Author ORCID Identifier

0009-0004-3853-3934

Date of Award

12-2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Neuroscience Institute

First Advisor

Daniel Cox

Second Advisor

Charles Derby

Third Advisor

Shawn Dotson

Fourth Advisor

Jordan Hamm

Abstract

Sensory systems are essential for the transduction of external stimuli into internal information that results in stimulus-specific behavioral output. The ability to detect and respond to potentially harmful environmental conditions is crucial for organismal survival. Thermosensory nociception, the process of detecting noxious thermal stimuli, supports adaptive behavioral responses, yet the molecular mechanisms underlying the transduction of these stimuli, particularly noxious cold detection, remains incompletely understood. Previous studies identified Class III multidendritic (CIII md) sensory neurons as the primary cold nociceptors in Drosophila larvae demonstrating that these neurons are both necessary and sufficient to elicit cold-evoked behavior.

CIII md neuron-specific transcriptomic profiling and immunohistochemical analyses identified promising candidate genes among canonical olfactory chemoreceptors, specifically odorant receptors (ORs). Targeted genetic mutations and RNAi-induced knockdown of ORs enriched in CIII neurons implicated Or65a and its co-receptor Orco in mediating cold nociception. Mechanistic assays assessing cold-evoked electrical activity and calcium dynamics demonstrated that Or65a or Orco knockdown reduced cold-evoked CIII calcium transients and neural activity shifting the firing pattern from bursting to tonic spiking. Importantly, these OR mutations did not affect the CIII-mediated gentle touch behavior, locomotion, or dendritic morphology, emphasizing their specific involvement in cold detection. Further, CIII-specific Orco expression in a whole larva Orco null mutant background rescued cold-evoked behavioral deficits demonstrating a specific requirement in these neurons.

In olfaction, serine residue 289 (S289) has been identified as a critical phosphorylation site leading to modulation of Orco-mediated activity. By expressing phospho-resistant (S289A) or phospho-mimetic (S289D) point mutations of the S289 residue, we could alter the phosphorylation state of Orco, which is reported to exhibit increased conductance via enhanced calcium permeability in while phosphorylated. We found that expression of the OrcoS289A phospho-resistant structure-function mutation decreased cold-evoked behavior implicating this residue and post-translational modification as a regulatory target for cold nociceptive behavior.

These discoveries are the first to demonstrate a non-canonical role of ORs in thermal nociception. Further, this research situates ORs with the closely related multimodal gustatory receptors (GRs) and ionotropic receptors (IRs), which have likewise been implicated in thermosensation, revealing functional roles that extend beyond labeled-line chemosensation.

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