Jeremy C. McIntyre, Ph.D. - US grants

Project Summary The proposed K99/R00 application incorporates a comprehensive research and training plan for studying ion channels and channelopathies in the olfactory system. Ion channels are critical for regulating excitability in many cell types including olfactory sensory neurons (OSNs). These proteins regulate the movement of ions across the cellular membrane. In OSNs ion channels are responsible for depolarizing the cell in response to odor stimulation, initializing an action potential and synaptic transmission. Channelopathies are a class of human genetic disorders in which ion channel function is disrupted leading to defects in multiple organ systems. Disruptions in ion channels are known to cause epilepsy, cardiac arrhythmias, blindness, deafness and alterations in pain sensitivity. Channelopathies in human patients can result from both loss-of- function and gain-of-function mutations in ion channel genes. It has recently been shown that channelopathies can result in anosmia in humans. Deletions of several different ion channels in mouse models also causes anosmia, indicating that olfactory signaling can be affected at multiple steps. The proposed research will analyze the ability of gene therapy to correct channelopathy induced defects in olfactory function at these different stages in signaling. Aim 1 of this proposal will use two mouse strains with targeted deletions in cyclic nucleotide gated (CNG) channel subunits. CNGA2 and CNGB1 are critical subunits of the olfactory CNG channel necessary for odor detection and their loss leads to anosmia. Using adenovirus, I will deliver functional copies of the missing gene to mutant OSNs to test the ability to restore olfactory function to anosmic animals. Restoration of the sense of smell will be analyzed with electrophysiological and behavioral methods. Aim 2 will investigate the ability of gene therapy to correct channelopathy induced defects in synaptic transmission due to loss of the sodium channel alpha-subunit Nav1.7 (encoded by Scn9a) in OSNs. Using olfactory specific Scn9a null mice, I will use adenovirus to express ectopic Scn9a in OSNs and analyze the restoration of synaptic transmission. In addition this aim will analyze the effects of gain-of-function mutations in Scn9a through adenovirus-mediated expression of identified mutant sodium channels in OSNs. This will test the effect of hyper-excitability on olfactory function. Finally Aim 3 will analyze the role of two voltage gated calcium channel (VGCC) alpha-subunits, Cav1.3 and Cav2.2, in olfactory function. Mutations in VGCCs, including CACNA1D (encoding Cav1.3), underlie channelopathy disorders affecting sensory function and are therefore potential causes of anosmia. An understanding of the function of these VGCCs in the olfactory system will help to direct the identification of novel mutations in anosmic patients. Together the results from these 3 aims will provide new insight into the mechanisms of olfactory signaling and the ability of gene therapy to correct defects in ion channel function. The results from the proposed research will be important for helping to develop therapies for patients with anosmia due to channelopathies. In addition these results may provide insight into developing treatments for other sensory defects such as vision and hearing loss.

