Thomas S. Hnasko, PhD - US grants

DESCRIPTION (provided by applicant): My long-term goal is to establish an independent laboratory at an academic research institution where I will have the freedom and resources to contribute to our understanding of the brain, and in particular, the neural mechanisms underlying addiction. My near-term goals are to demonstrate the resourcefulness and productivity necessary to achieve independence while mastering new skills and concepts that will allow me to thrive independently. Specifically, I will train on several new methodologies including 1) in vitro transport assays, 2) neuronal culture, 3) slice electrophysiology, and 4) tract tracing. I will also enroll in several classes designed to improve my technical literacy or enhance my ability to provide strong leadership &management. I plan to recruit one or more undergraduate student assistants which will both provide a mentoring opportunity and increase my efficiency. By the end of the mentored phase of this award, my goal is to be in a position to write a highly competitive R01 proposal, the crucial mechanism for funding an independent academic laboratory. Environment: Most of the training during the mentored phase of this proposal will occur at the University of California San Francisco (UCSF) under the direction of Dr. Robert Edwards. Robert is a world-renowned investigator at the forefront of vesicular transporter biophysics and cell biology. Robert's particular focus on in vitro approaches to the study of vesicular dopamine and glutamate transporters meshes perfectly with both the career development and research plan sections of this proposal. The physiology department at the Mission Bay campus of UCSF is an exciting setting to pursue this work with ample opportunity for collaboration and access to both standard and emerging technologies necessary to further this research plan. I also propose to spend 1-2 years training under the co-mentorship of Dr. Howard Fields who remains affiliated with UCSF but whose lab is now located at the Ernest Gallo Clinic and Research Center (EGCRC) in nearby Emeryville CA. Howard is a distinguished investigator whose main research interest is in unraveling the neural circuitry underlying addiction. Working with Howard my main goal will be to learn how to make electrophysiological recordings from fluorescent dopamine or glutamate neurons in acute slices made through the mouse midbrain. The EGCRC is a vibrant research environment where the primary mission is centered on understanding the neural mechanisms of addiction with the goal of treating and preventing drug abuse. Thus my time at the EGCRC will provide me with new skills in neurophysiology but also expose me to the work of a research community that broadly shares the goals described in this proposal. Research Plan: Dopamine neurons of the ventral tegmental area (VTA) are of fundamental importance to the initiation and development of drug dependence. However, their precise role in drug addiction remains unclear. Thus, a crucial step toward understanding the mechanisms by which drugs of abuse produce the molecular and chemical changes in brain circuitry that lead to addiction is to define the contribution of dopamine neurons to normal brain function. Emerging evidence suggests that dopamine neurons have the capacity to co-release the excitatory neurotransmitter glutamate and vesicular glutamate transporters (VGLUTs) are necessary for neurons to release glutamate. A central aim of this project is to use a mouse model that has been genetically engineered to lack the vesicular glutamate transporter (VGLUT2) specifically from dopamine neurons to assess the role of glutamate co-release by dopamine neurons on brain development and function. Electrophysiological, anatomical, biochemical &behavioral techniques will be employed. Emphasis will be placed on determining whether glutamate co-release contributes to the behavioral responses to drugs of abuse and, in particular, reward learning. In addition, the potential for VGLUT2 to induce vesicular acidification and promote vesicular dopamine accumulation will be assessed using the mice described above. A second aim is to characterize a second population of VGLUT2+ neurons in the VTA that do not express dopaminergic markers. These neurons represent an uncharacterized population of VTA neurons that may also play an important role in mediating responses to drugs of abuse. Using double-transgenic reporter mice, the electrophysiological properties, pharmacological responses, and anatomical projections of the VGLUT2+ neurons will be assessed and compared with their dopaminergic neighbors. The final aim will be to inject viral vectors engineered to express Cre recombinase into the VTA of conditional VGLUT2 knockout mice to assess the role of all VGLUT2+ VTA neurons in goal-directed behaviors. The overall goal of this proposal is to elucidate the function of glutamate-releasing VTA neurons, and in particular, assess their role in mediating the molecular and behavioral responses to drugs of abuse.

