Paul E. Micevych - US grants

The main objective of the proposed research is to elucidate the relationship between steroid effects on hypothalamic cholecystokinin (CCK) and the hypothalamic regulation of food intake (FI). Although the ventromedial nucleus of the hypothalamus (VMH) is considered the primary CNS site of both steroid and CCK effects on FI, we plan to identify all hypothalamic areas which contain steroid accumulating, CCK-positive neurons, to elucidate the functional nature of this interaction in vitro, and to determine the effects of steroids and CCK on FI. Steroid autoradiography and CCK immnunohisto-chemistry will be combined to determine which CCK-containing neurons bind steroids, providing a morphological basis for the interaction of steroids and CCK-neurons. Hypothalamic areas identified in the morphological studies as having an overlap of steroid-binding and CCK-immunoreactive staining will be microdissected from rats in which the reproductive cycle and pattern of FI have been monitored. The tissue will be used for in vitro superfusion experiments which will determine if steroids act primarily at the level of the CCK cell body or terminal. Moreover, this approach will enable us to detemine if steroids stimulate or inhibit the release of CCK or whether they modulate CCK release in response to other neuroactive agents such as opiates and GABA. Since tissue will be removed from intact or gonadectomized males, females, androgenized females and neonatally castrated males CCK levels and patterns of release can correlated with gonadal hormone environment and FI. A direct measure of the effects of steroids on CCK levels and FI will be done in parallel group of animals in which the cerebrospinal fluid and plasma levels of CCK will be monitored. Finally the effect of CCK or anti-CCK serum on the FI pattern of these animals will be assessed. It is expected that the study of the morphological and functional interaction of steroids and CCK neurons will identify the neurochemical structures which regulate feeding behavior and will prove particularly valuable in promoting the general understanding of this complex area of physiology.

The main objective of the present proposal is to understand how androgens reorganize the synaptic relations between androgen-sensitive spinal motoneurons and their afferents. The spinal nucleus of the bulbocavernosus (SNB) is a group of androgen-sensitive motoneurons which offer special advantages for studying this question. Steroids regulate the size and dendritic length of SNB neurons and there is recent evidence suggesting that androgens may also regulate the afferent input to the SNB. Thus, we plan to characterize the synaptic relationship of afferents with SNB cell bodies and processes, to identify neurons of origin of SNB afferents, and to determine whether afferent neurons accumulate steroids. Electron microscopic analysis of retrogradely labeled SNB neurons and their processes in males will provide an assessment of the organization of synaptic input to the cell body, and to proximal, middle, and distal dendrites of these motoneurons under varying androgen environments. Using a combination of retrograde and anterograde tract tracing, immunocytochemistry, and steroid autoradiography, we will characterize the chemical nature and origin of SNB afferents, and their ability to accumulate sex steroids. This analysis of SNB afferents is expected to shed significant light on the question of how steroids modulate the synaptic inputs to steroid-sensitive neurons in the adult CNS.

The main objective of the present study is to elucidate the relationship between the gonadal steroid environment and the organization and function of a steroid-sensitive cholecystokinin (CCK) circuit which is part of a sexually dimorphic limbic- hypothalamic system. This interconnected system, including the medial amygdaloid nucleus, bed nucleus of the atria terminalis, medial preoptic nucleus and the ventromedial region of the hypothalamus, is implicated in the integration of sensory and sex steroid cues involved in the CNS control of reproductive behavior. The sexually dimorphic distribution of the neuroactive peptide CCK in the limbic-hypothalamic system appears to be an important chemical marker for the action of steroids, especially since in this system the mechanism of steroid-regulation of CCK synthesis in males may be different from that in females. Initially, we propose to characterize the interaction between gonadal steroids and the CCK circuit in adult males by determining the metabolite of testosterone necessary to maintain the limbic-hypothalamic CCK circuit. To examine the steroidal influence on CCK release, in vitro superfusion will be utilized. In order to obtain a morphological signature of steroidal action on CCK cells, a combination of steroid autoradiography and CCK- immunohistochemistry will be used to determine if CCK cells in the male concentrate steroids. To begin to elucidate the integration of steroid information within the limbic-hypothalamic system, retrograde tract-tracing will be combined with steroid autoradiography and immunohistochemistry. The role of CCK in the male limbic-hypothalamic system will be tested by injecting CCK into discrete loci in this system. Finally, to understand the organizing-role of gonadal steroids during development, a series of rats will have their perinatal sex-steroid environment altered to produce sex-reversed adults in terms of their reproductive behavior. In two parallel groups of rats, the distribution of CCK- positive cells in the limbic-hypothalamic system of these rats will be determined and correlated with the effects of CCK on reproductive behavior. Together these studies will assess the role of CCK in male copulatory behavior and the effects steroids have on modulating the distribution and release of CCK in male rats. These proposed studies are expected to shed significant light on the important questions of how steroidal information is encoded and integrated in the CNS to influence reproductive behavior.

