Minghong Ma - US grants

DESCRIPTION (provided by applicant): The mammalian olfactory system has the remarkable ability to detect thousands of odors in the environment, with precise discrimination. As the primary neuronal element in the olfactory system, olfactory receptor neurons are responsible for detecting odor molecules in the environment, transforming chemical information to electrical signals and sending these signals to the brain. In contrast to rapid progress in molecular studies on olfaction, there is still a lack of functional information about peripheral coding mechanisms. Little is known about the odor response spectra of individual receptor neurons expressing a particular receptor gene, the overlap in response spectra between receptor neurons expressing different receptor genes, the spatial distribution of ORNs with distinct response spectra, and the relations between odor stimuli and receptor molecules. The long-term goal of this application is to address these critical issues by using the septal organ as a model system. The septal organ (organ of Masera) is a small patch of olfactory epithelium separated from the main olfactory epithelium by a region of respiratory epithelium, lying bilaterally near the base of the nasal septum at the entrance to the nasopharynx. Compared to the main olfactory epithelium, which contains ~2 million receptor neurons expressing ~1000 receptor genes, the septal organ is a much simpler system with only about 20,000 neurons expressing ~10 receptor genes. The specific aim of this project is to characterize the functional properties of receptor neurons in the septal organ, including signal transduction pathways, odor response spectra of different subtypes of receptor neurons and their spatial relations. The approaches involve making patch clamp recordings directly from the dendritic knobs of receptor neurons in an intact epithelial preparation, monitoring odor response activities of hundreds of neurons simultaneously with calcium imaging, and identifying key elements in signal transduction pathways by immunohistochemical staining. Carrying out this project will greatly enhance our knowledge about olfactory coding in general and provide the first physiological clues to the function of the septal organ, which is still a mystery.

Mammals have the remarkable power of detecting and discriminating thousands of odors. This largely relies on the various types of olfactory receptors expressed in the primary sensory neurons in the nose. Olfactory information is likely encoded by combinations of different receptors (neurons);i.e., a single receptor can respond to multiple odorants and a single odorant can be recognized by multiple receptors. However, very limited knowledge is available on how broadly or narrowly these receptors are tuned and on how these receptors are combined to encode odor mixtures. The rodent main olfactory system contains millions of sensory neurons expressing >1000 receptors and presents serious challenges for addressing these questions. Recently, we discovered that the mouse septal organ, a small patch of olfactory epithelium located in the ventral base of the nasal septum, mainly expresses a few defined receptors. Nearly 50% of the septal organ cells express the MOR256-3 receptor and approximately 43% express seven other receptors at varying levels. Furthermore, a single cell expresses only one receptor. The septal organ offers a unique opportunity to study how individual sensory neurons respond to single or mixed odorants in relation with the receptors they express and the central targets they innervate under physiological conditions. We will provide a relatively complete analysis of this kind by combining physiological, molecular, and histological approaches, and test specifically the following hypotheses. First, different olfactory receptors play different roles in odor detection and discrimination. Some olfactory receptors, exemplified by the major septal receptors, are much more broadly tuned than others and serve as general odor detectors in the nose. Second, different olfactory receptors behave differently in sensing odor mixtures. The broadly-tuned receptors tend to become less active when stimulated by a mixture and the narrowly-tuned receptors remain active as long as their preferred ligands are present. Third, different olfactory receptors (broadly vs narrowly-tuned) have different central projection patterns to the olfactory cortex. Overall, implementing such a project will greatly enhance our knowledge about olfactory coding and processing in general and shed light on the behavioral significance of the septal organ.