PROJECT SUMMARY Research in several species shows that rapidly stopping action activates the subthalamic nucleus (STN) of the basal ganglia (BG) ? and that the STN may be recruited via a hyperdirect pathway (HDP) from prefrontal cortical stopping nodes. Human research also shows that unexpected events (such as a surprising tones) also recruit the stopping system (including the STN), and interrupt working memory (WM). At the core of this proposal is the idea that WM is maintained via a recurrent cortico-BG loop, and that this can be interrupted via HDP recruitment of the STN. The interruption is induced via stop signals and unexpected events, and could function to `clear cognition'. This is a radical new theory: that a fronto-BG circuit for stopping underlies cognitive interruptions. Validating this theory has broad implications for understanding the BG, for mechanisms of cognitive interruptions, and for potential cognitive under-and-over flexibility in Parkinson's disease (PD) and ADHD. Here we propose to systematically test this theory using parallel human-mouse studies, based on extensive preliminary data and using several novel approaches, including event-related Deep Brain Stimulation (eDBS). Aim 1 (motor stop) will use STN eDBS in humans and optogenetic stimulation in mice to test whether the STN is causally important for stopping action. Aim 2 (cognitive stop) will use eDBS and functional MRI in humans and optogenetics in mice to test whether the STN is causally important for decrementing WM. Aim 3 (HDP) will use combinatorial optogenetics in mice and novel combinations of transcranial magnetic stimulation with concurrent local field recordings in human STN to test whether motor and cognitive Stops are implemented via HDP inputs to STN from frontal cortex. Throughout the proposal we focus on designing complementary experiments across species, so that what is learned in mice can directly inform our understanding of human neural circuit function and behavior.

PROJECT SUMMARY Synaptic dysfunction is a common feature of neuropsychiatric disease. For example, a hallmark of age-related neurodegenerative diseases such as Alzheimer?s and Parkinson?s is synaptic fibrilization and aggregation of key proteins that participate in synapse and cell loss. Maladaptive plastic changes in synapse structure and function underlie key aspects of behavioral and mood disorders ranging from addiction to depression, as well as neurodevelopmental diseases like schizophrenia and autism. It is for these reasons that many investigators across a range of neuroscience disciplines study the synapse, and the reason that new tools to study synapse structure and function within neural circuits of interest are sorely needed. Indeed, current tools to assess synapse structure in defined cell types are not readily compatible with state-of-the-art 3D volume approaches such as serial block face scanning electron microscopy, and are severely hampered by inadequate computational tools for quantitative assessment of these massive datasets. However, advances in molecular genetics, optics, engineering and computing provide new opportunities to develop information rich strategies to peer into the synapse. Here, we combine such advances to achieve a new state-of-the-art in imaging and analyzing microcircuit connectivity and synapse structure within neurotransmitter-defined neural networks. Specifically, we leverage the fact that the bulk of signaling across the synapse is mediated by a relatively small population of small molecule neurotransmitters that are synthesized and packaged into synaptic vesicles at the site of release in axonal compartments. The bulk of neurotransmission is thus dependent on just seven well- described vesicular transporters expressed in brain. Our overall goal is to build a rigorous, easily deployable, cell-type-specific, expandable, multi-functional toolkit for imaging and quantifying neurotransmitter-defined synaptic connections by both light and electron microscopy in mice. To accomplish this, we will use CRISPR/Cas9 to insert electron microscopy-compatible tags into native vesicular transporters (Aim 1), establish simplified procedures for their monochrome and ?multicolor? labeling in 3D ultrastructure (Aim 2), and computational tools for automated segmentation and quantitative analysis of key pre- and post-synaptic metrics (Aim 3). Though these Aims are independently meritorious, by synthesizing them we aim to generate a complete toolkit that will allow investigators to render neurotransmitter-defined circuit connections into 3D ultrastructure datasets with automated quantitative assessment of key features of pre- and post-synaptic structure.

