Teresa A. Milner - US grants

The goal of the proposed study is to establish whether there is a cellular basis for functional interactions between septohippocampal neurons containing acetylcholine and GABA and neurons containing other peptidergic and monoaminergic transmitters in the hippocampal formation and septal complex. This pathway and transmitters were chosen for study based on extensive physiological and pharmacological evidence for their role in learning and memory. Four specific studies are proposed which will employ electron microscopic dual labeling techniques for localizing (1) two antigens on single sections of rat brain using combinations of immunoperoxidase, immunogold and immunoautoradiographic labels and (2) single antigens using immunocytochemical labels combined with the histochemical demonstration of transport horseradish peroxidase or plant lectins for the identification of neuronal pathways. Study I will examine septal afferent terminals in the hippocampal formation with respect to coexistence and/or synaptic relations with cholinergic or GAB terminals. Studies II and III will determine the synaptic relations between cholinergic and GABAergic terminals in the hippocampal formation. In all three studies the synaptic relations of these terminals first will be examined with respect to all types of hippocampal neurons and subsequently with respect to known intrinsic transmitters such as GABA and the neuropeptides, somatostatin and neuropeptide Y. Moreover, septal afferent, cholinergic and GABAergic terminals will be examined in the hippocampal formation with respect to other subcortical afferents from monoaminergic neurons identified by antibodies to 5-hydroxytryptamine and the catecholamine synthesizing enzyme, tyrosine hydroxylase. Study IV proposes to study the synaptic relations between monoaminergic afferents and (1) septohippocampal neurons identified by retrograde transport of horseradish peroxidase following injections into the hippocampal formation, or (2) septal neurons identified by immunoreactivity for choline acetyltransferase or GABA. The results from the four studies should be complimentary and broaden the knowledge of interactions of the cholinergic and GABAergic components of the septohippocampal pathway in relation to (a) monoaminergic afferents in the septal complex and hippocampal formation and (b) neurons containing GABA, somatostatin and neuropeptide Y in the hippocampal formation. The studies will be carried out in adult male rats, but should provide basic information on the neuronal circuitry within regions implicated in specific memory disorders in man.

The endogenous opioid peptides (i.e., enkephalins and dynorphins) in the hippocampal formation are important in learning and memory and are affected following seizures. Four studies are proposed to examine the cellular relationships of opioid-containing neurons in the rat (or where relevant the guinea pig) hippocampal formation. The proposed studies will employ electron microscopic dual labeling techniques for localizing (1) two antigens on single sections of rodent brain using combinations of immunoperoxidase, immunogold and immunoautoradiographic labels and (2) single antigens using immunocytochemical labels combined with histochemical or immunocytochemical demonstration of anterogradely transported tract-tracers or intracellularly-filled neurons. Study I will examine the normal cellular relationships between enkephalin- and dynorphin-containing neurons within three intrinsic systems: (a) the granule cells of the dentate gyrus including their mossy fiber axons; (b) perforant path terminals arising from the lateral entorhinal cortex; and (c) interneurons. Also, the hypothesis will be tested that opioid- containing mossy fibers change in either number and/or ultrastructure following kainic acid induced seizures. Study II will test the hypothesis that enkephalins excite pyramidal cells by synapsing on inhibitory interneurons containing gamma-aminobutyric acid (GABA). Additionally, the synaptic relations between dynorphin-containing and GABAergic neurons will be examined. Study III will examine the interactions between opioid-containing neurons and glutamatergic pathways, particularly whether (a) opioid-containing terminals synapse on granule and pyramidal cells, (b) opioids and glutamate are co- localized in mossy fibers; and (c) the synaptic relations between terminals arising from the entorhinal cortex and dynorphin-containing granule cells. Study IV will test the hypothesis that septal cholinergic and brainstem catecholaminergic terminals are synaptically related to opioid-containing neurons. The experiments will be carried out in adult male rodents, but could yield information that is directly applicable to devising improved therapeutic measures to manipulate transmitter systems disrupted by disorders, such as seizures, in humans. Moreover, such studies may provide insight into the opioid dependence and tolerance phenomena, which possibly reside in the opioid system itself or in the interactions of opioid neurons with neurons containing GABA, catecholamines or glutamate.

