Weihong Lin, Ph.D - US grants

The focus of this proposal is to study the mechanisms underlying inhibitory olfactory responses in mouse olfactory receptor neurons (ORNs). Specific aims are: (1) to determine whether Ca2+-activated Kplus channels mediate odor-induced inhibitory responses; (2) to determine if heptaldehyde elicits exclusively excitatory or inhibitory responses in ORNs of transgenic mice (POMP-I7-IRES-RFP) where odor receptor I7 is expressed in mature ORNs. Odor- induced inhibitory responses and their dependence on K+ and intracellular Ca2+ will be examined utilizing a combination of Ca2+ imaging and perforated patch-clamp recording. The transgenic mice will be generated by injection of a cDNA construct of POMP-I7-IRES-RFP into fertilized mouse oocytes. The proximal 0.9 Kb of the olfactory marker protein (OMP) promoter will drive expression of the mouse I7 odor receptor. An internal ribosomal entry site (IRES) will be used to create a bicistronic mRNA encoding for I7 odor receptor and red fluorescent protein (RFP; Clontech). Since I7 responds to heptaldehyde specifically, this will result in a mouse with a large percentage of neurons responding to a specific odor, heptaldehyde. This mouse will be used to more stringently study mechanisms involved in inhibitory responses and the interaction between excitatory and inhibitory pathways in the same cells. Results gained from this study will expand our knowledge of odor detection and olfactory coding.

DESCRIPTION (provided by applicant): Mammalian olfaction encompasses two parallel signal-processing systems. The main olfactory epithelium (MOE) detects airborne odorants via the cAMP-signaling pathway. The vomeronasal organ (VNO) detects pheromones via phospholipase C (PLC)-dependent activation of TRP2 channels. However, pheromone detection is not exclusively mediated by the VNO. We have shown unexpectedly that pheromone 2-heptanone and 2,5-dimethylpyrazine (DMP) evoked field potentials in the MOE of cyclic nucleotide-gated channel subunit A2 knockout (CNGA2 KO) mice with a disrupted cAMP pathway, consistent with behavioral detection. When tested in wild type mice, these pheromone-induced responses in MOE were significantly less sensitive to inhibitors of the cAMP pathway, but more sensitive to a PLC inhibitor as compared to other odorants, indicating the presence of both cAMP-dependent and -independent mechanisms in control animals. Importantly, 2-heptanone and DMP activated a comparable subset of glomeruli in the main olfactory bulbs (MOB) in both CNGA2 KO and wild type mice. Activated glomeruli also included some necklace glomeruli, which are targeted by axons of olfactory neurons expressing the guanylyl cyclase D (GC-D) pathway. This proposal intends to further study transduction mechanisms of putative pheromones in the MOE and activated brain areas by MOE pheromonal inputs. Hypotheses are that both PLC- and/or GC-D-dependent signaling pathways mediate responses to 2-heptanone and DMP in CNGA2 KO mice; and the MOE responses to these pheromones activate the main olfactory cortex and brain areas that receive pheromonal inputs and regulate social and sexual activities. Aim 1. Determine whether PLC- and/or GC-D-dependent signaling pathways mediate responses to putative pheromones in the MOE. I will examine the involvement of these pathways by using Ca2+ imaging and a combination of pharmacological agents in both CNGA2 KO and control mice. Aim 2. Identify 2-heptanone and DMP-activated glomeruli in the MOB and -activated neurons in higher-order brain areas. Using Fos protein expression as an activity marker in immunolabeling, I will map systematically odor-activated glomeruli. I will determine activated brain areas by counting the number of Fos-positive neurons and comparing these numbers between mice exposed to 2-heptaone and DMP and the non-stimulated controls of the same genotypes in both CNGA2 KO and WT mice. Data generated from this study will correlate signaling pathways involved in MOE pheromone detection to central activity, and contribute to our overall understanding of the strategies used by the olfactory system to discriminate biologically relevant odorants and the influence of MOE on reproduction and social interaction.

