David Morilak - US grants

hypothalamic pituitary adrenal axis; stress; alpha adrenergic receptor; norepinephrine; receptor expression; mifepristone; restraint; messenger RNA; neuropharmacology; corticosterone; glucocorticoids; paraventricular nucleus; amygdala; hippocampus; hormone regulation /control mechanism; neural plasticity; adrenocorticotropic hormone; receptor sensitivity; beta adrenergic receptor; psychological stressor; digital imaging; laboratory rat; immunocytochemistry; in situ hybridization;

Over the past two decades, obtaining a better understanding of the physiological significance of co- localization of neuropeptide and classical neurotransmitters such as norepinephrine (NE) in the brain has been an important yet elusive goal in neuroscientific research. NE modulates a variety of processes involved in the response to stress. However, several observations have suggested that NE interacts in important ways with other modulatory neurotransmitters with which it may be co-localized, for the full manifestation of this modulatory influence. In this project, we propose using a multidisciplinary strategy of pharmacological, physiological, behavioral, neurochemical and anatomical approaches to investigate the functional interaction of NE and galanin (GAL), a neuropeptide with which it is prominently co-localized. It is thought that NE acts in limbic forebrain regions, such as central amygdala (CeA) and lateral bed nucleus of the stria terminalis (BSTL), to facilitate neuroendocrine and behavioral components of the stress response. We hypothesize that under conditions of intense activation of the noradrenergic system, GAL is released along with NE in the CeA and BSTL, and acts to buffer the modulatory effects of NE on these affective and hormonal components of the stress response. These hypotheses will be tested by exposing rats to acute immobilization stress, and selectively amplifying the stress-induced activation of the noradrenergic system by pretreatment with yohimbine. The first two aims are pharmacological investigations. In Aim 1, a GAL antagonist will be microinjected, alone or with an adrenergic antagonist, directly into CeA or BSTL of rats to test the hypothesis that GAL attenuates the facilitatory effect of NE on stress-induced ACTH secretion when stress-induced activation of the NE system is amplified by yohimbine. Similarly, Aim 2 tests the hypothesis that GAL attenuates the facilitatory influence of NE on stress-induced anxiety, as measured on the social interaction test and the elevated plus maze test. The last two aims are to investigate the neural mechanisms and substrates underlying these interactions. Aim 3 addresses the neurochemical substrates underlying these interactions by measuring GAL release in CeA and BSTL using microdialysis. Finally, in Aim 4, immunocytochemistry and in situ hybridization will be used io reveal the anatomical substrates for these interactions, by identifying cells in CeA and BSTL that are activated by stress and also express post-synaptic receptors for NE and GAL. In sum, the experiments outlined in this proposal represent a multidisciplinary investigation of the interaction of the neuropeptide GAL with the classic monoaminergic neurotransmitter NE, in modulating hormonal and affective components of the stress response in CeA and BSTL. By providing a basic neurobiological context within which to better understand the functional and regulatory interactions between monoamines and neuropeptides, these results may guide the future development of novel or more effective therapeutic strategies for the treatment of stress- related psychiatric disorders such as depression, anxiety or PTSD.

