Browsing by Subject "Olfaction"

Organisms are presented with a wide variety of environmental stimuli and must interpret and respond to these cues in to perform a wide variety of behaviors, such as foraging, mating, fleeing, and fighting. The ability of an organism to recognize various stimuli, such as pheromones, to identify mates or competitors through the activation of various circuits and molecular components in the brain is tightly regulated. In order to delineate how molecular changes occur in the brain during stimuli response we used Drosophila melanogaster as it has a well-defined nervous system. We focus in on the circuit which regulates sex-specific mating behaviors in male D. melanogaster. Sex-specific splicing regulates the expression of two genes known as fruitless (fruM) and doublesex (dsxM) in the courtship circuit. Here we demonstrate using in the fly olfactory system that Olfactory receptor 47b (Or47b) and Olfactory receptor 67d (Or67d) activity, through sensory experience, regulates the expression patterns of male-specific fruM through coincident activity of hormone binding transcription factors Gce and Met and histone acetyltransferase P300 activity. We also identify various genes which changes in various mutant and social contexts, including exon specific changes in fruitless transcripts as well as changes in the expression of hormone metabolism genes, and neuromodulators in antennae. Given these changes in neuromodulators and the known structure of the FruM and DsxM central circuits, we looked at changes in the chromatin state and expression levels and find changes in peripheral sensory neurons have downstream effects on higher order circuits. We identify that FruM regulates the chromatin structure of both itself and dsxM in whole brain lysates and that changes in chromatin structure depend on pheromone receptor and neurotransmitter activity across processing centers in the brain. Taken together, we identify potential candidates for future study, as well as lay the framework for understanding how sensory changes in the periphery have effects on various neuronal clusters in the brain.

Deciphering natural odor plumes with dynamic changes in odor concentrations presents a common challenge to all animals. A fundamental challenge in studying the organization principles of the olfactory system to encode odor concentration information is the lack of comprehensively identified sets of activated odorant receptors (ORs) across an odorant concentration range inside freely behaving animals. In mammals, this has recently become feasible with high-throughput sequencing-based methods that identify populations of odorant activated ORs in vivo. In this study, we characterized the mouse OR repertoires activated by two odorants, acetophenone (ACT) and 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), from 0.01% to 100% (v/v) concentrations. We also investigated the OR repertoires for structural derivatives of TMT (component of fox odor) such as 2­methyl-2-thiazoline (2MT) and 2,4,5-Trimethylthiazole (nTMT) and 2-sec-butyl-4,5-dihydrothiazole (SBT) for 1% and 100% (v/v) concentrations. We used a combination of in vivo, in situ and in silico approaches to investigate ORs with distinct sensitivities to the tested odorants. We examined Olfr923, which we identified to be one of the most sensitive ACT ORs based on our pS6-IP-Seq data. Using a mouse line that genetically labels Olfr923 positive axons, we provide evidence that ACT activates the Olfr923 glomerulus in the olfactory bulb. This study sheds light on the active process in which unique OR repertoires may collectively facilitate the discrimination of odorant concentrations. Together, these odorant receptors may shape the dynamic aspects of olfactory sensitivity and facilitate odorant intensity coding.

The external world is perceived via sensory receptors arranged in highly organized systems according to functional strategies, which in turn reflect features of critical importance to both the sense and the animal. The receptor organization and functional strategies of visual, auditory, and touch sensory systems have been mapped, but such a map for olfaction, the sense of smell, has remained elusive despite a concrete understanding of the fundamental principles of the system’s architecture.Sensory processing in olfactory systems is organized across olfactory bulb glomeruli, wherein axons of peripheral sensory neurons expressing the same olfactory receptor co-terminate to transmit receptor-specific activity to central neurons. Understanding how receptors map to glomeruli is therefore critical to understanding olfaction. High-throughput spatial transcriptomics is a rapidly advancing field, but low-abundance olfactory receptor expression within glomeruli has previously precluded high-throughput mapping of receptors to glomeruli. In Chapter 2, I describe the development and application of a novel method which combines sequential sectioning along the anteroposterior, dorsoventral, and mediolateral axes with target capture enrichment sequencing to overcome low-abundance target expression. This strategy spatially mapped 86% of olfactory receptors across the olfactory bulb and uncovered a relationship between olfactory receptor sequence and glomerular position.

Olfaction is one of the most primitive of our senses, and the olfactory receptors that mediate this very important chemical sense comprise the largest family of genes in the mammalian genome. It is therefore surprising that we understand so little of how olfactory receptors work. In particular we have a poor idea of what odorous chemicals are detected by most of the olfactory receptors in the genome, and for those receptors which we have paired with ligands, we know relatively little about how the structure of these ligands can either activate or inhibit the activation of these receptors. Furthermore the large repertoire of olfactory receptors, which belong to the G protein coupled receptor (GPCR) superfamily, can serve as a model to contribute to our broader understanding of GPCR- ligand binding, especially since GPCRs are important pharmaceutical targets.