Dysfunctional reward processing is a symptom common of major neuropsychiatric disorders including addiction, schizophrenia and depression. Perturbations in neuronal dopamine signaling have been implicated in the etiology of this symptom and is a target of current pharmacotherapies. Less is known about how neuropsychiatric symptoms manifest downstream of altered dopamine signaling, or the mechanisms by which dopamine signaling becomes perturbed in the first place. The ventral pallidum (VP) is a prime suspect that is receiving increasing attention. The VP is the major output of nucleus accumbens (NAc) and a key input back to the ventral tegmental area (VTA). Activity in VP is sensitive to hedonic stimuli and can potently drive motivated behaviors. Yet the VP is heterogeneous containing neurons with diverse neurotransmitters and projection targets. For example, VP glutamate and GABA neurons share similar projection patterns but drive opposite patterns of behavior on reinforcement tasks. Optogenetic activation of NAc D1-type and D2-type medium spiny neurons can similarly bi-directionally influence reinforcement, but how defined NAc and defined VP cell types are connected is unknown. In Aim 1 we employ optogenetic-assisted electrophysiology and reporter mice to establish the cell-type-specific connectivity between NAc and VP. Aim 2 will use in vivo calcium imaging to assess the intrinsic activity of defined VP populations in response to positive and negative valence stimuli and associated contexts. These studies will build on our recent work, address key knowledge gaps, and facilitate a programmatic assessment of the role of VP circuit heterogeneity in reward processing and neuropsychiatric illness.

PROJECT SUMMARY The ventral tegmental area (VTA) is a core component of the neural circuitry that drives goal-directed behavior, and a primary target through which drugs of abuse modulate behavior. Although generally regarded as a dopaminergic nucleus, about half of VTA neurons signal through release of the amino acid neurotransmitters GABA and glutamate. These neurons are also targets of drugs of abuse but much less studied. Recent evidence from our lab and others has begun to show that, like VTA DA neurons, activity in VTA GABA and glutamate neurons can profoundly shape motivated behaviors. Furthermore, sub-populations of VTA neurons release more than one of these three recycling transmitters, including neurons that co-release dopamine and glutamate, or glutamate and GABA. The goals of this proposal are to identify how transmitter co-release from VTA contributes to the processes underlying behavioral reinforcement, and how co-releasing neurons functionally integrate into the mesolimbic neural circuits that regulate motivated behavior. We will use an array of genetic approaches in combination with behavioral, anatomical, and electrophysiological assays in mice to selectively probe the connectivity and function of discrete VTA circuits. Together this continuing research program comprises a thorough plan to define the form and function of novel classes of VTA neurons and enhance our understanding of how intrinsic VTA heterogeneity shapes behaviors relevant to neuropsychiatric disease.

PROJECT SUMMARY Withdrawal is a major obstacle in overcoming opioid dependence and addiction. Identifying the neural circuits involved and how opioids modulate their activity is essential for developing new therapeutic strategies. The medial habenula (MHb) expresses particularly high levels of mu-opioid receptor (MOR) and emerging evidence implicates the MHb as a hotspot for the physical and affective symptoms of opioid withdrawal and relapse. Despite this, there is remarkably little information on how MOR signaling influences MHb projection neurons, or how chronic MOR signaling could induce physiological changes in MHb circuits that contribute to withdrawal. MHb neurons project principally to the interpeduncular nucleus (IPN) where they can release acetylcholine (ACh) or glutamate; and some of these neurons can co-release both glutamate and ACh. While cholinergic- and glutamatergic-defined MHb neurons have been implicated in processes underlying addiction, the respective roles and relative importance of these co-transmitters remain unclear. Based on prior literature and preliminary data we posit an important role for glutamate and ACh co-release from MHb to IPN in mediating effects of opioid dependence, including withdrawal. In this exploratory proposal we aim to test how acute and chronic MOR activation influences activity in MHb and synaptic transmission in IPN and contributes to opioid dependence.