DESCRIPTION (provided by applicant): Chronic stress is a factor in many psychiatric diseases, such as depression, PTSD and other anxiety disorders. The brain noradrenergic (NE) system is important in arousal and acute stress reactivity, and is implicated in the etiology of stress-related psychiatric disorders. In the previus grant period, we showed that increasing NE transmission acutely in the prefrontal cortex (PFC) of rats facilitates cognitive flexibility on an attentional setshifting test (AST). By contrast, chonic unpredictable stress (CUS) compromised cognitive flexibility. We also showed that after CUS, elevating NE acutely still facilitates PFC function, but blocking NE receptors in the PFC during CUS protects cognitive flexibility. This suggests that after chronic stress, evoking NE activity acutely is still beneficial in the short term, but this comes at a cost. Over time, this repeated facilitation takes a toll, compromising the same circuits that are facilitated acutely, ultimately inducing a cognitive deficit. The purpose of this project is to identify the mechanisms by which the processes that mediate cognitive flexibility in PFC are compromised by repeated NE modulation during chronic stress. We focus on glutamate neurotransmission in the PFC, both pre- and post-synaptically. In Aim 1, we will first determine the adrenergic receptor subtype by which repeated elicitation of NE activity during CUS compromises cognitive flexibility and function of the medial prefrontal cortex (mPFC), which mediates cognitive set-shifting, and the orbitofrontal cortex (OFC), which mediates reversal learning. Rats will be exposed to 2 weeks of CUS, with or without local administration of selective ?1- or ß-adrenergic receptor antagonists into mPFC or OFC prior to each stress session. They will be tested drug free for cognitive performance on the AST after CUS is terminated. The effective antagonist will be used in all subsequent aims. In Aim 2, we will use microdialysis to measure CUS-induced changes in, and adrenergic antagonist protection of glutamate release in PFC in response to acute stress, excitatory afferent activation, or during cognitive performance on the AST. In Aim 3, changes in post-synaptic PFC response to glutamate activity will be assessed. We will measure changes in fos induction in the PFC in response to activation of excitatory glutamate afferents from the thalamus, hippocampus and contralateral PFC. We will identify the glutamate receptors responsible for different aspects of cognitive flexibility in PFC, then measure CUS-induced changes in expression, phosphorylation and membrane localization of those receptors, and their protection by adrenergic antagonist treatment during CUS. We will measure changes in expression of PSD95, a post-synaptic protein important in regulating glutamate synaptic plasticity. And in Aim 4, we will assess changes in downstream JAK2-STAT3 signaling and its modulation of cognitive flexibility. These results will help us better understand the mechanisms of stress-induced pathology that underlie dysregulation of PFC function and cognitive capability, and will help us identify novel therapeutic targets for treatment of stress-related psychiatric disorders in patients who are only partially-responsive or resistant to existing approaches.

[unreadable] DESCRIPTION (provided by applicant): Changes in monoaminergic neurotransmission contribute to time-dependent modifications in behavior, affect and cognition that comprise both the antidepressant and anxiolytic effects of chronic antidepressant drug treatment, including selective norepinephrine (NE) reuptake inhibitors such as desipramine (DMI). Elevation of tonic levels of noradrenergic activity has been implicated in arousal, vigilance, and attention, which could improve inhibitory symptoms of depression. However, anxiety is also a prominent component of depression, and phasic, acute stress-evoked activation of noradrenergic neurotransmission enhances anxiety-like behavioral responses to stress. Therefore, it is unclear how enhancing NE transmission could contribute to anxiolytic as well as antidepressant effects. Nonetheless, selective NE reuptake inhibitors are effective in resolving anxiety-related symptoms as well as inhibitory symptoms of depression. Thus, in this project, we hypothesize that chronic NE reuptake blockade differentially regulates tonic- and phasically-activated noradrenergic transmission to account for this dual effect. To test this, we propose a series of experiments using microdialysis, together with a series of behavioral pharmacological studies, to examine time-dependent neurochemical changes in noradrenergic neurotransmission, and corresponding changes in behavioral measures of attentional set-shifting capability and acute anxiety-like behavioral reactivity on the elevated plus-maze and defensive burying tests, after chronic treatment of rats with DMI. We predict that tonically elevating noradrenergic activity in the medial prefrontal cortex will enhance arousal and attention, but that a concurrent attenuation of phasic, stress-activated NE neurotransmission in limbic regions such as the bed nucleus of the stria terminalis and lateral septum via autoreceptor-mediated inhibition, will reduce acute anxiety-like behavioral stress reactivity. Key observations made following DMI treatment will also be verified using other selective NE reuptake inhibitors, reboxetine and atomoxetine, which lack the potential non- selective post-synaptic antagonist activity of DMI. Relevant to the NIMH mission, a better understanding of how these regulatory processes contribute to both antidepressant and anxiolytic efficacy of AD drugs may offer novel insights for future development of more effective, more specific or more rapid treatment, or even ultimately to the prevention of serious mental health problems such as depression and anxiety disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cognitive dysfunction related to changes in prefrontal cortex are prevalent