DESCRIPTION (provided by applicant): Cilia, microtubule-based protrusions from the cell surface, are cellular organelles present in nearly all mammalian cells. By converting environmental signals into intracellular responses, cilia serve many critical functions including perception of smell. Recent years have witnessed rapid progress in understanding the cell biology and signal transduction events in the cilia of various cell types. However, little is know about what factors shape cilia morphology and how the morphology impacts the function of cilia. We recently discovered that olfactory cilia vary drastically in length and consequently in physiological signals. Prompted by this novel finding, we aim to address these issues using the olfactory system as a model. Odor detection starts with the binding of odorants to specific odorant receptors (ORs) located in the cilia of olfactory sensory neurons (OSNs) in the nose. The mouse olfactory epithelium harbors several million OSNs, each of which expresses one type of OR out of a repertoire of ~1200. Therefore, a few thousand OSNs express any given OR. These neurons are scattered in one of a few broadly-defined zones in the olfactory epithelium, but their axons converge typically onto two discrete glomeruli in the olfactory bulb. Contrary to the common belief that mature OSNs have rather homogeneous morphology, we found that OSNs expressing the same OR significantly differ in cilia length. From the posterior to the anterior nasal septum, the cilia length gradually increases from ~1 mm to ~20 mm with the longest cilia (~50 mm) found in the dorsal recess. Remarkably, the heatmap of the cilia length roughly matches the odorant absorption pattern in the nasal cavity, as determined by airflow rate and odorant solubility. Prompted by this novel finding, this proposal aims to address several key issues regarding the structure, function, and modulation of OSN cilia by combining immunostaining, electrophysiological, genetic and computational approaches. Specifically, three hypotheses will be tested. First, the cilia length of OSNs is positively correlated with the odorant absorption pattern in the nasal cavity. Second, the cilia length of OSNs can be increased by enhanced sensory inputs and/or neuronal activity. Third, OSNs expressing the same OR but with different cilia morphology carry non-redundant information into the brain. The results will offer new insights into the structure and function of sensory cilia and advance our understanding of the coding and processing of odor information by the olfactory system.

Smell is a powerful sense that can trigger intense emotion, stereotyped behaviors and durable memories. The sense offers an extraordinary opportunity to connect atomic-level objects (odorant molecules and smell receptors in the nose) to neural responses. This project will predict which smell receptors in the nose are activated by a given odor. To accomplish this goal, the team of investigators will apply computational approaches to develop chemical structure-based receptor models and test these models using odor molecules interacting with olfactory receptors. The success of the project will enable the team to understand more precisely how the brain perceives the external environment. The results will also have widespread and diverse industrial applications, including rational design of new flavors and fragrances and development of new biosensors for detecting various chemicals. Furthermore, this project will make broader impacts in training and educating high school, undergraduate, and graduate students in various disciplines as well as in outreaching activities.

The complexity of the odor molecules, the large number of the smell receptors and combinatorial activation of the receptors make understanding odor coding an enormous challenge. This collaborative proposal represents the first of its kind that combines computational approaches with experimental measurements at both the receptor and the neuron level. Affinity calculations between odorants and the receptors, as well as the receptors' activation, will be obtained by nanosecond-scale simulations. Atomic-level simulations, initially assessed by experiments, will predict which odors would activate the receptors of interest. Comparisons between experimental findings and computational predictions will lead to a comprehensive computational model that converges with experimental data.

A companion project is being funded by the French National Research Agency (ANR).

We propose to create a flexible, interdisciplinary, Jointly Sponsored NIH Predoctoral Training Program in the Neurosciences (JSPTPN) to prepare exceptional predoctoral students in their first two years of graduate school for productive careers in basic neuroscience research and related fields. Our proposed program trains students to work towards understanding the operation of the nervous system, including education and research opportunities to identify and ameliorate many dysfunctional and disease conditions such as stroke, epilepsy, traumatic brain injury, neurodegenerative disorders, and addiction. This program is based in the Neuroscience Graduate Group (NGG), an interdisciplinary PhD program that includes faculty from 22 Departments in 6 Schools of the University of Pennsylvania plus the affiliated Children's Hospital of Philadelphia. Graduate education in biomedical sciences at Penn is based on this kind of interdepartmental Graduate Group and is overseen by the Office of Biomedical Graduate Studies (BGS). BGS ensures effective curricular development, quality control, and uniform admission standards across all relevant Graduate Groups, including the NGG. Direct management of the proposed training program is done by a five-person Executive Committee that sets and reviews policy and selects trainees. Faculty membership is governed by: 1) expertise in a relevant field of study, 2) significant contribution to training, 3) commitment to the goals of the program, and 4) extramural funding to support trainees. Junior faculty receive extensive guidance on mentoring. Admission of students to the NGG is vetted by a BGS-wide admissions committee. Subsequent admission to the proposed JSPTPN will be decided by its Executive Committee. Support for each trainee will encompass their first 21 months in graduate school. The Training Program will consist of two years of coursework plus at least two lab rotations. All students will take a yearly course on the responsible conduct of scientific research and will participate in several newly developed training components that focus on instruction in the scientific method and statistical methodology. Students will also receive training through seminars, journal clubs, annual retreats, scientific meetings, paper and poster presentations, and social events that encourage interactions. Successful completion of a comprehensive Candidacy Examination marks the start of independent research toward the dissertation. Thesis research is conducted under the supervision of a faculty advisor and is monitored by a Thesis Committee and the NGG Academic Review Committee. The dissertation defense takes place when the thesis advisor and committee agree that the work is complete. Most graduates move on for advanced (postdoctoral) training and pursue an academic career. Based on the number of potential trainees, we request support for 12 predoctoral trainees/year for the next 5 years.

