Anatol C. Kreitzer, Ph.D. - US grants

DESCRIPTION (provided by applicant): Long-term changes in synaptic strength are thought to represent a cellular correlate of learning and memory. In the hippocampus, long-term potentiation (LTP) and long-term depression (LTD) have been studied extensively, and a number of different mechanisms have been elucidated, some of which coexist at the same synapses. In the striatum, changes in synaptic function are thought to correlate with behaviors such as habit formation, as well as with pathologies such as Parkinson's and Huntington's disease and drug addiction. However, the mechanisms, or even the types of plasticities present at these synapses, have not been fully described. The goal of this research proposal is to examine LTD in both the dorsal striatum and its ventral extension, the nucleus accumbens. A number of studies have found various and conflicting results regarding LTD in these areas. To examine the types of LTD present at synapses in both dorsal striatum and nucleus accumbens, various induction protocols and experimental paradigms will be tested. Specific mechanisms of LTD induction and expression will then be studied using electrophysioiogical recordings combined with molecular techniques such as viral-mediated gene transfer. These studies will provide a framework for understanding how long-term changes in synaptic strength in the striatum are related to such health issues as addiction and neurodegenerative disease.

Why does this program need a communication/administration core? The original request for proposals (RFP) specifically mandated investigators at different institutions. Indeed, the RFP stipulated that the number of investigators from one institution could not exceed two. For this reason, the original proposal included R. Edwards, R.B. Kelly (both UCSF) and D. Sulzer (Columbia). We were forced to exclude M. von Zastrow (UCSF) because he would have been the third person at UCSF. Since these restrictions were dropped on resubmission, we added M. von Zastrow, but then replaced R.B. Kelly with T. Ryan (Cornell Med), and thus had two investigators in New York City as well as the two in San Francisco, With three independent institutions involved, and two different departments even at UCSF, the program has presented a significant challenge to communication and administration. Although we are now replacing T. Ryan with a UCSF-affiliated investigator, A. Kreitzer holds a position in the private Gladstone Institutes, and his project thus also requires an outside contract, with the associated complications.

The importance of ventral midbrain (VM) dopamine (DA) synaptic activity is clear from their roles in motor, learning and behavioral disorders, including drug dependence. These synapses contribute to the normal execution of motor sequences, learning, and habit formation learning by mediating short- and long-terhi plasticity at two levels in the basal ganglia, at axon terminals in the striatum, and from somatodendritic areas Together, those actions determine which medium spiny neurons (MSN) synapses transfer activity to substantia nigra reticulara (SNr) nigrothalamic neurons that integrate basal ganglia circuitry and drive the cortex to control behavior, DA released by synaptic vesicle exocytosis from axon terminals acts at multiple pre- and postsynaptic sites that together alter striatonigral dii-ecf and striatopallidal indirect MSN activity. Transmission from sonnatodendritic regions is poorly understood, in part because they lack conventional synaptic vesicles but rather presunned neurosecretory organelles that express VMAT2. Somatodendritic DA release also appears to haVe multiple targets that control nigrothalamic activity, but little is known currently about such synapses. Our hypothesis is that somatodendritic DA release enables frequency-dependent selection of synaptic terminals ofthe striatonigral direct pathway. Our |ab has developed opticaltechniques to measure activity at individual synaptic terminals, including, fluorescent false neurotransmitters (FFNs) that characterize DA release and the synaptic vesicle fusion probe FM 1-43 to striatonigral synapses. Our preliminary evidence indicates that somatodendritic DA release selects striatonigral synapses via presynaptic D1 receptors in a frequency-dependent manner, an effect not detectable using classical methods. We will work with the Edwards lab using mutant mice that Should lack somatodendhtic DA release, the Krietzer lab who have developed means to selectively activate DA, striatonigral and pallidonigral pathways, and the von: Zastrow lab, who are exploring means to interfere with Dl signalling on the MSN neurons, including by amphetamine (AMPH), RELEVANCE (See instructions): The role of dopamine neurotransmission in drug dependence is well established, but the precise means by Which it alters synapses leading to this disorder is unclear, particularly in the midbrain where dopamine release is required but is released using different molecular mechanisms than in most of the rest of the central nervous system. We wiN determine how this release occurs and its relevant synaptic effects.

