Paul G. Mermelstein - US grants

DESCRIPTION: From organisms using the most basic of nervous systems to the intricate circuits found within the human brain, a fundamental requirement of neuronal function is that it be able to modulate its activity based upon previous experience. In many neurobiological systems, specific patterns of electrical activity will alter the responsiveness of a cell. Long-term changes in cellular excitability due to synaptic stimulation require the synthesis of new protein. This can occur through several mechanisms, including the activation of transcription factors leading to changes in gene expression. Mammalian neurons utilize a variety of second messenger systems sensitive to synaptic stimulation in order to trigger activity-dependent gene expression. Moreover, these signaling proteins regulate many different transcription factors. Using rodent hippocampal neurons, we have been particularly interested in the regulation of cAMP response element binding protein (CREB)- and nuclear factor of activated T-cells (NF-AT)-dependent transcription following brief episodes of heightened synaptic activity, as this is an established model for learning and memory. Under these conditions, both transcription factors are regulated by calcium. Yet remarkably, CREB- and NF-AT-dependent transcription is activated only following the opening of L-type voltage-gated calcium channels. Increases in intracellular calcium via other avenues are ineffective in inducing changes in gene expression. The experiments outlined within this proposal will determine the mechanism by which L-type channels are privileged in signaling to CREB and NF-AT as well as examine the physiological significance for having L-type, and not other calcium entry pathways, responsible for CREB- and NF-AT-dependent transcription. It is our central hypothesis that clustering of L-type channels results in localized calcium signaling within the regions of a neuron containing the signaling machinery responsible for triggering activity-dependent gene expression. Moreover, we believe CREB and NF-AT are differentially regulated by synaptic activity because the second messenger systems responsible for their activation exhibit differences in calcium-sensitivity. Further, we will look to determine the downstream genes being regulated by CREB- and NF-AT-dependent transcription. We will use DNA transfection and manipulation, field stimulation, pharmacology and DNA microarray techniques to achieve our goals. Our results will have implications in clinical disorders arising from dysfunction in activity-dependent gene expression such as Rubenstein-Taybi syndrome, epilepsy and drug addiction, and may help in our understanding of illnesses that result in memory deficits including Alzheimer's disease.

DESCRIPTION (provided by applicant): The striatum contributes to our ability to learn sensorimotor tasks. It is also heavily involved in the wanting, seeking and self-administration of addictive drugs. Additionally, striatal dysfunction has been linked to a variety of neurological disorders. A major component to all these long-term alterations in brain function is modifications in striatal gene expression and protein synthesis. The diversity of transcription factors that direct these changes in neuronal plasticity are just now being appreciated. Outside of the nervous system, NFATc transcription factors are critical mediators of immune responses, vascular and cardiac development, and muscle growth. Recently, these transcription factors were discovered within brain, and it is the central hypothesis of this proposal that NFAT-dependent transcription is a critical mediator of striatal plasticity. Specific Aim 1 will characterize the expression of the 4, calcium/calcineurin-sensitive NFATc isoforms within the different neuronal subpopulations of the striatum. Specific Aim 2 will elucidate the intracellular signaling pathways triggered by dopamine receptor stimulation (D1- and D2-class) that lead to alterations in NFAT-dependent transcription. Specific Aim 3 will determine whether acute and/or chronic cocaine exposure leads to heightened NFAT-dependent transcription, and also whether inhibition of NFAT results in a diminution of cocaine-induced behavioral sensitization. In sum, utilizing modern cellular and molecular techniques, this study will reveal which NFATc proteins are expressed in the striatum, the stimuli that activate of NFAT-dependent transcription, and whether heightened NFAT activity can be linked to neuronal changes observed following exposure to addictive drugs. Ultimately, this project will lead to a better understanding of the role NFAT-dependent transcription plays in shaping long-term changes in brain function.