PROJECT SUMMARY Substance abuse and misuse pose significant costs to society and represent a global disease burden. Relapse after a period of drug-free abstinence is one of the most profoundly debilitating aspects of addiction, occurring in 40?80% of individuals. Understanding the neurobiological mechanisms of drug taking and relapse will ultimately lead to better therapeutic interventions. The ventral striatum is a network of brain structures implicated in compulsive drug-seeking and includes the ventral pallidum, nucleus accumbens (NAc), and olfactory tubercle (OT). The OT, like the NAc, is a site of massive innervation of dopaminergic neuron terminals from the ventral tegmental area in the midbrain. Rodents self-administer psychoactive substances and electrical current into the OT, and more readily administer cocaine into the OT than even NAc. Further, our lab has uncovered that the activity of OT neurons robustly reflects reward-guided behaviors and rewards. Despite this evidence pointing towards a role for the OT in mediating reinforcement, little is known about the OT, and at present, the OT is not included in mainstream models of the reward system. The short-term goal of the parent project is to build off both our published and unpublished studies positioning the OT in the reward circuitry to determine mechanisms whereby the OT exerts control over cocaine seeking and taking. Our overall hypothesis is that there is a functional organization amongst ventral striatum subregions which influences drug seeking. The goal of this administrative supplement is to provide summer research opportunities to a NIDA research intern. The intern will engage in projects using in vivo physiological methods to demonstrate manners whereby OT neurons, including OT medium spiny neurons, represent drug seeking (Aim 2), and those projects employing cell-specific optogenetic methods to determine the regulation of reinforcement and drug-seeking by OT neurons (Aim 3). The results of this project will answer long-standing questions about the fundamental circuitry of the OT and its significance in the context of motivated behavior and drug-seeking. Together this project will contribute to our long-term goal of generating a more complete model of the brain's reward system.

Project Summary Breathing patterns have strong impacts on emotions in humans. Voluntary control of respiration, especially via nasal breathing as practiced in yoga and meditation, is effective in reducing anxiety, stress, or even panic attacks. The effects of breathing patterns on emotions are thought to be related to respiration-entrained brain rhythms, which have been recognized for decades, but their sources and functions remain elusive. One potential source of respiration-entrained brain activity is the olfactory system. Nasal airflow activates intrinsically mechanosensitive olfactory sensory neurons (OSNs) in the nose, which carry the information to the olfactory bulb (OB) and subsequently to the olfactory cortical regions including the anterior olfactory nucleus/taenia tecta (AON for simplicity). It is well known that the neural activity along the olfactory pathway is highly correlated with respiration. Interestingly, recent studies indicate that many non-olfactory cortical and limbic structures including the medial prefrontal cortex (mPFC) also display nasal airflow-dependent, respiration-entrained oscillations in both rodents and humans. A potential role of respiration-entrained neural activity has been examined in the context of learned fear, an emotional state inferred by quantifiable freezing behavior in rodents. During fear retrieval after tone-foot shock pairing, mice freeze to the conditioned tones while breathe at a steady rate (~4 Hz), which is correlated with a predominant 4-Hz oscillation in the mPFC, a region critical for expression of conditioned fear. Disruption of peripheral olfactory inputs significantly reduces the 4-Hz oscillation in the mPFC and leads to prolonged freezing periods. However, the neural circuits underlying the effects of olfactory inputs on the mPFC activity and fear-related behaviors remain largely unresolved. We recently discovered that the mPFC receives direct inputs from the AON, a major target of the OB tufted cells, which receive stronger peripheral inputs and display robust respiration-entrained activity. The central hypothesis of this proposal is that the OB tufted cells?AON?mPFC pathway is the critical neural circuit in modulating the mPFC respiration-related rhythm and relevant behaviors. Multidisciplinary approaches (gene editing, ex vivo and in vivo electrophysiology, optogenetics, circuit tracing, and behavior) will be combined to pursue three specific aims. We will (1) dissect out this neural pathway in a cell-type specific manner, (2) determine functional properties of this pathway, and (3) determine behavioral effects of optogenetic manipulations of this pathway. Overall, the current study will provide critical insights into olfactory modulation of respiration-entrained brain activity and behavior.