DESCRIPTION (provided by applicant): Mammalian olfaction encompasses two parallel signal-processing systems. The main olfactory epithelium (MOE) detects airborne odorants via the cAMP-signaling pathway. The vomeronasal organ (VNO) detects pheromones via phospholipase C (PLC)-dependent activation of TRP2 channels. However, pheromone detection is not exclusively mediated by the VNO. We have shown unexpectedly that pheromone 2-heptanone and 2,5-dimethylpyrazine (DMP) evoked field potentials in the MOE of cyclic nucleotide-gated channel subunit A2 knockout (CNGA2 KO) mice with a disrupted cAMP pathway, consistent with behavioral detection. When tested in wild type mice, these pheromone-induced responses in MOE were significantly less sensitive to inhibitors of the cAMP pathway, but more sensitive to a PLC inhibitor as compared to other odorants, indicating the presence of both cAMP-dependent and -independent mechanisms in control animals. Importantly, 2-heptanone and DMP activated a comparable subset of glomeruli in the main olfactory bulbs (MOB) in both CNGA2 KO and wild type mice. Activated glomeruli also included some necklace glomeruli, which are targeted by axons of olfactory neurons expressing the guanylyl cyclase D (GC-D) pathway. This proposal intends to further study transduction mechanisms of putative pheromones in the MOE and activated brain areas by MOE pheromonal inputs. Hypotheses are that both PLC- and/or GC-D-dependent signaling pathways mediate responses to 2-heptanone and DMP in CNGA2 KO mice; and the MOE responses to these pheromones activate the main olfactory cortex and brain areas that receive pheromonal inputs and regulate social and sexual activities. Aim 1. Determine whether PLC- and/or GC-D-dependent signaling pathways mediate responses to putative pheromones in the MOE. I will examine the involvement of these pathways by using Ca2+ imaging and a combination of pharmacological agents in both CNGA2 KO and control mice. Aim 2. Identify 2-heptanone and DMP-activated glomeruli in the MOB and -activated neurons in higher-order brain areas. Using Fos protein expression as an activity marker in immunolabeling, I will map systematically odor-activated glomeruli. I will determine activated brain areas by counting the number of Fos-positive neurons and comparing these numbers between mice exposed to 2-heptaone and DMP and the non-stimulated controls of the same genotypes in both CNGA2 KO and WT mice. Data generated from this study will correlate signaling pathways involved in MOE pheromone detection to central activity, and contribute to our overall understanding of the strategies used by the olfactory system to discriminate biologically relevant odorants and the influence of MOE on reproduction and social interaction.

DESCRIPTION (provided by applicant): Olfactory perception of intraspecific chemosignals provides sensory information about social and sexual status, genetic makeup, and species identity. In this proposal we describe experiments to investigate activities induced by these chemosignals and their underlying transduction mechanisms. Previously we have shown that the main olfactory system employs both cAMP- and phospholipase C- mediated pathways in pheromone signaling. Genetic knockout of the cyclic nucleotide-gated channel subunit 2 (CNGA2), which eliminates the cAMP signaling, does not eliminate pheromone-induced activation. In our preliminary study, we find that a novel signaling component, Transient Receptor Potential channel M5 (TRPM5), is present in a subset of mouse mature OSNs involved in olfactory signaling of pheromones and urinary components. Knockout of both CNGA2 and TRPM5 results in profound aberration in the main olfactory epithelium and bulbs, indicating the functional importance of TRPM5 in olfactory signaling. We hypothesize that TRPM5 is involved in signaling of intraspecific chemical cues in the main olfactory system. The proposed experiments fall into 3 aims. 1) We will test whether functional TRPM5 is crucial for intraspecific signal-evoked glomerular activation patterns. We will examine intraspecific chemosignals-induced activation in olfactory bulbs use immunolabeling of Fos protein as a neuronal activation marker and construct glomerular activation maps using a glomerular mapping program. We will then determine whether activated glomeruli receive input from the TRPM5-expressing OSNs, and whether knockout of TRPM5 alters the activation patterns. 2) We will test whether TRPM5-expressing OSNs provide sensory input to sustain tyrosine hydroxylase activity found in single CNGA2 knockout mice and whether double- knockouts of both CNGA2 and TRPM5 result in abnormality and neuronal death in the main olfactory system using anatomical and immunocytochemical approaches. 3) We will test whether TRPM5 is involved in signal transduction of intraspecific chemosignals in OSNs. We will characterize electrical and Ca2+ responses to pheromones and urine components in OSNs from TRPM5-GFP, TRPM5 knockout-GFP, CNGA2 knockout-TRPM5GFP mice and determine the functions of TRPM5 and associated pathways using Ca2+ imaging and electrophysiological recording methods. Taken together, the proposed studies will provide fundamental knowledge on signaling processing of intraspecific chemical cues and functional roles of TRPM5 in the main olfactory system. PUBLIC HEALTH RELEVANCE: In this application, we propose to investigate olfactory signaling mechanisms and the abnormality resulted from dysfunction of ion channels in the olfactory epithelium. Olfactory deficits are known to associate with aging and diseases. Our studies will provide knowledge ultimately useful for clinical treatment of olfactory dysfunctions.