[unreadable] DESCRIPTION (provided by applicant): Estradiol plays a critical role in sexual maturation and receptivity. These effects are well characterized: primarily mediated through intracellular estrogen receptors, estradiol initiates changes in gene expression, leading to alterations in cell excitability. But estradiol also influences neuronal plasticity via rapid actions initiated at the plasma membrane. Many intracellular signaling pathways are modulated by estradiol via this unconventional method, influencing various brain functions such as learning and memory, sensorimotor control and nociception. Yet, the mechanism by which estradiol acts at the membrane remains unknown. Preliminary data suggest that estradiol, through interactions with a membrane localized receptor, activates both group I and group II metabotropic glutamate receptors, leading to alterations in several major signaling cascades. This previously undefined mechanism of estradiol action can theoretically account for the majority of unexplained actions of the steroid hormone. The central hypothesis of this proposal is that through activation of metabotropic glutamate receptors, estradiol has profound influences upon brain function. Specific Aim 1 will characterize the effects of estradiol, through activation of metabotropic glutamate receptors, upon CREB and NFATc4, as these two transcription factors are essential regulators of many behaviors. Moreover, we will quantify mRNA expression for genes regulated by CREB and NFATc4 following estradiol administration. Specific Aim 2 will delineate the mechanism by which estradiol activates metabotropic glutamate receptors. Experiments will detail estrogen receptor specificity, gender specific differences in estradiol sensitivity, and interactions between estrogen and glutamate receptors. In sum, this study will attempt to decipher a long standing mystery regarding the actions of estradiol upon the central nervous system. It will potentially provide a unifying theory detailing a principal mechanism by which estradiol can rapidly trigger changes in cell excitability and ultimately lead to a better understanding of how estradiol modulates brain function. [unreadable] [unreadable]

c. Imaging and Tract Tracing Core - Co-Directors: Tim Ebner, Glenn Giesler and Paul Mermelstein Overview and Significance: Purpose of the Core: The Imaging and Tract Tracing Core is designed to allow researchers from diverse experimental backgrounds to take advantage of modern imaging techniques. The Imaging and Tract Tracing Core is intended to strengthen the research of the investigators by providing access to experimental approaches, equipment and skilled personnel that would not normally be readily available. The Core provides researchers with a diverse array of imaging techniques, allowing investigators to probe, at both cellular and systems levels, normal functioning as well as genetically altered changes in brain function. Importance of Core Services: There are three major Divisions in the Imaging and Tract Tracing Core: Confocal Imaging, Tract Tracing/Histology and Activity-Dependent Optical Imaging. The Confocal Imaging Division allows studies ranging from the imaging of structures in single layers of cells to the examination of deeper tissue, in fixed slices and in vivo. The Division provides investigators access and training in single and multiphoton microscopy. The Tract Tracing/Histology Division allows investigators to examine genetic changes in the nervous system at both the cellular and systems level using anterograde and retrograde tracing methods. The Activity-Dependent Optical Imaging Division provides the equipment and technical expertise for in vivo imaging of neuronal activity in mice. Optical imaging of activity-dependent signals includes flavoprotein autofluorescence, voltage sensitive and calcium dyes, pH imaging, and the hemodynamic intrinsic signal. Necessity of the Core: The visualization of nervous system structure and function is one of the most powerful approaches to understanding questions being raised in neuroscience today. Individual laboratories at the University of Minnesota have expertise in specific imaging techniques, but no laboratory has the capability to apply all of these methods in a cohesive manner. In addition, while there are several confocal microscopes on campus, access is limited due to heavy use and for the vast majority of investigators, training is unavailable. The readily available expertise and equipment of the Core allows NINDS investigators to test questions in an integrative fashion, providing greater depth to their findings and driving their research forward.