in depression and anxiety disorders. Chronic stress is a risk factor in these illnesses, interacting with alterations in serotonergic function;and drugs that block the reuptake of serotonin (SSRIs) are used in the treatment of these disorders. However, it is not known how chronic stress affects serotonergic activity in prefrontal cortex, nor how that may contribute to deficits in executive function and cognitive flexibility. In this pilot project, an attentional set-shifting test (AST) will be used to assess a role for serotonin (5-HT) in chronic stress-induced deficits of cognitive flexibility in rats. Two weeks of chronic stress induced a selective deficit in reversal learning on the AST, which has been linked to orbitofrontal cortex, and which may be modulated specifically by 5-HT. Aim 1 will be to characterize the duration of this cognitive deficit, as well as anxiety-like behavior, following two weeks of chronic stress, and also to assess the deficit after 5 weeks of stress. This will determine the design of the chronic drug treatment studies to be used in aim 3. Aim 2 will test the hypothesis that stress-induced cognitive deficits in reversal learning on the AST are associated with reduced 5-HT activity in orbitofrontal cortex. Changes in 5-HT release during behavioral testing will be measured using microdialysis, and changes in post-synaptic 5-HT receptor binding density will be measured by quantitative autoradiography. Aim 3 will test the efficacy of chronic treatment with the SSRI escitalopram, delivered by osmotic minipump, in alleviating the stress- induced cognitive deficit. First, the ability of escitalopram to prevent the cognitive deficit will be tested, by administering drug during the 2-week treatment. Next, the ability of escitalopram to reverse the cognitive deficit will be tested in one of two designs, depending on the outcome of aim 1. Drug will be given beginning after treatment is complete and continued for 3 weeks until testing, or drug will be given beginning after 2 weeks of stress, continuing both drug and stress treatment until testing. The results of this project will add to our understanding of the neural mechanisms underlying chronic stress-induced psychopathology, and the mechanisms by which therapeutic drugs may exert their effects. They will hopefully lead ultimately to a more comprehensive proposal to explore the mechanisms underlying specific cognitive deficits induced by different stressors, modeling different components of depression and anxiety, possibly involving different neurotransmitter systems and sub-regions of prefrontal cortex, and perhaps predicting preferential response to different classes of therapeutic drugs. PUBLIC HEALTH RELEVANCE: This project will add to our understanding of how chronic stress is related to psychiatric illnesses such as depression or anxiety disorders, and how therapeutic drugs such as antidepressants may exert their effects. Further, the results may improve the treatment of these disorders, by suggesting that a more careful and precise evaluation of the specific cognitive deficits exhibited by a patient might better predict the most effective treatment strategy.

DESCRIPTION (provided by applicant): This proposal is submitted by the Mood Disorders Translational Core Center (MD-TCC), within the Center for Biomedical Neuroscience at the University of Texas Health Science Center at San Antonio. The MD-TCC is comprised of a core group of preclinical and clinical investigators from the Departments of Pharmacology and Psychiatry who share an interest in research into the mechanisms and treatment of depression and other mood disorders. This is an active and interactive group, with ample evidence of collaborative publication and funding. The proposal is for funding to support the recruitment of two high-caliber new faculty members into this group over two years, requesting $250,000 per year in direct costs for each to support their salary and benefits as well as salary and benefits for one new laboratory research personnel slot for each, equipment and supplies as appropriate to their new research programs. One of these new faculty will be a preclinical scientist with a primary appointment in the Department of Pharmacology, and the other will be a translational/clinical scientist with a primary appointment in the Department of Psychiatry. Both will have been trained in an environment of collaborative translational research. We plan to try to coordinate the recruitment of these two scientists such that their own research interests will not only complement and integrate them into the MD-TCC, but will also complement and Integrate with each other. Possible areas in which we plan to target our search could include genetics and epigenetics of depression;etiology and treatment of co-morbid cardiovascular disease and depression;neuroimmune function and brain cytokine signaling in depression;factors affecting vulnerability and resilience in development and aging. The senior members of the MD-TCC will not only provide the new faculty members with ample opportunities for productive collaboration, but will also provide mentoring, advice and guidance in many aspects of their career development, and will provide a peer group assisting them in the development and refinement of research programs that will be competitive for renewable external funding, most notably from NIMH. The translational research activities of the MD-TCC, and those of the new faculty that this center grant will support, are consistent with the goals of the NIMH strategic plan, and of the Recovery Act. PUBLIC HEALTH RELEVANCE: Depression is a debilitating and costly disorder. Consistent with the goals of the NIMH Strategic Plan, the MD-TCC brings together preclinical and clinical researchers at the UTHSCSA, to foster the movement of basic science discoveries into clinical research, and ultimately to improving the treatment of depression. This proposal advances that purpose by supporting the recruitment of two new faculty into this group.