The main objective of the present study is to characterize the estrogenic regulation of prepro-cholecystokinin (pCCK) messenger RNA (mRNA) in cells of the limbic-hypothalamic circuit. This interconnected circuit, which includes the medial preoptic nucleus (MPN) and the ventromedial nucleus of the hypothalamus (VMH), is implicated in the integration of sensory and hormonal cues that are involved in the CNS control of reproductive behavior. Estrogen stimulates the expression of pCCK mRNA and CCK peptide and reversibly decreases CCK receptor binding levels in the VMH. The effects of exogenous CCK on reproductive behavior are estrogen dependent and site-specific; in the VMH, CCK inhibits and in the MPN, CCK facilitates lordosis behavior. The proposed research is a systematic examination of estrogenic modulation of pCCK mRNA levels in the limbic- hypothalamic circuit. Initially, we will determine whether estrogen activation of preproCCK mRNA expression is differentially regulated in cells projecting to the MPN compared with those that project to the VMH. We hypothesize that estrogen acts trans-synaptically to regulate pCCK mRNA expression and we propose to study the mechanisms through which estrogen upregulates pCCK message and peptide in the CNS. Experiments will determine if pCCK mRNA levels are regulated by estrogen when electrical activity is disrupted (with local infusions of tetrodotoxin) or when protein synthesis is disrupted (with anisomycin implants). These experiments will test also whether elevating second messengers with phorbol ester and forskolin will augment estrogen induced pCCK mRNA levels. Morphological evidence will be obtained for estrogenic stimulation of early response genes in pCCK cells and whether this is a trans-synaptic effect of estrogen. In the MPN, exogenous CCK facilitates lordosis but the type and distribution of CCK receptors has not been determined. a CCK receptor autoradiography study will map and characterize the distribution of CCK binding sites in the medial preoptic area. This experiment will help elucidate the observed facilitatory effects of endogenous CCK in the MPN. CCK-B receptors are found in the VMH, and the use of a selective CCK-B receptor antagonist, will allow us to examine the role of endogenous CCK in the modulation of lordosis behavior. We have shown that pCCK levels, in adulthood, are determined by the gonadal steroid environment. The adult steroid pattern appears at puberty. The last experiments will characterize the effects of estrogen during pubertal development of the limbic-hypothalamic CCK circuit and correlate the changes in expression of pCCK mRNA with induction of lordosis behavior. Together the proposed research will enhance our understanding not only of the limbic-hypothalamic CCK circuit but also contribute to our understanding of mechanisms involved in gonadal steroid trans-synaptic regulation of CNS neurochemistry.

neurochemistry; mental retardation; histology; biomedical facility; receptor binding; autoradiography; histochemistry /cytochemistry; immunocytochemistry; electron microscopy; microscopy; in situ hybridization;

bioimaging /biomedical imaging; histochemistry /cytochemistry

DESCRIPTION (applicant's abstract): Estrogen has profound effects on the structure and function of the CNS. Recent evidence that estrogen has neuroprotective effects and enhances spine density in the hippocampus have made the study of estrogenic activation of the CNS an interesting and timely topic. Much of our understanding of estrogen's actions in the brain is derived from studying steroid responsive cells and circuits that regulate reproductive behaviors. The present proposal is focused on the interaction of two transmitter systems, cholecystokinin (CCK) and endogenous opioid peptides (EOP) in a classic steroid sensitive system that regulates lordosis, the limbic-hypothalamic circuit. These transmitters are highly regulated by estrogen, and both alter reproductive behavior. Our working hypothesis for this proposal is that estrogen alters the interaction among the EOP and CCK systems. Preliminary results suggest that delta opioid receptors mediate estrogen-induced CCK expression, and mu opioid receptors mediate CCK release. We propose five experiments to test parts of this general hypothesis. In the first experiment, we will determine the dependence of estrogen-induced CCK release on increased CCK mRNA expression. The second experiment, using receptor and GTPgammaS binding, will characterize the functional coupling of mu- and delta-opioid receptors in response to estrogen stimulation. The third experiment, using colocalization studies, will provide morphological evidence for direct versus indirect opioid effects on CCK cells. The fourth experiment, using microdialysis and behavioral testing, will provide direct evidence of a functional interaction of CCK and EOP. Finally, in experiment five, using both in vitro and in vivo assays, we will test the interaction of opioids at the CCK receptor and vice versa. These experiments will not only tell us about the actions of steroids in specific circuits, but they will also provide important information about the interactions of neuropeptide circuits regulating behavior.