DESCRIPTION (provided by applicant): The cerebellum is essential for the control of movement and plays an important role in cognitive processes. Within the cerebellum, estrogens have long been demonstrated to affect synaptic circuitry and function. Yet to date, the mechanisms by which estradiol can alter cerebellar output have remained a mystery. Using state-of-the-art optical imaging techniques, we have found estrogen receptor signaling to be essential for the maintenance of the parallel fiber synapses connecting cerebellar granule cells to Purkinje neurons. Further, we have found that estrogen receptor regulation of the parallel fiber synapse to be dependent on locally synthesized estradiol. These effects are observed in both male and female mice. Since parallel fibers dominate the cytoarchitecture of the cerebellar cortex, and are central elements in modulating cerebellar function, we believe these data are the beginnings to our understanding of a new fundamental mechanism of estrogen action. Using the combined expertise and experiences of two complementary investigators at the University of Minnesota, our goal is to characterize this phenomenon. Specific Aim 1 will determine whether electrical activity within the parallel fiber-Purkinje cell network drives local neuroestrogen production. Specific Aim 2 will identify the estrogen receptor(s) responsible for maintaining the efficacy of synaptic neurotransmission between the cerebellar granule neurons and Purkinje cells. This Aim will also determine whether the estrogen receptors are located pre- or postsynaptically. Specific Aim 3 will evaluate the functional impact of diminishing estrogen signaling on cerebellar-mediated locomotor behavior. By characterizing the critical regulatory mechanisms for estrogen receptor support of cerebellar glutamatergic neurotransmission, we will have uncovered a heretofore unknown aspect of nervous system function. In addition, these results will have important implications in the use of estrogen receptor modulators to benefit health and its impact in the treatment of disease.

Estrogen receptors were originally characterized as intracellular receptors, acting only in brain regions that regulate reproductive behaviors. Recent discoveries have identified that various neurons throughout the nervous system exhibit responses to estradiol through the activation of surface membrane receptors. These membrane estrogen receptors are now known to lead to alterations in learning and memory, sexual receptivity, motor control and pain. The nervous system is not unique in this regard, as membrane estrogen receptors affect the reproductive organs, the cardiovascular system and bone. The goal of this project is to determine how estrogen receptors are adapted to act as surface membrane signaling molecules. The project willl test the hypothesis that palmitoylation of the estrogen receptor is the essential regulatory step in converting intracellular receptors into membrane signaling proteins. There are 23 different enzymes known to palmitoylate proteins. The principal goal is to identify which of the 23 enzymes is responsible for estrogen receptor palmitoylation, and then to determine the physiological and behavioral impact of manipulating estrogen receptor palmitoylation within brain. Identifying the proteins that palmitoylate steroid receptors provides an innovative experimental system that will challenge dogma and train personnel to integrate various in vivo and in vitro model systems. State-of-the-art cellular/molecular methods will be integrated with established behavioral approaches. Using specific assessment tools, Dr. Mermelstein will monitor the technical, intellectual and instructional development of his trainees. Dr. Mermelstein and his students will also participate in multiple outreach activities, including the development of a Neuroscience Minor as part of the undergraduate curriculum at the University of Minnesota.

DESCRIPTION (provided by applicant): In comparison to men, women are at an increased risk to abuse drugs. Across the spectrum of addiction, women show heightened intake of addictive substances, with greater craving, leading to an increased likelihood of addiction and relapse. These responses peak during the follicular phase of the menstrual cycle when estrogen levels are at their highest. These findings have been recapitulated in the female laboratory rat, where estradiol heightens multiple measures of drug responsiveness and abuse. Remarkably, the mechanisms by which estradiol mediates enhanced vulnerability to drug addiction are completely unknown. We propose a novel molecular mechanism mediating the actions of estradiol on nucleus accumbens neurons. Specifically we hypothesize that estradiol stimulation of estrogen receptor ¿ (ER¿) localized to the surface membrane of nucleus accumbens neurons activates metabotropic glutamate receptor 5 (mGluR5) signaling. Activation of ER¿/mGluR5 signaling by estradiol, in turn, affects nucleus accumbens spine structure, nucleus accumbens glutamatergic neurotransmission and ultimately responsiveness to drugs of abuse. Collectively, these studies will provide a foundation for developing novel therapeutic approaches targeted to treating drug addiction in women.

