Catherine S. Woolley - US grants

DESCRIPTION: A significant proportion of women with epilepsy experience increased seizure frequency during phases of the menstrual cycle in which estradiol levels are elevated. This is termed catemenial epilepsy. Animal models of epilepsy also demonstrate that estradiol increases seizure susceptibility. Previous work in the adult female rat has shown that estradiol induces new dendritic spines and axospinous synapses on CA1 pyramidal cells in the hippocampus, a key brain structure in the generation and propagation of seizure activity. Furthermore, estradiol-induced dendritic spines and synapses are correlated with increased excitability of hippocampal neurons and decreased hippocampal seizure threshold. This correlation suggests that estradiol-induced seizure susceptibility in women with catamenial epilepsy may be due, at least in part, to hormone-mediated alterations in hippocampal synaptic connectivity. The studies in this proposal will use the adult female rat to test the hypothesis that estradiol facilitates seizure activity through alteration of hippocampal synaptic structure and physiology. The proposed experiments will use light and electron microscopy, electrophysiological recording from hippocampal slices and behavioral seizure testing to better understand how estradiol-induced changes in synaptic connectivity affect hippocampal neuronal excitability and behavioral seizure susceptibility. Three hypotheses will be tested: 1) Estradiol up-regulates a subpopulation of NMDA receptor-specific excitatory synapses; 2) Estradiol up-regulates GABAA receptor-mediated inhibition; 3)Estradiol-induced changes in hippocampal synaptic structure/function are necessary for estradiol-induced seizure facilitation. These studies will further understanding of estradiol's effects on hippocampal synaptic structure and function. In women with catamenial epilepsy, estradiol-induced changes in hippocampal synaptic connectivity could provide a structural mechanism for the increased seizure frequency seen with elevated estradiol during certain phases of the menstrual cycle. As such, this proposal will lend insight into a mechanism of catamenial epilepsy and suggest a novel role for hormone-mediated structural plasticity in control of seizure susceptibility.

The requested funds will be used to purchase a Zeiss LSM 510 laser scanning confocal microscope that will be the cornerstone instrument in a Life Sciences Imaging Facility at Northwestern University. Current imaging instruments at Northwestern are outdated and/or not capable of performing the types of imaging studies Northwestern researchers require. The new confocal microscope will be capable of high resolution, high sensitivity analysis of triple label fluorescence and 3-dimensional reconstruction from thick specimens. Items to be purchased are: a basic laser scanning confocal system with Argon and dual HeNe lasers, three photomultiplier tubes, transmitted light detection and an integrated computer system; an inverted microscope stand equipped for epifluorescence and differential interference contrast optics, Z-axis stage control; an anti-vibration table; and 3-dimensional reconstruction software. This confocal microscopy system represents a significant improvement over currently available imaging instrumentation at Northwestern. This new system will allow triple labeling studies and high resolution, 3-dimensional reconstruction of fine cellular process and subcellular compartments that are not possible with our current confocal microscope. This equipment will be used in a variety of developmental and cell biological studies, including: 1) "Estrogen-induced hippocampal seizure susceptibility," which involves imaging of neuronal axons and dendrites in rat hippocampus; 2) "Regulation of the Ci protein by signal transduction," which involves imaging Drosophila embryos and imaginal discs; 3) "Genetic control of cell fate diversity in Drosophila," which involves imaging Drosophila embryos and larvae; 4) "Control of olfactory and gustatory neuron regeneration," which involves imaging mouse embryos and explant cultures; 5) "Mechanism of activin action in granulosa cells" and "Inhibin actions on reproductive target cells," which involve imaging whole, unfixed mouse ovaries. All of these projects depend critically co-localization experiments in which it is essential to maximize resolution and color separation of individual fluorescent labels. Furthermore, many of these studies require the ability to assay the subcellular distribution of biomolecules and perform 3-dimensional reconstruction of labeled structures. These requirements necessitate a confocal microscope with the highest resolution, sensitivity and flexibility. Based upon technical considerations and demonstration of various confocal microscopy systems, we have determined that the Zeiss LSM 510 laser scanning confocal microscope is the best choice to meet the imaging needs of Life Science researchers at Northwestern University.