DESCRIPTION (provided by applicant): The main olfactory epithelium (MOE) in the mammalian nasal cavity serves two distinct functions. First, the MOE detects almost an infinite number of airborne odor molecules and initiates the sense of smell. Second, the MOE metabolizes and removes inhaled xenobiotics, which include high levels of odorous irritants, air pollutants and microorganisms. This latter, epithelial defense function is critically important, no only for maintaining the proper function of the MOE, but also for protecting vital organs, such as the lower airway, lungs and brain by limiting xenobiotic access to these organs. However, fundamental information about mechanisms of xenobiotic detection and the subsequent pathways coordinating MOE defense is missing, which limits our understanding of olfactory dysfunction in respiratory diseases associated with xenobiotic insults. We recently have identified a population of transient receptor potential M5-expressing microvillous cells (trpM5-MCs) in the MOE. Our initial study shows that trpM5-MCs are chemosensitive and cholinergic, meaning they are capable of releasing acetylcholine (ACh) to modulate intracellular Ca2+ levels of neighboring supporting cells. We hypothesize that trpM5-MCs detect xenobiotics and subsequently coordinate activities of the MOE defense network. We will test this central hypothesis by pursuing the following four specific aims. Aim 1 will determine the response profiles of trpM5-MCs and the key elements necessary for xenobiotic detection. Aims 2 and 3 will characterize intracellular and intercellular pathways that allow trpM5-MCs to coordinate and regulate MOE defense network activities. Aim 4 will determine morphological and functional deficits in the MOE of mice with defective trpM5-MCs. The rationale for the proposed research is that knowledge of the mechanisms of xenobiotic detection and the pathways leading to regulation of xenobiotic clearance is important for the understanding of epithelial defense and will enable innovative approaches to target the initial development of xenobiotic-associated respiratory diseases. We expect to obtain fundamental knowledge about xenobiotic detection mechanisms and subsequent pathways that coordinate and regulate MOE defense. We also expect that defects in xenobiotic detection in trpM5-MCs will impair the MOE defense network, resulting in structural and functional deficits in the epithelium. This proposed research is innovative, because it represents a new approach to understand the relationship between xenobiotic exposure and respiratory diseases. The significance of these studies is that they lay the groundwork for future research on xenobiotic-induced pathological changes that may ultimately lead to new treatments for xenobiotic exposure-related olfactory dysfunction and respiratory diseases.

Abstract The abuse use of electronic cigarette (e-cig) or vaping is a worldwide health problem. Major symptoms of e-cig, or vaping, product use-associated lung injury (EVALI) include cough and shortness of breath. In general, sensory irritation induced by chemical or mechanical stimulation in the lower airway, such as trachea triggers reflexive cough and dyspnea. However, we lack a direct assessment of sensory activation induced by e-cig constituents, tetrahydrocannabinol (THC) and vitamin E acetate that potentially cause EVALI. Furthermore, there is a lack of research data whether chronic vaping neurogenically hyper-sensitizes the airway. In our parent grant project, we electrophysiologically record event-related potentials (ERP) in the nasal mucosa to objectively assess sensory irritation evoked by e-cig flavorings, nicotine, PG/VG and flavored popular e-liquids. Our data have demonstrated dose-dependent sensory activation evoked by these e-liquid constituents. We hypothesize that certain e-liquid constituents cause sensory irritation in the trachea, which subsequently triggers reflexive cough and dyspnea, and that long-term e-cig vapor exposure hyper-sensitizes the airway mucosa. We will test our hypotheses by pursuing research: 1) measuring ERP from mouse tracheal mucosa to evaluate sensory irritation caused by e-liquid constituents, THC and vitamin E acetate, 2) monitor and correlate tracheal mucosal sensory irritation with respiratory alteration, and 3) determining whether 2-week vapor exposure sensitizes the tracheal mucosa resulting in potentiated responses to e-liquid constituents and tussive stimuli. We have accumulated sufficient data from our parent grant project that will enable us to speed up this proposed research substantially by narrowing the tested stimulus concentration ranges. Importantly, we have successfully measured in vivo ERP responses to some e-liquid flavorings, nicotine, and tussigenic capsaicin, and acetic acid from tracheal epithelium that has intact nerve innervation. This direct recording from trachea mucosa presents an innovative approach for the risk assessment of e-cig vaping. Together, our preliminary data promise for time-sensitive, significant results towards a better understanding of the health effects of e-cig vaping and causal factors of EVALI and help guide FDA regulation of e-cig manufacture and sale.