Browsing by Author "Matsunami, Hiroaki"

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, the sense of smell, is one of the least well understood sensory systems. Central to the lack of understanding has been an inability to reliably control the stimulus, odorants. Here I developed analytical frameworks to further our understandings of how to quantify odor molecules based on responses elicited from olfactory receptors. I also identified a novel subset of olfactory receptors which displayed sexual dimorphism, plasticity, and served to respond to semiochemicals in mice. Finally, I identified a single candidate rabbit olfactory receptor to respond to a rabbit mammary pheromone.

The song system of the zebra finch (Taeniopygia guttata) is highly sexually dimorphic, where only males develop the neural structures necessary to learn and produce learned vocalizations in adulthood. During early development, both males and females begin to develop their song system in a monomorphic manner, which diverges shortly after the onset of a critical sensory learning phase and results in reduced cell survival and proliferation in females, and accelerated cell proliferation in males. Estrogen has long been known to be involved in coordinating sexual development of the perinatal brain and nestling female zebra finches treated with estrogen do not exhibit this female-specific atrophy of the song system. How estrogen influences the development of the song system, and what it is doing at the molecular level has not been examined utilizing current generation sequencing technology.

In this dissertation, I tested whether estrogen manipulation impacts the transcriptomic profiles of telencephalic song learning nuclei in males and females. I treated animals with either vehicle, exemestane (an estrogen synthesis inhibitor), or 17-β-estradiol from the moment of hatching until time of sacrifice. I collected the song learning nuclei and their surrounding brain regions during the onset of sensory motor learning for transcriptomic analysis or during adulthood after collecting behavior. I found that of the 4 telencephalic song nuclei examined during the onset of the sensorimotor learning period at post hatch day 30, Area X was the most sexually dimorphic and the most impacted by estrogen administration. HVC was less sexually dimorphic and less impacted by estrogen manipulation. RA and LMAN had limited sexually dimorphic features, with little impact on their transcriptomes with estrogen manipulation. Additionally, I found that chronic estrogen depletion in males delayed male specific plumage development and resulted in impaired song learning. This supports the notion that while estrogen is sufficient in preventing atrophy of the song system in female zebra finches, it is not necessary for the gross development in males and may instead refine normal song development.

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.

The chemical cues that provide an olfactory portrait of mammalian individuals are in part detected by chemosensory receptors in the vomeronasal organ (VNO). By and large, the pertinent receptor-cue combinations used for olfactory communication are unidentified. Here we identify members of the formyl peptide receptor (FPR) family of G protein coupled receptors as candidate chemosensory receptors in the VNO of mice. We demonstrate that N-formylated mitochondrially encoded peptides presented by the major histocompatibility complex (MHC) molecule H2-M3 stimulate a subset of the VNO sensory neurons (VSNs). We show that one VNO localized FPR, Fpr-rs1, is differentially activated by strain specific variants of N-formylated peptides. We show that N-formylated peptides can function as chemosignals in a strain selective pregnancy block. We propose that this link between self-recognition peptides of the immune system and chemosensory pathways provides a possible molecular means to communicate the nature of an individual's maternal lineage or strain.

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.

Pheromones are chemicals from conspecifics that affect innate behavior or hormonal changes. In mammals, the vomeronasal organ (VNO) is thought to play a prominent role in detecting pheromones; the vomeronasal sensory neurons (VSNs) express three families of seven-transmembrane G-protein coupled receptors (GPCRs): the V1Rs, V2Rs, and FPRs, in two molecularly and spatially-distinct regions. In mice, VSNs that express the V2Rs are thought to detect peptide cues, including MHC-presenting peptides, major urinary proteins (MUPs), and exocrine gland-secreting peptides (ESPs). They are thought to be involved in various pheromone-mediated behaviors and physiological changes, such as mating, aggression, and selective pregnancy block. In order to understand how pheromones are detected by the vomeronasal receptors, it is essential to know which receptors are activated by a given chemical. However, identifying cognate ligands for the V2Rs has been challenging, partly because they are poorly localized to the surface of heterologous cells. Here, we show that the calreticulin chaperone family members play a crucial role in trafficking V2Rs. A calreticulin homologue, calreticulin4 is specifically expressed in the VNO, while calreticulin expression level is low. Depleting calreticulin expression in HEK293T cells allows V2Rs to be trafficked to the cell surface, whereas expression of calreticulin4 does not block the trafficking of the V2Rs. Using this knowledge, we have established a heterologous cell system to functionally identify the V2Rs and demonstrate that the ESP family members can differentially activate the V2Rs. We also show the large extracellular domain of the V2Rs plays a crucial role in ligand selectivity. Our results provide a platform to characterize ligand selectivity of the V2Rs and suggest that a unique mechanism involving calreticulins regulates the functional expression of the V2Rs.