E-cigarettes are increasingly used by teenagers, who are particularly vulnerable to the addictive properties of nicotine. With the exclusion of menthol, the use of flavor additives has been banned from traditional cigarettes, while e-cigarettes (e-cigs) are marketed in over 7,000 different flavors. We hypothesize that flavorants enhance nicotine reward through sensory and/or natural reward mechanisms. As a consequence, flavored e-cigs may promote nicotine experimentation, dependence, and eventually, the use of regular cigarettes. Despite the fact that the FDA has recently regulated the sales of flavored e-cigs to minors, no flavor ban has been implemented, and adolescents could still obtain flavored e-cig products through friends or unscrupulous sellers. Furthermore, understanding how flavors influence e-cig use is important for the implementation of regulatory rules that can reduce potential disease and death deriving from the consumption of this increasingly popular tobacco product. The overall goal of this application is to compare the rewarding and reinforcing properties of flavored vs. non-flavored e-cigs in adolescent and adult mice, and identify the neural substrates responsible for the enhancing effects of flavorants. Our hypothesis is that flavored e-cigs are more rewarding than non-flavored e-cigs and that flavor additives promote and sustain nicotine seeking in adolescents due to the combined influence of flavorants and nicotine on the ventral striatum. The first goal is to determine whether flavorants enhance adolescent nicotine reward and promote nicotine self- administration, and compare their effects in adult mice. The second goal is to understand how e-cig flavorants are encoded in the ventral striatum during acquisition of e-cigarette vapor preference and self-administration. The third goal is to determine whether exposure to e-cig flavorants alters synaptic plasticity in the ventral striatum and whether it modifies cellular responses to nicotine. Our experiments will combine behavioral approaches with tetrode recordings during behavior and brain slice electrophysiology to determine how e-cigarette nicotine vapor affects reward-associated brain areas, and whether flavorants (e.g. fruit or mint) have additive or synergistic effects.

Project Summary The striatum is an evolutionarily conserved structure involved in cognitive and limbic regulation of motor control. Striatal circuits are implicated in the initiation and execution of ethologically relevant motor output, ranging from exploratory actions to highly stereotyped motor patterns. Dysfunction of these circuits leads to motor control abnormalities that frequently manifest as excessive repetitive behaviors. Self-directed grooming, a highly stereotyped repetitive motor pattern, is observed in virtually all animals, serving vital functions in hygiene maintenance, thermoregulation, de-arousal, stress reduction, and social communication. Abnormally repetitive grooming is a central behavioral phenotype observed in numerous models for neurological and neuropsychiatric diseases. A better understanding of the neural control of grooming may thus yield fundamental insights into how the brain controls repetitive motor output in both normal and diseased conditions. Our preliminary work suggests that an understudied population of interneurons within the olfactory tubercle (OT; the most ventral part of the striatum), predominantly in the Islands of Calleja (IC), is involved in mediating this behavior. The striatum has a fairly uniform cellular composition, with ~95% of the neurons being spiny projection neurons (SPNs), classified as D1- or D2-type according to the dopamine receptors they express. One exception to this uniformity is the existence of evolutionarily conserved IC, clusters of densely- packed, GABAergic granule cells, which express the D3 dopamine receptor. By means of optogenetic manipulations, we have shown that activation of OT D3 neurons initiates robust grooming behavior via arrest of other alternative ongoing behaviors. In contrast, inactivation of these neurons halts ongoing grooming. These findings lead to the central hypothesis that OT D3 neurons play critical roles in controlling grooming behavior. Through an array of modern neuroscience approaches (optogenetics, ex vivo and in vivo electrophysiology, fiber photometry, neural circuit tracing, and behavior), we will pursue three specific aims to determine (1) in vivo activity patterns of OT D3 neurons and SPNs in grooming and other behaviors, (2) contributions of OT D3 neurons to grooming in relation to other brain regions, and (3) the effects of dopamine release into the OT on grooming behavior. Overall, this project will provide insights into the neural circuitry of the IC/OT D3 neurons and its role in neurobiological control of a highly important motor pattern.