PROJECT SUMMARY/ABSTRACT Overview: For 30 years, this institutional training program at the University of Minnesota has focused on the preparation of graduate students and postdoctoral fellows for research and study in the field of drug addiction. The faculty mentors are all members of the University interdepartmental Graduate Program of Neuroscience. The 13 current trainers span four departments within the Academic Health Center. Our trainers' research programs are strongly supported by extramural funds. Each shares the common interest in understanding the changes that occur to the nervous system with drug abuse. Objective: The proposed program is to support six predoctoral trainees and three postdoctoral fellows: the same number of trainees supported by the current funding period. The vast majority of predoctoral trainees are students in the interdepartmental Graduate Program of Neuroscience. This program has been ranked as one of the top three graduate programs within the entire University. Exceptional graduate students in departmental graduate programs (e.g. Pharmacology) that minor in Neuroscience are also eligible. Graduate students become eligible for support at the end of their first year, once they select a faculty mentor who is part of the training program. For postdoctoral fellows, a strong publication history and previous training in addiction research are the most important criteria for selection to the training grant. Of the eligible postdoctoral fellows working in the laboratories of our trainers, only the top third are selected. Rationale and Design: The motivation for this training program is for all trainees to experience the breadth of topics, scientific approaches, and methodologies taken to study drug addiction. Additionally, research programs across investigators are highly collaborative. This enrichment is intended to maximize the trainees' potentials for rewarding careers in scientific thought and research. Active classroom activities and journal clubs, along with a seminar series and a program retreat reinforce the cohesive community. Additionally, the University's committed support to the neurosciences (over 100 million dollars in just the last five years) is unequalled at the institution. While breadth is important, each trainee has his/her own individual development plan. Trainees work with both their mentor as well as additional trainers to further their specific career goals. This is true for postdoctoral fellows as well, where both the PI as well as additional trainers monitor progress. Notably, the graduate students and postdoctoral fellows are not the only ones receiving preparation. Our trainers undergo additional instruction, to ensure they are better mentors beyond just laboratory advisors, which ensures an optimal environment for training.

DESCRIPTION: The profound detrimental impact to society from individuals abusing drugs is well established. To meet the challenge of developing new and effective treatments to help those that have succumbed to the temptation of drug use and abuse, we need to inspire the next generation of students to pursue research careers in the field. The need is particularly acute among populations of students who are currently underrepresented in the field of drug abuse. Published analyses indicate that exposing undergraduate students, especially early in their career, to laboratory research is an extremely effective way for developing their interest in research as a profession. Since 1989, the University of Minnesota of has recognized and met this challenge by offering summer residential research programs in the biomedical sciences. This proposal is to fund a drug abuse component to these summer programs in which we will train five undergraduate students who have completed their freshman or sophomore years in college. We will recruit students nationally, focusing on students from groups that are underrepresented within the biomedical research profession. We will provide them with a 10-week intensive research experience that will include professional mentoring (academic survival skills and preparation for graduate school) as well as workshops on research ethics. Our goal is to inspire a new generation of drug abuse researchers. In turn, we expect these individuals to become part of the research infrastructure dedicated to solving medical problems of nervous system dysfunction

? DESCRIPTION (provided by applicant): Across all stages of drug use and abuse, women exhibit heightened responsiveness and hence greater vulnerability to the properties of addictive drugs. This is especially true regarding the increased susceptibility women exhibit towards drug relapse. In women, maximal vulnerability peaks during the follicular phase of the menstrual cycle when estrogen levels are at their highest. These findings have been recapitulated in female rodents, where estradiol heightens multiple measures of drug responsiveness and abuse. The mechanisms by which estradiol mediates enhanced vulnerability to drug relapse are completely unknown. The two PIs of this proposal have been independently studying the neurological underpinnings of relapse, and the mechanisms by which estrogens affect neuronal activity. Now working together, we have generated a unifying theory, whereby estradiol activation of estrogen receptor ? (ER?), localized to the surface membrane of medium spiny neurons of the nucleus accumbens shell (NAcSh), activates metabotropic glutamate receptor 1a (mGluR1a) signaling. Activation of ER?/mGluR1a signaling by estradiol is hypothesized to in turn promote mGluR5-induced relapse, a process mediated through the long-term depression of NAcSh afferents from the infralimbic cortex. To test this theory, the studies outlined in this proposal will utilize a recently developed mouse model of estradiol facilitation of drug relapse, taking advantage of electrophysiological, optogenetic and viral technologies. Collectively, these studies will provide a foundation to better understand and develop novel therapeutic approaches to treat drug relapse in women.