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.

Olfactory sensory neurons (OSNs), which detect a myriad of odorants, are known to express one allele of one olfactory receptor (OR) gene (Olfr) from the largest gene family in the mammalian genome. The OSNs expressing the same OR project their axons to the main olfactory bulb where they converge to form glomeruli. This “One neuron-one receptor rule” makes the olfactory epithelium (OE), which consists of a vast number of OSNs expressing unique ORs, one of the most heterogeneous cell populations. However, the mechanism of how the single OR allele is chosen remains unclear along with the question of whether one OSN only expresses a single OR gene, a hypothesis that has not been rigorously verified while we performed the experiments. Moreover, failure of axonal targeting to single glomerulus was observed in MeCP2 deficient OSNs where delayed development was proposed as an explanation for the phenotype. How Mecp2 mutation caused this aberrant targeting is not entirely understood.

In this dissertation, we explored the transcriptomes of single and mature OSNs by single-cell RNA-Seq to reveal their heterogeneity and further studied the OR gene expression from these isolated OSNs. The singularity of sequenced OSNs was ensured by the observation of monoallelic expression of X-linked genes from the hybrid samples from crosses between mice of different strains where strain-specific polymorphisms could be used to track the allelic origins of SNP-containing reads. The clustering of expression profiles from triplicates that originated from the same cell assured that the transcriptomic identities of OSNs were maintained through the experimental process. The average gene expression profiles of sequenced OSNs correlated well to the conventional transcriptome data of FACS-sorted Omp-positive cells, and the top-ranked expression of OR was conceded in the single-OSN transcriptomes. While exploring cellular diversity, in addition to OR genes, we revealed nearly 200 differentially expressed genes among the sequenced OSNs in this study. Among the 36 sequenced OSNs, eight cells (22.2%) showed multiple OR gene expression and the presences of additional ORs were not restricted to the neighbor loci that shared the transcriptional effect of the primary OR expression, suggesting that the “One neuron-one receptor rule” might not be strictly true at the transcription level. All of the inferable ORs, including additional co-expressed ORs, were shown to be monoallelic. Our sequencing of 21 Mecp2308 mutant OSNs, of which 62% expressed more than one OR genes, and the expression levels of the additional ORs were significantly higher than those in the wild-type, suggested that MeCP2 plays a role in the regulation of singular OR gene expression. Dual label in situ hybridization along with the sequence data revealed that dorsal and ventral ORs were co-expressed in the same Mecp2 mutant OSN, further implying that MeCP2 might be involved in regulation of OR territories in the OE. Our results suggested a new role of MeCP2 in OR gene choice and ratified that this multiple-OR expression caused by Mecp2 mutation did not accompany delayed OSN development that has been observed in the previous studies on the Mecp2 mutants.

The carotid body (CB) is a major arterial chemoreceptor containing glomus cells that are activated by changes in arterial blood contents including oxygen. Despite significant advancement in the characterization of their physiological properties, our understanding on the underlying molecular machinery and signaling pathway in CB glomus cells is still limited.

To overcome these limitations, in chapter 1, I demonstrated the first transcriptome profile of CB glomus cells using single cell sequencing technology, which allowed us to uncover a set of abundantly expressed genes, including novel glomus cell-specific transcripts. These results revealed involvement of G protein-coupled receptor (GPCR) signaling pathway, various types of ion channels, as well as atypical mitochondrial subunits in CB function. I also identified ligands for the mostly highly expressed GPCR (Olfr78) in CB glomus cells and examined this receptor’s role in CB mediated hypoxic ventilatory response.

Current knowledge of CB suggest glomus cells rely on unusual mitochondria for their sensitivity to hypoxia. I previously identified the atypical mitochondrial subunit Ndufa4l2 as a highly over-represented gene in CB glomus cells. In chapter 2, to investigate the functional significance of Ndufa4l2 in CB function, I phenotyped both Ndufa4l2 knockout mice and mice with conditional Ndufa4l2 deletion in CB glomus cells. I found that Ndufa4l2 is essential to the establishment of regular breathing after birth. Ablating Ndufa4l2 in postnatal CB glomus cells resulted in defective CB sensitivity to hypoxia as well as CB mediated hypoxic ventilatory response. Together, our data showed that Ndufa4l2 is critical to respiratory control and the oxygen sensitivity of CB glomus cells.