STROBE) for its high-efficiency and biological relevance to screen for potential taste neurons. In total, 123 driver lines were selected and screened through optogenetic neuronal activation. One line, in particular, R70C07-GAL4, was chosen for the further characterization of its role in feeding inhibition. It predominantly labels two clusters of cell bodies that are bilateral to the brain’s primary taste center, the subesophageal zone (SEZ). It is predicted to be responsible for aversive feeding behaviour. To test this, we created split-GAL4 lines to narrow down this population before GRASP and additional optogenetic activation experiments were performed to confirm the identity of neurons that are responsible for altering feeding behaviour. Upon activation, one subset of the lateral SEZ population was revealed to induce significantly aversive feeding behaviour, while another induced appetitive feeding. Surprisingly, both subsets show a positive GRASP signal with both sweet and bitter gustatory receptor neurons (GRNs). The evidence presented here demonstrates that clear feeding preferences can be made even by activating a population of neurons that communicates with GRNs of opposing valence. This raises the question of whether feeding decisions are instigated by the relative activity between pathways of opposing taste valences, and if this ratio of activity is already encoded at the level of second-order taste neurons, possibly challenging the extension of the labelled-lines theory into higher-order neurons.
Although feeding behavior is a matter of life and death for animals, the genetic factors that control it remain poorly understood. We have identified a novel regulator of hunger-induced behavior through comparison of transcriptomic changes in the fruit fly Drosophila melanogaster and the yellow fever mosquito Aedes aegypti. Head mRNA from each insect was sequenced at a roughly equivalent level of starvation. Using data gleaned from the protein orthology database OrthoDB, we looked for gene pairs in which both A. aegypti and D. melanogaster orthologs were significantly regulated by starvation. This identified Juvenile Hormone esterase (Jhe) as a possible modulator of hunger-induced behavior. Pan-neuronal knockdown of Jhe resulted in increased food consumption and caused enhancement of starvation-induced sleep suppression in Drosophila. These behavioral phenotypes were not caused by a developmental or metabolic defects, and were reproduced by feeding adult Drosophila methoprene, a synthetic Juvenile Hormone analog. Application of precocene I, an inhibitor of Juvenile Hormone biosynthesis, reversed the phenotype. Our analysis suggests that Jhe (and Juvenile Hormone by extension) is a novel and biologically relevant regulator of hunger-induced behavior.
The sense of taste plays a critical role in animal behaviour. The ability to taste prior to ingestion allows an animal to differentiate between beneficial substances, such as calorie-rich foods, from those that may be toxic and which often taste bitter. For animals to avoid harmful substances, exposure to bitter substances must not only activate bitter-sensing neurons, but also suppress the response of acceptance-mediating neurons. The ability to choose appropriately between consumption and avoidance requires functional neuronal circuits in the brain that start with the processing of taste input from the environment and end with a behavioural output. γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the insect and mammalian brain. We show evidence of functional connections between GABAergic interneurons (those that release GABA) and taste sensory neurons in the fly brain. Neurons that detect palatable substances (i.e. sugars) express high levels of a metabotropic GABAB receptor (GABABR), whereas those that sense unpalatable substances (i.e. bitter compounds) express little to no GABABR. Using a behavioural assay (proboscis extension reflex) and calcium imaging, I investigated how GABAergic inhibition shapes sweet neuron output and contributes to the integration of competitive taste stimuli. When GABABR is knocked down in sugar neurons, the behavioural response to sugars is elevated. Pharmacological assays show that GABAergic activation suppresses the sweet neuron response while GABAergic blockade potentiates the response. In flies expressing GABABR knockdown, suppression of sugar neuron activity by bitter exposure is relieved both behaviourally, by increased acceptance of bitter mixtures, and cellularly, by increased calcium response. Our model proposes that GABA acts via GABABR in the fly taste circuit to produce ecologically relevant responses to both attractive and repellent energy sources.