DESCRIPTION (applicant's abstract): Estrogen has profound effects on the structure and function of the CNS. Recent evidence that estrogen has neuroprotective effects and enhances spine density in the hippocampus have made the study of estrogenic activation of the CNS an interesting and timely topic. Much of our understanding of estrogen's actions in the brain is derived from studying steroid responsive cells and circuits that regulate reproductive behaviors. The present proposal is focused on the interaction of two transmitter systems, cholecystokinin (CCK) and endogenous opioid peptides (EOP) in a classic steroid sensitive system that regulates lordosis, the limbic-hypothalamic circuit. These transmitters are highly regulated by estrogen, and both alter reproductive behavior. Our working hypothesis for this proposal is that estrogen alters the interaction among the EOP and CCK systems. Preliminary results suggest that delta opioid receptors mediate estrogen-induced CCK expression, and mu opioid receptors mediate CCK release. We propose five experiments to test parts of this general hypothesis. In the first experiment, we will determine the dependence of estrogen-induced CCK release on increased CCK mRNA expression. The second experiment, using receptor and GTPgammaS binding, will characterize the functional coupling of mu- and delta-opioid receptors in response to estrogen stimulation. The third experiment, using colocalization studies, will provide morphological evidence for direct versus indirect opioid effects on CCK cells. The fourth experiment, using microdialysis and behavioral testing, will provide direct evidence of a functional interaction of CCK and EOP. Finally, in experiment five, using both in vitro and in vivo assays, we will test the interaction of opioids at the CCK receptor and vice versa. These experiments will not only tell us about the actions of steroids in specific circuits, but they will also provide important information about the interactions of neuropeptide circuits regulating behavior.

DESCRIPTION (provided by applicant): Our long term goal is to understand how sex steroids regulate G protein-coupled receptor mediated neurotransmission that underlies hormonal activation of behavior. We have demonstrated that membrane-initiated estradiol signaling (MIES) activates a circuit regulating lordosis behavior, a measure of sexual receptivity. Estradiol sequentially activates estrogen receptor-a (ERa), releasing NPY onto NPY-Y1 receptors in the ARH, which stimulates ¿-endorphin release into the medial preoptic nucleus activating ¿-opioid receptors (MORs). The present proposal builds on our discovery that MIES requires the metabotropic glutamate receptor, mGluR1a, to initiate cell signaling and that estradiol modulates membrane levels of ERa. We propose to test the hypothesis that MIES, which allows for hormonal-neural integration, regulates ERa trafficking to and from the membrane, and spinogenesis needed for sexual receptivity. Three Specific Aims are proposed: 1) MIES modulates ERa-mGluR1a levels in the membrane which will be tested through a series of pharmacological studies that systematically block or activate MIES and intracellular pathways. 2) Lordosis behavior depends on MIES-induced trafficking of ERa-mGluR1a to the membrane. Manipulations that alter ERa-mGluR1a trafficking in the ARH will be tested on MOR internalization, a measure of circuit activation, and lordosis behavior. 3) MIES integrates hormonal and neuronal signals inducing morphological reorganization of the ARH underlying sexual receptivity. We will determine how estradiol, by activating different intracellular signaling pathways, induces dendritic spine formation and maturation needed for lordosis behavior. These experiments have salience for understanding not only reproductive behavior, but estradiol cell signaling throughout the nervous system through their interaction with mGluRs.