In this dissertation, I explore the relationship between olfactory receptors and their ligands, both by manipulating the ligands presented to the olfactory receptors, as well as by altering the structure of the receptor itself by mutagenesis. Here we report the probable requirement of a hydrated germinal-diol form of octanal for activation of the rodent OR-I7 receptor by ligand manipulation, and the successful in vitro modeling and manipulation of ketamine binding to MOR136-1. We also report the results of a large-scale screen of 1190 human and mouse olfactory receptors for receptors activated by volatile general anesthetics, which has lead to the identification of 32 olfactory receptor-volatile general anesthetic pairs.

In mammals, the perception of smell starts with the activation of odorant receptors (ORs) by volatile molecules in the environment. Mammalian genomes typically encode large numbers of ORs, with approximately 400 intact ORs in human and more than 1000 in mouse. Central to the question of how olfactory stimuli are represented at the peripheral level is defining the ligand selectivity and activity regulation of ORs.

Processing of chemosensory signals in the brain is dynamically regulated in part by an animal’s physiological state. The Matsunami lab previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. In chapter 2, I demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system.

Understanding odor coding requires comprehensive mapping between odorant receptors and corresponding odorants. In chapter 3, I present a high-throughput in vivo method to identify repertoires of odorant receptors activated by odorants, using phosphorylated ribosome immunoprecipitation of mRNA from olfactory epithelium of odor-stimulated mice followed by RNA-Seq. This approach screens endogenously expressed odorant receptors against an odorant in one set of experiments, using awake and freely behaving mice. In combination with validations in a heterologous system, we identify sets of odorant receptors for two odorants, acetophenone and 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), encompassing 69 receptor-odorant pairs. I also identified shared amino acid residues specific to the acetophenone or TMT receptors, and developed a model to predict receptor activation. This study provides a means to understand the combinatorial coding of odors in vivo.

During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to

generate defined motor outputs, or behavior. This integration requires developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown.

Here we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs) involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless. During development, the putative chromatin modulator Alhambra (Alh) represses the expression of both fru and of specific olfactory receptors, helping to coordinate and establish both the sensory and circuit identities of the ORNs involved in sex-specific behaviors. In contrast, the maintenance of fru expression and thus the identities of

these ORNs in adults utilize signaling from olfactory receptors through Cam Kinases and the histone acetyl transferase p300/CBP. Our results highlight feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs.These mechanisms might underlie innate and adaptable aspects of odor-guided sex- specific behaviors.

Understanding how we detect our environment is crucial to understanding how life evolved and now functions. Volatile chemicals from our surroundings are sensed by our olfactory system, a primitive sense that organisms have relied on for survival for millions of years. Mammals express a large family of odorant receptor (OR) genes in the sensory neurons in the nose that mediate this chemosensation. Each mature olfactory sensory neuron (OSN) expresses a single allele of a single OR gene at one time although in the absence of a functional gene OSNs can switch to another OR gene. A functional OR can inhibit the expression of another OR by co-opting the unfolded protein response (UPR). How OSNs make their initial OR gene choice and the mechanisms by which the ORs interact with UPR factors remain unknown.

In this study, I make use of a double knock out mouse that has RTP1 and RTP2, proteins required for the efficient surface trafficking of ORs in heterologous cells, to study the gene regulation of ORs during a large-scale perturbation of the trafficking of ORs to the cell surface. We initially generate and validate the RTP1 and RTP2 double knock out mouse (RTP1,2DKO) and show that consistent with our heterologous expression system, the mutant mice have OR trafficking defects. These OR trafficking defects give rise to higher rates of cell death and the mutant mice have fewer mature OSNs. Surprisingly we identified a subset of ORs that were overrepresented in the RTP1,2DKO animals. Some of these ORs can target the cell surface in the absence of the RTPs. This finding gave rise to two cohorts of ORs, those that are underrepresented in the mutants and presumably dependent on the RTPs for cell surface trafficking and ORs that are overrepresented in RTP1,2DKO. We show that OSNs expressing underrepresented receptors were more likely to be unable to terminate UPR had a higher tendency to switch the OR it was expressing. Using these two cohorts we showed that the trafficking of ORs to the cell surface is a crucial step in the stabilization of the expression of the OR. In the absence of this cell surface trafficking the OSN is unable to terminate the UPR pathway and either undergoes cell death or OR gene switching.