Project Summary Substance use disorders are a significant public health issue, costing tens of thousands of lives and hundreds of billions of dollars annually . Considerable efforts have been expended to elucidate the neural mechanisms underpinning the acute and chronic actions of drugs of abuse on the nervous system, with the hope that better understanding of these mechanisms will lead to novel therapeutics. Much of this research has targeted receptors on neurons that are localized to cell bodies, axons, or dendrites; however, neurons also contain primary cilia, which are microtubule-based organelles that project from the cell bodies of all neurons. The importance of cilia function for human health is highlighted by the number of diseases caused by cilia dysfunction, several of which are associated with cognitive and motivational deficits. Neuronal cilia express a variety of G protein-coupled receptors (GPCRs), several of which are rarely expressed outside of cilia. Notably, several of these receptors, including the receptor for melanin-concentrating hormone (MCHR1) and the orphan GPCR, GPR88, have been shown to modulate responses to drugs of abuse. Despite what we know about cilia, our understanding of how cilia regulate neuronal function and behavior is still limited, and, in particular, there has been no prior research on interactions between neuronal cilia and drugs of abuse. The long-term goal of this research is to determine how ciliary signaling contributes to integration of neuromodulatory signals that regulate short- and long-term responses to drugs of abuse. As a first step toward this goal, the objective of our R21 proposal is to determine the role of primary cilia on dopaminergic and GABAergic neurons in the VTA and nucleus accumbens, respectively, in regulation of cocaine-induced behavioral plasticity and reward. This will be accomplished through molecular-genetic approaches to target cilia loss on specific neuronal types, in combination with behavioral pharmacological approaches. The proposed experiments will allow us to test our central hypothesis that neuronal cilia within mesolimbic circuitry are critical regulators of cocaine-induced plasticity and reward. We will test this hypothesis through two Specific Aims. Experiments in Aim 1 will use novel transgenic mouse strains to determine a) how cilia loss on dopaminergic and/or GABAergic neurons affects locomotion and locomotor sensitization induced by acute and repeated cocaine, respectively; b) whether locomotor alterations can be rescued by virally- mediated restoration of cilia in targeted brain regions, and c) how repeated cocaine alters cilia morphology and MCHR1 and GPR88 expression in mesolimbic brain regions. Experiments in Aim 2 will use similar approaches to determine whether cilia on dopaminergic and/or GABAergic neurons are necessary and sufficient for the rewarding effects of cocaine using a conditioned place preference task. The proposed research is innovative, as neuronal cilia have heretofore not been assessed in the context of drugs of abuse. The proposed research is significant, as cilia represent a unique neuronal signaling environment, a better understanding of which could lead to novel targets for therapies aimed at reducing substance use.

Project Summary The sense of smell is essential for maintaining full human health and quality of life. It plays an important role in the detection of environmental dangers as well guiding decisions such as what foods to eat. However, olfactory processing is influenced by the physiological state of an organism. Both sleep deprivation and changes in satiety are connected with changes in the function of the olfactory system. Physiological changes such as these are integrated in the hypothalamus, where different neuropeptides are expressed by specific populations of neurons. These peptides can regulate transitions between wakefulness and sleep, or promote feeding behaviors. One peptide that functions in both promoting feeding and sleep is melanin-concentrating hormone (MCH). Neurons expressing MCH project to several areas of the brain including the olfactory bulb (OB), where the MCH receptor, MCHR1, is expressed. This connection represents a previously understudied pathway providing a potential mechanism for sleep or satiety induced changes in olfactory function. The proposed research will investigate the role of MCH signaling and hypothalamic MCH neurons in contributing to odor processing. The aims of this proposal will test the central hypothesis centrifugal MCH neurons integrate physiological states and regulate olfactory function. Aim 1 will use molecular and biochemical techniques to investigate changes in MCH levels in the OB in response to food restriction. It will also use complementary mouse models to determine the cellular targets of hypothalamic MCH neurons in olfactory regions. Aim 2 will investigate the effects of MCH on the activity of mitral cells in the olfactory bulb, and how changes in MCH effect odor threshold detection and cross-habituation in animals that lack components of the MCH signaling pathway. It will also test how activation of hypothalamic MCH neurons modulates these behaviors. Using AAV mediated approaches, we will target MCHR1 removal specifically in the OB to isolate its contribution to regulating behavioral changes. Finally, in Aim 3 we will investigate how disruption of primary cilia, the cellular site of MCHR1 localization, on neurons in the OB impacts an animal's ability to detect and discriminate odors. Completion of the proposed studies will provide new mechanistic insight into the role of the lateral hypothalamus in regulating olfactory function. The results from the proposed research will be important for understanding how changes in satiety or in wakefulness can impact the sense of smell. It will also provide insight into mechanisms of sensory dysfunction that occur in some ciliopathy patients. Completion of this project will establish future experiments to address the molecular mechanisms of MCH modulation in the OB.