DESCRIPTION (provided by applicant): Cognitive dysfunction is a major component of depression, and may in fact underlie many of the mood symptoms. Specifically, a deficit in cognitive flexibility, associated with hypoactivity in the medial prefrontal cortex (mPFC), creates negative biases about the self, the world and the future. Chronic stress is also a major risk factor for depression, and we have shown previously that performance on an attentional set-shifting test (AST), which is a measure of prefrontal-dependent cognitive flexibility in rats, is compromised by chronic stress. It is also facilitated by the monoamines, norepinephrine (NE), and serotonin (5-HT), and is responsive to antidepressant drugs that block the reuptake of NE and/or 5-HT. Such drugs are effective antidepressants, but their effectiveness is limited, as partial response and treatment resistance remain significant problems for the treatment of depression. As an alternative to pharmacotherapy, evidence-based psychotherapy, primarily involving cognitive therapy or cognitive-behavioral therapy (CBT), has efficacy comparable to antidepressant drug treatment, and the combination of pharmacotherapy and psychotherapy has efficacy greater than either alone. But little is known about the mechanisms involved. The cognitive set-shifting test, used extensively in the previous funding period to investigate antidepressant drug mechanisms, bears conceptual similarity to CBT, in that it requires rats to modify previously established contingencies based on feedback from a changing environment. Thus, to extend this work, the four specific aims comprising this competing renewal will address the neurobiological mechanisms underlying evidence-based psychotherapy, using cognitive set-shifting as a rat model of CBT. In aim 1, the antidepressant-like effectiveness of this model of CBT will be further established. Chronic unpredictable stress (CUS) will be used to create a deficit of cognitive flexibility. The cognitive set-shifting test will be used as the model of psychotherapy. And to avoid learning effects, a second measure of prefrontal-dependent cognitive flexibility will be used as the dependent measure, the extinction of cue-conditioned fear. In aim 2, based on our past results with antidepressant drugs, the role of 11-adrenergic receptors and 5-HT2A serotonin receptors in mPFC in the beneficial effects of CBT will be investigated, using local microinjections into mPFC. In aim 3, the effectiveness of adjunct treatment with drugs that block reuptake of NE and 5-HT, in combination with CBT, will be studied. And in aim 4, the generality of this model will be tested by switching the roles of the two behavioral tasks - extinction will serve as the model of psychotherapy that engages prefrontal cortex, and set-shifting will be the dependent measure of cognitive flexibility that is compromised by chronic stress. This project addresses the neurobiological mechanisms underlying the efficacy of evidence-based psychotherapy, using a novel rat model of CBT, in the face of chronic stress- induced deficits of cognitive flexibility. The results will therefore generate new knowledge upon which to develop strategies that are more customized, rapid, specific and effective in the treatment of depression. PUBLIC HEALTH RELEVANCE: These preclinical laboratory studies will address neurobiological mechanisms underlying the efficacy of evidence-based psychotherapy, using cognitive set-shifting as a rat model of cognitive-behavioral therapy. They will also address mechanisms underlying the increased efficacy of adjunct treatment, combining antidepressant drugs with psychotherapy. Understanding these processes will lead to new knowledge by which to develop strategies that are more customized, more rapid, more specific and more effective in the treatment of depression.

The Integrated Graduate Program in Neuroscience at UTHSCSA provides training in a range of neuroscientific research areas and approaches, from molecular, cellular, and neurochemical to systems, behavioral and clinical neuroscience. With 30 training faculty drawn from the mentoring faculty in the Neuroscience Program within the Integrated Biomedical Sciences Graduate Program (IBMS) at UTHSCSA, this Training Program offers a course of study tailored to the individual needs and interests of students who come to us from a variety of backgrounds in the basic biological sciences, including biology, biochemistry, chemistry, molecular biology, psychology and neuroscience. An interactive and collegial community of educators and researchers creates a challenging yet supportive environment within which our students can develop into successful neuroscientists. Program Goals are to provide our students: I) a curriculum that gives them a broad foundation of fundamental concepts in Neuroscience; II) skills and knowledge necessary to conduct high-quality Neuroscience research; and III) training in ethical behavior and responsible conduct, and professional skills that will prepare them for successful careers as independent neuroscientists. A set of 8 Student Learning Objectives that service these goals form the basis for a program evaluation and monitoring system that informs ongoing evolution of our program, including changes, additions and improvements to curriculum, professional development and enrichment activities, and other elements of the program. The progress of our students is facilitated and monitored by a robust administrative and organizational structure, and experienced program leadership. Students enter the IBMS, and are fully supported by the graduate school during their first year. In the first semester they take a common biomedical sciences course and conduct lab rotations. They join a lab and begin the Neuroscience curriculum in the spring semester. The students to be supported by this T32 training grant are selected at the end of the first summer, to be supported during their second year of study, during which they complete the core Neuroscience course curriculum, conduct a Clinical Practicum in Neuroscience, develop their research project, and engage in several enrichment and professional development activities before completing the Qualifying Exam at the end of year 2. In the year following support, students present their dissertation proposal and submit individual fellowship applications. Other advanced-stage T32s on our campus may support post-QE Neuroscience students working in relevant areas while they navigate the fellowship submission and revision process. Our program has been successful, with a nearly 90% retention and completion rate, and essentially 100% of students who completed their degree proceeded to research-intensive or research-related positions. We also have a strong history of successful outcomes training students from under-represented populations, while maintaining national competitiveness. Renewed funding of this training grant will continue the growth and development of our program, and enhance our ability to train our students for the challenges of the future.