DESCRIPTION (provided by applicant): Human and animal studies show that females are more susceptible to drug addiction than males. The hormone estrogen appears to interact with underlying sexual dimorphism in addiction-related neural circuitry to produce females'greater susceptibility to addictive drugs. The nucleus accumbens is central in the neural circuitry of addiction, and chronic exposure to drugs such as cocaine produces persistent increases in dendritic length and dendritic spine density on accumbens neurons. These drug-induced dendritic changes suggest alterations in synaptic connectivity of the nucleus accumbens that could be related to the process of addiction, but this has never been tested directly. Furthermore, analogy to estrogen's effects on dendritic spines and synapses in the hippocampus suggests that estrogen might potentiate drug-induced dendritic changes in females, which could contribute to their greater susceptibility to addiction. However, whether cocaine-induced dendritic and synaptic changes in the nucleus accumbens occur differently in males and females has never been tested. We will use a combination of light and electron microscopy, western blotting, and whole-cell patch camp electrophysiology in rats to investigate whether and how chronic cocaine-induced changes in the dendritic arbor of nucleus accumbens neurons are paralleled by synaptic changes, and whether and how such dendritic/synaptic changes occur differently in males and females. There are 3 specific aims: 1) Use c-Fos, FosB, deltaFosB, and P-CREB immunostaining and western blots to map cocaine-responsive subregions of nucleus accumbens in male and female rats;2) Use whole-cell patch clamp recording to determine the effect(s) of chronic cocaine exposure on functional synaptic connectivity in the nucleus accumbens of male and female rats;3) Use light and electron microscopy to determine the effect(s) of chronic cocaine exposure on structural synaptic connectivity in the nucleus accumbens of male and female rats. These studies will determine whether chronic exposure to drugs of abuse such as cocaine produces long- lasting changes in the structure and/or function of addiction-related brain circuitry, and whether drug-induced changes in the structure and function of addiction-related brain circuitry differ between males and females. The results will contribute to an understanding of gender differences in drug addiction.

DESCRIPTION (provided by applicant): The long-term goal of this research is to identify molecular targets for new mental health therapeutics, particularly for affective disorders. We will approach this goal by defining cellular mechanisms of acute, estrogen receptor beta (ERbeta)-dependent regulation of synapses in the hippocampus of adult male and female rats. That acute estrogen-ERbeta signaling in the hippocampus plays a critical role in mental health is based on four convergent lines of research: First, fluctuating levels of estrogen in women are associated with changes in memory, mood, and affect. Second, animal studies support a role for estrogen in regulating affective behaviors and indicate that activation of ERbeta in the hippocampus is anxiolytic and anti-depressive. Third, ERbeta agonists delivered directly into the hippocampus can reduce behavioral measures of anxiety and depression rapidly, within 10-20 minutes. Fourth, a key estrogen, 17beta-estradiol (E2), is produced locally in the hippocampus of males and females, providing a physiological source of E2 that could act acutely to influence affective behavior in both sexes. Together, these findings indicate that understanding the mechanisms of acute E2 actions in the hippocampus could point to molecular targets for future, focused efforts to develop new therapies for the treatment of anxiety and depression. Previous studies of acute E2 actions in the hippocampus have concentrated on the postsynaptic elements of synaptic transmission. We recently discovered that E2 acutely potentiates hippocampal excitatory synaptic transmission through at least one, ERbeta-dependent, presynaptic mechanism. We propose to build on these results by investigating the molecular mechanisms of acute E2 regulation of synaptic transmission, explicitly addressing both pre- and postsynaptic mechanisms. We will use a combination of two-photon imaging, whole-cell patch-clamp electrophysiology, synaptic biochemistry, and behavioral studies to pursue three specific aims: 1) To establish whether acute E2-induced synaptic potentiation is pre- and/or postsynaptic; 2) To investigate presynaptic mechanisms of acute E2- induced synaptic potentiation; 3) To test the role of kinases in acute E2 effects on synaptic plasticity and affective behaviors. Because there is evidence that males and females are differentially sensitive to acute effects of E2 on hippocampal physiology, we will study both sexes to investigate how cellular mechanisms of acute E2 action differ between the sexes. Discovery of sex-specific mechanisms of E2 action and their role(s) in behavior could lead to sex-specific treatments for affective disorders.