PROJECT SUMMARY (See instructions): This is a renewal application of a highly successful project that has previously elaborated postsynaptic regulation of opioid and dopamine receptors. The studies proposed in the renewal will examine also the presynaptic compartment, specifically focusing on Dl-type dopamine receptors that regulate the direct pathway GABAergic output from striatum / nucleus accumbens, and to precise evaluation ofthe regulation of synaptic vesicle exocytosis from axons. The Specific Aims of the proposed studies are: Specific Aim 1. Determine whether rapid DIR trafficking events are restricted to the somatodendritic surface or occur also in axons of MSNs. Specific Aim 2. Define the effects of extracellular DA dynamics and electrical activity on Dl R endocytosis in MSNs. Specific Aim 3. Identify functional consequences of DIR trafficking on pre- and post- synaptic signaling. The proposed experiments will define the basic properties of dopaminergic regulation ofthe direct pathway, which controls natural motivated behavior and is critical for the reinforcing effects of addictive drugs. The studies also directly test, and mechanistically elucidate, presynaptic regulation by Dl receptors. This is a fundamental area of cellular neuroscience that is may reveal new approaches for manipulating addictive drug action or treating addictive disorders.

The reward pathway links adaptive behavior to the appropriate environmental cues through changes in the release of dopamine. Drugs of abuse bypass the requirement for adaptive behavior by increasing dopamine directly, triggering plasticity that results in addiction. However, the mechanisms that control dopamine release remain poorly understood. Unlike other classical transmitters, dopamine acts as a neuromodulator, and it has remained unclear how it can convey a signal with the temporal resolution required to associate environmental cues with reward. In addition, dopamine undergoes release from dendrite as well as axon, and dendritic dopamine release has been implicated in plasticity of the reward system- The long-term objective of this proposal is thus to understand how the regulation of dopamine release contributes to the role of dopamine neurons in physiology and behavior. The strategy is to characterize the mechanisms that regulate dopamine release: 1) Determine the relationship between dopamine and glutamate release by midbrain dopamine neurons. Dopamine neurons form glutamate synapses in vitro, suggesting a mechanism for rapid, precise, synaptic signaling by these cells. Using VGLUT2 conditional knock-out mice, we have now demonstrated a dual role for glutamate co-release in vivo in dopamine storage and as an independent signal for postsynaptic neurons. We will now use live imaging of cultured midbrain dopamine neurons to characterize further the relationship between release ofthe two transmitters. Since VGLUT2 is highly expressed by ventral tegmental area (VTA) dopamine neurons early in development, we will also determine how the VGLUT2 cKO influences monoamine release and synapse formation in vitro, and extend the analysis in vivo using brain slices. 2) Characterize the properties of dendritic dopamine release. Using a pHluorin-based reporter for the vesicular monoamine transporter VMAT2, we have obsen/ed exocytotic events in the dendrites of cultured midbrain dopamine neurons. We will now use imaging to assess the role of release from dense core vesicles and endosomes, and its regulation by action potentials, calcium and synaptic input We will also examine the signals on VMAT2 responsible for its trafficking. The analysis of glutamate co-release by dopamine neurons will explore novel exocytotic pathways relevant to the role of dopamine neurons in reward, but also to many other neuronal populations which have recently been shown to mediate co-release. Similarly, the analysis of dendritic dopamine release will illuminate a process that may contribute to retrograde signaling and plasticity at many synapses.

A group of brain nuclei collectively known as the basal ganglia are involved in learning to perform complex behavioral tasks. A major instructive signal for learning these tasks is the brain chemical dopamine, which is thought to signal important environmental cues related to rewards or positive outcomes, thereby allowing the brain to more effectively learn how to perform tasks that lead to reward. Unfortunately, addictive drugs hijack this system by directly causing the release of dopamine, thereby signaling a false reward signal, and leading to reinforcement ofthe behaviors associated with drug administration itself. By understanding how dopamine causes plastic changes in the brain that lead to addictive behaviors, we hope to be able to develop treatments for this devastating neurological condition. This project takes a unique and novel approach to this problem. In Aim 1, we apply new tools and methods that allow for highly selective stimulation of defined cell populations. We will perform electrophysiological recordings in brain slices taken from one ofthe least understood parts ofthe basal ganglia: the substantia nigra pars reticulata (SNr). The SNr is one ofthe two major output regions ofthe basal ganglia, and is therefore in a privileged position to control the signals that leave the basal ganglia and regulate cortical and subcortical motor control systems. Although early studies demonstrated its sensitivity to dopamine signaling and its importance in animal paradigms of addiction, little progress has been made, due to technical difficulties in disentangling the function ofthe complex brain circuits that are integrated in this region. In Aim 2, we will develop and exploit a new paradigm for addiction that involves optogenetic self-stimulation ofthe direct pathway circuit. This behavior is highly reinforcing and increases in frequency over many days, and may share key mechanistic features with psychostimulant addiction. We will dissect the mechanisms ofthis behavioral reinforcement in the SNr, and then in Aim 3, we will perform in vivo recordings to determine how direct pathway strength is modified during the acquisition of a highly-reinforced behavior involving direct pathway activation. RELEVANCE (See instructions): Drug addiction and other compulsive behaviors represent major public health problems that impact not only those addicted, but society as a whole. Here, we utilize cutting edge technology to dissect the function of brain areas involved in addiction, with the goal of developing new therapies to treat these maladaptive behaviors.