DESCRIPTION (provided by applicant): The proportion of underrepresented minorities (URMs) earning doctorate degrees in the basic biomedical sciences has increased modestly over the past few decades. Our proposed Program, the South Texas Advanced Research Training Undergraduate Program (START-UP) is a response to a Funding Opportunity Announcement from the Blueprint Program for Enhancing Neuroscience Diversity through Undergraduate Research Education Experiences (BP-ENDURE). As such, the overall goal of our Program is to encourage and prepare junior and senior undergraduate URMs from the San Antonio and South Texas Region to enter doctoral programs in neuroscience, to complete them successfully, and become well-trained and competitive neuroscientists. To accomplish this, a comprehensive program is proposed for the URM students accepted into the program, involving extensive research experiences in the laboratories of successful neuroscientists, and opportunities to develop and improve their writing, speaking, and time management skills. Students will be recruited into START-UP from five partner institutions in San Antonio, namely Our Lady of the Lake University, St. Mary's University, Trinity University, University of the Incarnate Word, and the University of Texas, San Antonio. Collectively these schools have 24,527 undergraduates who are URMs (based on ethnicity), of whom 2,947 are Science Majors. Also, these schools have a high number of students from low income families, many of whom are the first in their families to attend college. Faculty contacts have been established at each school to assist us recruit suitable students into START-UP. Thirty-one training faculty have been identified (including three from UTSA), who are appropriate to mentor these students in their laboratories. The students will participate in laboratory research for an average of 12 hours per week during the two academic semesters, and 40 hours/week during a 10-week intensive summer research exposure. Students will also have an opportunity to work in one of seven major neuroscience programs at institutions outside of San Antonio during the summer. In addition to their laboratory research, the students will also attend seminars and journal clubs, research retreats, and have exposure to neuroscientists from other institutions. The students will all receive instruction on the responsible conduct of research. The Co-Directors of the Program are Drs. Alan Frazer and David Weiss, experienced scientists and administrators, who have run programs similar to START-UP previously. They will be members of an Executive Committee that will oversee all aspects of the Program. There is a formal evaluation plan for the Program, as well as an outcomes assessment process. Further, a plan is described to disseminate nationally all materials developed for the design and implementation of START-UP.