? DESCRIPTION (provided by applicant): Neurosteroids, steroids synthesized de novo in the brain, have been implicated in functions ranging from stress to depression, anxiety, and cognition. One neurosteroid is progesterone, a classic sex hormone involved in the regulation of reproduction. We have previously shown that peripheral estradiol (E2) increases hypothalamic neuroprogesterone (neuroP) synthesis, which initiates the luteinizing hormone (LH) surge. The LH surge triggers ovulation and the luteinization of the ruptured ovarian follicle - critical event in reproduction. This estrogen positive feedback is regulated by kisspeptin neurons of the hypothalamic rostral periventricular nucleus (RP3V). We propose to use immortalized, female, adult mouse hypothalamic kisspeptin neurons, mHypoA-51s, to examine the E2 and neuroP signaling underlying the LH surge. Preliminary data indicate that mHypoA-51 cell line is a good model for RP3V kisspeptin neurons: both have estrogen receptor-? (ER?), progesterone receptor (PR) and kisspeptin. Like native kisspeptin neurons, E2 in mHypoA- 51s induces expression of kisspeptin and PR, which is trafficked to the membrane. Surprisingly, subsequent stimulation of mHypoA-51s with P4 or astrocyte conditioned media induces calcium release and augments kisspeptin expression. Our overall hypothesis is that E2 induced PR mediates neuroP activation of kisspeptin neurons to induce the LH surge. Three sub-hypotheses will be tested in vitro and in vivo to characterize the integration of estrogenic and progestational signaling underlying estrogen positive feedback: (1) nuclear ER? induces PR, which is trafficked to the membrane and activated by neuroP; (2) neuroP activation of membrane localized PR stimulates kisspeptin release; and (3), neuroP signaling in kisspeptin neurons regulates the LH surge. Together the proposed studies will test a novel mechanism of progesterone signaling in the CNS, through which integration of peripheral and central steroidal information regulates reproduction.

Purpose and Objectives Genetic and environmentally-induced developmental disorders can adversely affect central nervous system (CMS) development and function. The mechanisms of pathogenesis and neural repair as well as the translational research that will lead to the development of therapeutic treatments for regeneration of the damaged CNS have been increasingly the subject of intense study by researchers, including the members of this Center. To match the complexity of these problems one needs a multidisciplinary approach. Thus, it was to be useful to our neuroscientists to come to a single integrated neuroscience core where they could be advised by a team of expert faculty and staff of the proper approach, technique and equipment to use, to investigate their neuroscience research problems. To achieve this objective 3 cores were merged (Cell Biology, Cytology and Imaging). Expensive state-of-the-art instruments like the Zeiss Laser Scanning Confocal Microscope LSM 510 META, and the Leica Laser Cell Capture Microdissection system were purchased. Additional faculty were recruited to offer broad neuroscience expertise which together with contributions provided by other MRRC and Campus Cores, would satisfactorily address the vast needs of projects listed in the cores user table. Here the rationale for the services provided will be presented. The Neuroscience and Imaging Core provides expertise, services and equipment to support cellular and molecular neuroscience research: [unreadable] Stem Cell/ Cell Culture/Cell Transplant [unreadable] Zebrafish Facility [unreadable] Neurocytology [unreadable] Immunocytochemistry [unreadable] In Situ Hybridization [unreadable] Confocal Microscopy (Zeiss 510 META) [unreadable] Laser Cell Capture from tissue slices and cell cultures First, the in vitro/in vivo approaches of cell culture and cell transplantation will be discussed. The discovery that the adult CNS possesses the potential to regenerate via multipotent stem cells, has given rise to increasing interest on studying neural stem cells either to understand pathogenesis in neurodegenerative diseases in animal models or as potential therapeutic tools for degenerative diseases. The purpose of this core is to fulfill the need of incorporating new technology aimed at the study of the restoration of CNS function. The Core combines various specialized domains, including the culture of specific neural cells, neural stem and ES cells, their propagation, proper differentiation, identification and their use for neural transplantation. These approaches have proven to be promising in the field of CNS repair. The core integrates the state-of-the-art methodology on the preparation of embryonic stem cells (ES) and neural stem cells, as well as neuronal and glial cell cultures. Cell care and their proper identification and selection are of the essence to insure reproducible data. The selection of adequate tools such as live fluorescent labeling of cells to be used in transplant studies determines the long-term outcome of this type of studies. Investigators will be advised on the design of transplant studies and characterization of grafted cells. Zebrafish have become an important vertebrate animal model, not only for investigation of neural development but also for studies of behavior, neurodegenerative, metabolic and other disease processes, including cancer. Developmental studies in particular capitalize on this model, because of the transparency of embryos, enabling visualization of specific populations of neurons and their axons using green fluorescence-tagged transgenes, and the ability to do high throughput genetic screens. Moreover, the sequencings of zebrafish transcriptomes and the genome are near completion. A large set of mutants that affect nervous system development are already available. As such, there is a high level of interest in the Zebrafish model by members of the MRRC, as well as the overall biomedical research community at UCLA. In fact, a large centralized zebrafish facility in the Life Sciences building at UCLA is currently in the planning. Such a facility is needed to accommodate UCLA investigators who currently use, or expect to use this model, and for the recruitment of new faculty. The MRRC has enthusiastically supported this proposal, which should handle much of the space needs for large screens and the holding of major zebrafish stocks. However, much of the experimental work will need to be done either in individual laboratories, or more efficiently, in a common facility in each building shared by the investigators who use the model. Use of a common facility will be much more cost- and time-efficient because it will circumvent the need for each laboratory to learn the methods and provide routine care of fish, set up breeding, etc. It will also free up space in individual laboratories for other projects. To this end, we have dedicated a room for this purpose in the new Neuroscience Research building that will house most MRRC investigators. This room is approximately 350 ft2, and is capable of holding 3-5 racks of fish. This room will be managed by Dr. James A. Waschek with the help of Paul Zhao, SRA. Dr. Waschek recently returned from nine months of sabbatical leave at the laboratory or Dr. Stephen Ekker, at the University of Minnesota, who has used the zebrafish model for several years. Under Waschek's supervision, the technician will maintain all lines of fish, set up breedings, and provide embryos for individual investigators. The research associate will also teach embryo injection techniques to individual investigators.