DESCRIPTION (provided by applicant): This is an application for renewal of support for a successful training program for students admitted to the UCSF graduate program in Neuroscience. The goal of this training program will continue to be to provide the best possible education and training of students in concepts and methods of modern neuroscience in order to give these students the skills, knowledge and enthusiasm needed for them to make creative contributions to Neuroscience during their entire careers. The Neuroscience Program currently has 72 faculty members in 18 different basic science and clinical departments and affiliated institutes. Program membership is restricted to faculty who contribute to program activities and faculty memberships are periodically reviewed. Virtually all areas of neuroscience are encompassed by the research interests of our faculty who have received numerous honors, including Nobel and other highly prestigious prizes. Our faculty includes many members of the National Academy of Sciences, the Institute of Medicine and the American Academy of Arts and Sciences. Over the past 3 decades this program has recruited many of the most able and imaginative students interested in Neuroscience. Many of these have now initiated or are well established in careers at leading American or international universities and elsewhere. Several of our older graduates already have careers of distinction recognized by prestigious awards, such as MacArthur and McKnight faculty awards. Our more recent graduates have received also prestigious awards, including Helen Hay Whitney postdoctoral scholarships, and Harold Weintraub and Donald B. Lindsley Awards for outstanding Ph.D. theses. During their first year, trainees will receive a strong foundation in modern neuroscience as well as scientific writing and oral presentation skills through an introductory 2-quarter-long core course, intensive mini- courses, a grant-writing workshop, and oral defense of a written minor proposal. The minicourses are sponsored in collaboration with other biomedical science graduate programs and are intended to stimulate creative theses at the interface of disciplines as well as provide training in how to efficiently identify the major obstacles to progress and opportunities in a new research area. The trainees will attend a weekly seminar series and weekly student-faculty journal club. They will also complete at least three laboratory rotations in more than one area of Neuroscience before choosing a thesis laboratory. The program introduces students to our many laboratories through faculty and advanced student/postdoc presentations at our annual retreat and a series of dinners for first year students with small groups of faculty. During their second year, students will complete a Scientific Ethics course, begin advanced literature-based special topics courses for 2nd year and more senior students, and continue attendance at seminars, journal clubs, and the annual retreat. They will also write and orally defend a thesis proposal in order to advance to candidacy. All trainees will receive comprehensive research training in the laboratory of their Ph.D. supervisor and this will be their major commitment after advancement to candidacy although they are expected to continue participating in program intellectual activities. Students meet no less than annually with their thesis committees and at least every six months with their thesis committee chair, who is not their research supervisor. Student progress is also supervised by a Student Progress Oversight Committee that reviews thesis committee reports. At every stage of their training, students have access to activities sponsored by the UCSF Graduate Education in Medical Science Program (GEMS) which is an HHMI and School of Medicine-funded program that sponsors courses and activities aimed at increasing the medical literacy and knowledge of graduate students. The UCSF Office of Career and Professional Development sponsors many workshops for our students to help them develop their leadership, time management, oral and written presentation and other skills. This office also sponsors many seminars and information sessions aimed at increasing the knowledge of our students about their future career options. This training grant has been the single most important pillar underlying our program's success over the past 30 years. This training grant is used to support students during their first and second years of study before they have advanced to candidacy and initiated full-time Ph.D. thesis research. Continued support is essential for us to continue to provide graduate students with the education they will need to become leaders in their field in the future.

Abstract Behavior is motivated by reward, and the most powerful rewards are those that satisfy a physiologic need. For decades, neuroscientists have studied the midbrain dopamine system to understand reward and hypothalamic circuits to understand sensing of internal needs. But how these two neural systems are interact to give rise to behaviors like eating and drinking remains poorly understood. Recently, we have used approaches for simultaneous neural recording and manipulation to observe directly the communication between these two systems. We have also mapped the signals they each receive from the gut in response to ingestion of food and fluids. This has revealed that hunger and thirst powerfully modulate the dopamine system, but do so in different ways and likely involve distinct circuit mechanisms. We propose here to build on these findings to systematically delineate how these neural circuits for need and reward interact in the brain. In Aim 1, we investigate how these circuits represent internal needs, by recording their dynamics at multiple levels of analysis under different physiologic states, and further measuring how those dynamics are influenced by targeted circuit manipulations. In Aim 2, we investigate how these circuits use information about bodily needs to drive learning about food, by monitoring and manipulating their activity during the learning process. In Aim 3, we investigate how these circuits use information about internal state to drive motivation, by monitoring and manipulating their activity during tasks where animals must evaluate competing needs and rewards. These studies will provide fundamental insight into the mechanisms by which information about body needs is utilized by the brain to generate learning and motivation.