Cognitive impairment has a serious detrimental impact on the quality of life for prostate cancer survivors treated by androgen deprivation therapy (ADT). Neuroimaging studies have shown structural and functional deficits in the medial prefrontal cortex (mPFC) and hippocampus (Hipp), which mediate higher order cognitive processes, including cognitive flexibility and spatial cognition, that are impaired after ADT. In this project, we will investigate mechanisms that underly the cognitive impairments we have now shown to be induced by androgen deprivation in rats. Also, as there is currently no satisfactory treatment for cognitive impairment after ADT, we will test the efficacy of vortioxetine, a novel multi-modal antidepressant drug that has been shown to have specific and unique positive effects on cognitive impairment in depression, in potentially reversing cognitive impairment after ADT. Similar to SSRIs, vortioxetine blocks the serotonin transporter, but it also has direct actions on several pre- and post-synaptic serotonin receptors that give it additional efficacy against symptoms of depression that are often resistant to treatment, including cognitive impairment. To assess mPFC-mediated cognitive function in rats, we will use the Attentional Set-shifting Test (AST). And to assess spatial cognition mediated in the hippocampus (Hipp), we will use the Novel Object Location (NOL) test. We have already shown that ADT induces an impairment in cognitive set-shifting. Thus in aim 1A, we will complete the pilot study in which we have preliminary data showing a spatial cognition deficit in the NOL test as well. In addition, there are many factors to consider in the full context of treating prostate cancer. Thus, in the rest of aim 1, together with ADT and vortioxetine, we will investigate the interacting influences of factors including age-related cognitive decline; an alternate method of inducing ADT with the GnRH antagonist degarelix; and chemotherapy with docetaxel. Indeed, because of recent changes to standard of care, we will also include docetaxel in all subsequent aims. In aim 2, we will then study neural processes related to functional plasticity that may underly the cognitive effects of ADT and vortioxetine, measuring changes in electrical response evoked in mPFC by stimulating afferents from the mediodorsal thalamus (MDT) and ventral Hipp, an indication of synaptic efficacy and functional integrity of cortical circuits. In aim 3, we will study processes related to structural plasticity, measuring changes in dendritic complexity and synaptic spine density and morphology on pyramidal cells that drive the behavioral output of the mPFC and Hipp. Effects of androgens are mediated by gene transcription and protein expression. Thus, in aim 4, we will assess changes in gene expression in the mPFC and Hipp by microarray analysis, then use a ?candidate factor? approach to investigate changes in mRNA and protein expression and phosphorylation of specific plasticity-related signaling molecules. The results of this project may identify new targets for treating cognitive impairment after ADT, and they may reveal new mechanisms underlying the efficacy of vortioxetine.

Chronic stress is a factor in many psychiatric illnesses that share dysregulation of the prefrontal cortex (PFC), and impaired executive function mediated in the PFC. Current therapies are inadequate, and residual cognitive symptoms often persist. This may be because multiple PFC circuits are dysregulated, disrupting multiple cognitive processes. If different mechanisms are affected in different PFC circuits, treatments that are beneficial to one may be ineffective or even detrimental to another. Over the years, we have studied chronic stress-induced cognitive impairment in the medial PFC and the orbitofrontal cortex (OFC) using different stress paradigms, but we have never compared these sub-regions directly. We have, however, observed differences in signaling mechanisms and functional plasticity suggesting that they may respond differently to chronic stress. We have also seen that chronic unpredictable stress (CUS) induces cognitive deficits in both regions. Thus, we will now utilize CUS in this proposal for competing renewal to directly compare and contrast the circuit-level dysregulation underlying cognitive impairment induced by CUS in the mPFC and OFC. In four specific aims, we will investigate the generality of effects by assessing changes in different behaviors mediated in these regions relevant to stress- related psychiatric disorders. We will study differential changes in afferent-evoked responses in mPFC and OFC, then use optogenetics to directly manipulate functional plasticity in those pathways to determine if changes seen after stress are sufficient for stress-induced cognitive deficits, and if opposing them is therapeutic. And we will study differences in signal transduction and structural anatomical plasticity that may underlie the differential changes in functional response induced by CUS in the mPFC and OFC. In Aim 1, we will assess differences in functional plasticity in the mPFC and OFC after CUS by measuring stress-induced changes in electrical responses elicited by stimulation of afferent input from the mediodorsal thalamus and ventral hippocampus to the mPFC, and from mediodorsal thalamus and basolateral amygdala to the OFC. In Aim 2, we will use opto- genetics to test the effects of directly potentiating or attenuating afferent-evoked responses in these same circuits on behaviors mediated in the mPFC and OFC. We predict that attenuating responses in the mPFC and potentiating responses in the OFC will mimic the effects of stress, whereas eliciting the opposite effect in each region will be beneficial in rescuing CUS-induced cognitive deficits in stressed animals. In Aim 3, we will assess changes in dendritic complexity and spine density on PFC pyramidal cells after stress, and test the role of new spine formation in the effects of optogenetically-induced plasticity. In Aim 4, we will test the differential roles of plasticity-related signaling pathways in the mPFC and OFC. We predict that CUS will attenuate PI3K-Akt signaling in mPFC and attenuate JAK signaling in OFC. And we predict these deficits will be restored by opto- genetically-induced plasticity that rescues PFC-mediated cognition. By revealing how stress dysregulates PFC circuits mediating executive function, these results may inform new, targeted strategies to improve treatment.