DESCRIPTION Sexual dysfunction in women is primarily characterized by low levels of sexual arousal and desire. Because no effective treatments exist for disorders of sexual desire in women, the FDA was pressured to fast-track approval for the drug Addyi despite the absence of clinical evidence that the drug provided any therapeutic benefit. Underlying the inability to construct a rational approach to developing therapeutics for disorders of sexual desire in women is the lack of research on the mechanistic basis for female sexual desire in pre-clinical models. We have developed a Syrian hamster model of sexual desire that captures several essential elements needed to translate the findings to women. We developed a procedure to evaluate the rewarding properties of female sexual behavior, an element reported to be lacking in women with low sexual desire. Women with low sexual desire also do not initiate sexual contacts with their spouse or partner. In this regard we discovered an experimental approach to test the female hamster's willingness to initiate sexual contacts with a male. Based on these hamster studies we demonstrated that the rewarding consequences of sexual interactions with a male feed forward to increase the female's sexual contacts. We further identified the mesolimbic dopamine system, and prefrontal glutamatergic afferents, centered on the nucleus accumbens, as a critical neural node mediating the rewarding effects of sexual behavior in females. The research in this proposal takes three specific approaches to establish a programmatic understanding of the neurobiology of female sexual desire. In the first aim we will use inhibitory DREADDs to determine the relative contribution of dopamine and glutamate afferents to the nucleus accumbens to the development of sexual reward and initiation of sexual interactions with the male in our hamster model. We will also examine the contributions of these afferents to changes in dendritic spine morphology consequent to female sexual experience. The second aim takes a discovery approach to examining three possible intracellular signaling pathways mediating the effects of sexual experience on behavioral and morphological plasticity. The last aim will pharmacologically manipulate the individual signaling pathways to identify which of these pathways are potential molecular targets for therapeutic development to treat problems of sexual desire in women. Collectively this research will take a systematic approach to developing a neurobiology of female sexual desire with an eye to the rational development of effective therapies.

PROJECT SUMMARY/ABSTRACT Historically, research in the field of steroid hormone signaling has been primarily focused on the transcriptional effects mediated by steroid hormone receptors (SHRs) acting within the nuclei of cells. It is now recognized that the same nuclear SHRs that trigger changes in cellular physiology through the regulation of gene expression can be post-translationally modified and subsequently localized to the cytosol and/or surface membrane to mobilize a variety of intracellular signaling pathways. In addition, new SHRs that function solely outside the nucleus have been identified. These extranuclear effects of SHRs regulate a myriad of biological processes relevant to human health and disease. SHRs rarely act in isolation, but rather synchronize cytoplasmic and nuclear signaling pathways via complex networks of interacting molecules that serve to optimize physiological endocrine functions. Conversely, dysfunctional endocrine systems have a profound impact on health and can lead to various disease states. Not surprisingly, the field has rapidly expanded, such that hundreds of scientific papers are published on the topic each year. Thus, to better understand the myriad of SHR-regulated signaling pathways and their physiological and pathological impact, it is essential to bring together researchers across the field of endocrinology to disseminate new findings, generate novel hypotheses, find consensus on current issues and best approaches, and uncover new and/or unifying themes. To do so, the members of the FASEB scientific research group ?Rapid Signaling and Genomic Steroid Hormone Actions in Health & Disease will come together for the first time to with the participants of the international ?Rapid Responses to Steroid Hormones? (RRSH) to explore emerging data regarding the integration of nuclear and extranuclear SHR function. The title of this joint conference is ?The Rapid Signaling and Genomic Steroid Hormone Actions in Health and Disease.? The major aims of this conference are to (1) highlight recent research discoveries in the context of integrated SHR actions relevant to health and disease (2) promote the career development of emerging scientists and trainees to ensure the continued vibrancy of the field and (3) further existing research interactions to foster new partnerships that will advance knowledge and foster innovation.