ABSTRACT Our long-term goal is to understand steroid regulation of social interaction by studying limbic-hypothalamic neural circuits that control female sexually receptivity, lordosis. We demonstrated that within this circuit, estradiol membrane-initiated signaling activates an arcuate nucleus (ARH) to medial preoptic nucleus (MPN) projection responsible for transient ?-opioid receptor (MORs) activation needed for full sexual receptivity. These MOR neurons in turn project to the ventromedial nucleus of the hypothalamus (VMH) modulating lordosis behavior. The present proposal is based on these results and on results in male mice that show that the posterodorsal medial amygdala (MeApd) modulates social behavior. Because in females, MeApd projections affecting the social behavior, lordosis, are not well defined, we seek to characterize specific MeApd projections that modulate the ARH-MPN-VMH circuit underlying steroid activation of female sexual receptivity. We will test the general hypothesis that the MeApd gates estradiol-initiated opioid inhibition to modulate sexual receptivity. Three Specific Aims are proposed to functionally dissect this circuit: 1) Selective activation of intrinsic ARH NPY neurons, and MPN-projecting POMC terminals inhibit lordosis. This experiment formally tests that the sequential activation of NPY and POMC neurons inhibits lordosis. NPY-Cre and POMC-Cre neurons will be infected with a stimulatory Cre-dependent channelrhodopsin (ChR2) adeno-associated virus (AAV) and photostimulated in behaving female mice. 2) The MeApd differentially modulates sexual receptivity through glutamatergic projections to the MPN, and GABAergic to the VMH. We will use AAV-directed expression of ChR2, to stimulate, or halorhodopsin, to inhibit, glutamatergic and GABAergic MeApd outputs. Our expectation is that glutamatergic inputs to the MPN enhance the MOR-mediated inhibition of lordosis (asocial behavior), while GABAergic inputs to the VMH promote receptivity (social behavior), as measured by the lordosis response to stimulus males. 3) Sexual receptivity involves a switch from glutamatergic to GABAergic neuron activation in the MeApd. These experiments will determine if in females, as in males, the MeApd controls social behavior through shift from glutamatergic (asocial) to GABAergic (social) outputs. First, we will determine the pattern of glutamatergic and GABAergic neuron activation in the MeApd induced by estradiol, and estradiol + progesterone is associated with the change from nonreceptive/asocial behavior to receptive/social behavior. Finally, we will sequentially inhibit glutamatergic and GABAergic MeApd neurons in gonadally intact, cycling vGLUT-Cre and vGAT-Cre mice using a Cre-dependent halorhodopsin-AAV. Together, these experiments will provide a functional circuit analysis of how steroid signaling in the limbic-hypothalamic circuit leads a prototypic social interaction, lordosis behavior.