DESCRIPTION (provided by applicant): The long-term goal of this research is to identify molecular targets that could be used for new mental health therapeutics, particularly for affective disorders that differ between the sexes. We will approach this goal by identifying synaptic proteins that are acutely regulated by estradiol (E2) and/or agonists selective for the ¿ form of the estrogen receptor (ER¿ in the hippocampus of adult male and female rats. The rationale for this approach is as follows: First, the hippocampus is a brain region implicated in affective behaviors and E2 or ER¿ agonists infused directly into the hippocampus acutely decrease anxiety-related behaviors in females but not in males. Second, there is strong evidence that E2 is produced locally as a neurosteroid in the hippocampus of both sexes, providing a physiological source of E2 that could acutely modulate affective behavior in vivo. Third, we have found that E2 and ER¿ agonists have differential effects on synaptic transmission in the hippocampus of males and females with the same time course as their effects on anxietylike behavior in females. These findings support the idea that sex differences in acute ER¿-dependent synaptic modulation in the hippocampus could contribute to sex differences in anxiety-like behaviors. An essential step in moving these behavioral and electrophysiological studies toward future, focused efforts to develop novel mental health therapeutics is to identify specific molecular mechanisms that act downstream of acute E2-ER¿ signaling to modulate hippocampal synaptic physiology and behavior. Here, we propose to use two unbiased and complementary proteomics approaches to identify synaptic proteins that are acutely regulated by E2 and/or ER¿ agonists in the hippocampus of male and female rats. In Aim 1, we will use two dimensional difference in gel electrophoresis followed by mass spectrometry to identify synaptic proteins that are differentially regulated by acute ER¿ activation in the hippocampus of males vs. females. In Aim 2, we will use strong cation exchange chromatography and titanium dioxide enrichment of phosphopeptides followed by mass spectrometry to identify synaptic proteins that are differentially phosphorylated by acute ER¿ activation in the hippocampus of males vs. females. Upon completion of this project, we will have a broad and unbiased profile of synaptic proteins the levels and/or phosphorylation of which differ between males and females, and/or that are differentially regulated by acute ER¿ activation in the hippocampus of males vs. females. Because we will use treatments and timing that are the same as in ongoing behavioral and electrophysiological studies, we will be able to relate specific molecular changes that we discover with proteomics to sex differences in regulation of anxiety-like behaviors and of synaptic function in the hippocampus. Discovery of sex-specific mechanisms of synaptic protein regulation in the hippocampus could identify biomarkers for susceptibility to affective disorders and ultimately lead to sex-specific treatments.

DESCRIPTION (provided by applicant): The Neurobiology of Information Storage Training Program (NISTP) is based in the Northwestern University Interdepartmental Neuroscience program (NUIN), emerging from within a multidisciplinary group of highly interactive investigators who have carried out research at different levels, and have successfully engaged in collaborative research on cellular, molecular, structural, network and system determinants of information storage. After the first 2 cycles of NIMH-supported training grant activity it is clear that NISTP is part of the fabric of the neuroscience community at Northwestern. Since its inception in 2003, NISTP has evolved by continually adding elements that enrich the training experience. Training includes a formal didactic component in the form of a special course focusing on the latest research in information storage neurobiology, three special lecture series each with the purpose of bringing the most outstanding scientists to interact with the trainees, a mock study section which provides both professional training and is a springboard for submission of an NRSA proposal (a NISTP requirement), the annual retreat that fosters a wide-range of survival skills with maximum trainee - faculty interaction, two different journal clubs reviewing recent research in the neurobiology of information storage, and a 'Buddy Program' that encourages trainees to see the 'bench-to-bedside' application of their fundamental research. With these value added components, we believe that trainees emerge from the training program both poised to advance research in fundamental biological mechanisms of learning and memory and well-positioned to develop novel translational applications.

PROJECT SUMMARY This proposal seeks to map and study the function of a likely widespread and potentially powerful modulatory system in the brain that is currently `under the radar' of both basic and clinical neuroscience: the neurosteroid estrogen system. Although estrogens have been studied for decades as reproductive hormones that act through nuclear receptors to regulate gene expression, compelling evidence has accumulated for an additional, distinct system of estrogen action in the brain. In this system, estrogens are produced directly within the brain, of both sexes, where they act through extranuclear receptors to regulate neurophysiology and behavior on a time scale of minutes. Although relatively little is known about the neurosteroid estrogen system, especially in vivo, what is known indicates tremendous potential for neurosteroid estrogens to influence brain functions broadly, from cognition to neuropsychiatric disorders to seizures in epilepsy. Despite this potential, however, current research aimed at understanding where and how neurosteroid estrogens operate in the brain is severely limited by a lack of reliable research tools. We propose to address this problem using a two-pronged approach. First, in a collaborative project, we will generate multiple lines of gene-targeted mice to visualize and map the distribution of key components of the neurosteroid estrogen system: P450 aromatase (estrogen synthase) and three estrogen receptors (ERs), ER?, ER?, and G protein-coupled ER-1 (GPER1), in both males and females. Second, we will adapt approaches for in vivo microdialysis to directly measure steroid levels in specific regions of the male and female brain to identify sites and circumstances of neurosteroid estrogen synthesis. Microdialysis will be followed by targeted manipulation of aromatase activity to investigate the functional consequences of neurosteroid estrogen synthesis. Together, these studies will address five key questions about the neurosteroid estrogen system: (1) Where in the brain are neurosteroid estrogens synthesized? (2) Under what circumstances are they synthesized in vivo? (3) Through what receptors, located where, do neurosteroid estrogens signal? (4) What are the downstream physiological and behavioral consequences of neurosteroid estrogen signaling? (5) How is the neurosteroid estrogen system the same or different in males and females? At the completion of this project, we expect to have generated the first comprehensive maps of the neurosteroid estrogen system in both the male and female brain and to have identified specific behaviors/functions that are influenced by neurosteroid estrogens. Moreover, we expect that this work will transform the field by providing powerful new tools to overcome limitations of current approaches and permit reliable investigation of the neurosteroid estrogen system for the first time.