PROJECT SUMMARY: Structural Circuits Core Modern tissue clearing and imaging methods provide the potential for a wealth of information regarding nervous system anatomy and function. However, to best utilize such methods, understanding the options for tissue clearing, and the advantages and disadvantages of various imaging methods need to be appreciated. Furthermore, data handling, analysis, and storage all provide significant obstacles when attempting to make full use of the capabilities of these modern techniques. The goal of the Structural Circuits Core (SCC) is to help researchers best utilize ultramodern technology for the anatomical mapping of brain circuitry involved in drug addiction. In order to do so, the SCC partners with the University Imaging Center (UIC), the University of Minnesota Informatics Institute (UMII), the Minnesota Supercomputing Institute (SCI) and the Data Repository for the University of Minnesota (DRUM). The SCC provides the infrastructure for automated use of brain clearing technology paired with meso- and micro-scale imaging of the central nervous system. Importantly, the SCC helps tailor experiments to best utilize the most appropriate methodological and technological approaches, and, integrating with the Addiction Connectome Core, provides standardized resources for the analysis, storage, and distribution of these imaging datasets. The SCC not only allows investigators to quickly and thoroughly interrogate their own data, but also allows quantitative comparisons across laboratories through a common format structure and custom designed software.

Despite the many initiatives designed to increase diversity among neuroscience researchers, the sad fact is that there has been a decline in the proportion and absolute numbers of minority researchers in the field of neuroscience. This failure is disturbing at a number of levels, but perhaps most notably highlights the inability of more senior investigators to effectively mentor junior investigators in their discipline. The loss of diversity among career scientists means a loss of diversity of thought, which in turn limits the generation of new ideas and scientific progress in the neurosciences. The inability of the multiple training programs to increase diversity is not an indictment of those programs, but rather it illustrates limitations in program approach. As a result, we propose to take a different route to addressing this problem. Our program is based on published findings that underrepresented individuals are lost in the system at transition points in their training, i.e., graduate school to postdoctoral training, postdoctoral training to faculty members, and tenure for young faculty. From self-report data, a principal contributing factor regarding attrition is that individuals feel isolated by not having meaningful ethnic and/or racial peer groups. In this R25 program we propose to take a longitudinal approach to mentoring in which we establish peer groups across each stage of professional development and utilize these peer groups to provide interactive mentoring within a community. We will recruit from across the nation a cohort of trainees from underrepresented backgrounds, consisting of graduate students, postdoctoral fellows, and early stage investigators. For each of these cohorts we will provide longitudinal training in professional skills and mentoring. Through both mentor/mentee and peer-to-peer/peer-to-near peer mentorship structures, we will tailor professional development consistent with the progression of the trainee, while simultaneously creating a network of support amongst its participants. A key element of the program will be to teach the participants in the program how to be effective mentors themselves as a means to maintain the longitudinal development of program participants. Finally, we will provide access to high-level research cores and laboratories to enhance the scientific impact of the participants? research. Participants will meet in Minnesota for one week each summer for professional training and guidance. For topics that span across career stage, the cohort will work together. There will also be specialized training sessions specific to academic rank. In addition to the on-site summer training, there will be two additional formal training events. In the fall, our trainees and mentors will meet at the annual Society for Neuroscience meeting to participate in both professional development and a social event. In the winter, there will be an on-line video teleconference discussion following the group?s completion of an on-line professional development session. Overall, our goal is to fundamentally alter mentored training, and significantly enhance workforce diversity in the academic pursuit of research on neurological disorders.