The C1 adrenergic neurons in the rostral ventrolateral medulla (RVL) are critical for maintaining sympathetic tone. Ultrastructural studies conducted during the presently funded period have provided anatomical evidence that catecholamines and opioids can both directly and indirectly [through gamma-aminobutyric acid (GABA) interneurons] modulate C1 adrenergic neurons via separate, as well as, interrelated mechanisms. Moreover, they have demonstrated that C1 adrenergic terminals co-localize glutamate and form monosynaptic contacts with sympathetic preganglionic neurons (SPNs) located in the intermediolateral cell column (IML) of the thoracic spinal cord. Physiological and pharmacological studies have suggested several models for the receptor-mediated mechanisms by which catecholamines and opioids may modulate C1 adrenergic neurons. However, anatomical support for such models is lacking. Thus, the present project takes advantage of the recent cloning and generation of antisera to catecholamine, opioid and glutamatergic receptors and proposes 3 studies which aim to directly examine the receptor-mediated regulation of C1 adrenergic neurons by these transmitters in the rat, using quantitative light microscopic and electron microscopic immunocytochemistry. Study I will localize alpha2A -adrenergic receptors (alpha2A Ars) in the RVL that may be the substrates for antihypertensive actions of alpha2 -adrenergic drugs. Subsequent studies will differentiate two models of modulation of C1 neurons by determining whether alpha2A Ars in the RVL are located either (a) within C1 neurons at sites postsynaptic to terminals contacting them, including those containing GABA, or (b) in GABAergic terminals presynaptic to reticulospinal neurons. Study II will test the hypothesis that both the diverse physiological effects of opiates in the RVL as well as the heterogeneous cellular relations between endogenous opioid (enkephalin) terminals and C1 adrenergic neurons can be explained by a differential localization of gamma- and sigma-opioid receptors (Ors) at pre- or postsynaptic sites, respectively. In addition, both studies will test the premise that chronic administration of clonidine (Study I) or morphine (Study II) will alter the number and/or cellular distribution of alpha2A Ars or sigma- or gammaORs, respectively, in the RVL. Finally, since C1 adrenergic neurons contain both adrenergic receptors and glutamate, Study III will determine if adrenergic terminals that contact identified SPNs in the IML contain alpha2A Ars at presynaptic sites and/or N-methyl-D- aspartate (NMDA)-type glutamate receptors at pre- and/or postsynaptic sites. These receptors may mediate and/or modulate the activity of the C1 reticulospinal pathway. These studies will yield information that may lead to improved pharmacological treatments for, and further the understanding of the mechanisms of, hypertension.

DESCRIPTION (provided by applicant): Ritalin (methylphenidate; MPH) is one of the most commonly prescribed drugs for children with attention deficit hyperactivity disorder (ADHD). Over the past decade, ritalin usage has increased in the United States such that children diagnosed with ADHD often are maintained on the drug throughout late childhood and adolescence. Little is known however, regarding the long term consequences of therapeutic doses of MPH on brain development. Developmental MPH exposure may profoundly affect synaptogenesis, myelination and gliogenesis in several brain regions. Of particular note is the process of synaptogenesis, which occurs postnatally in several regions of the brain that are associated with learning and memory (e.g., hippocampus and cerebral cortex). Thus, the present proposal seeks to generate data to aid in evaluating the safety of therapeutic maintenance of MPH in children and adolescents. To achieve this goal, two aims are proposed: (1 ) to develop an animal model that reflects the clinical maintenance of MPH in children and (2) to assess the effects of long-term developmental exposure to therapeutic doses of MPH in this model on the adult brain. Initially, this model will utilize the maximum therapeutic dosage and duration of MPH that is used to treat ADHD in children. Long-term exposure to therapeutic doses of MPH then will be assessed in the forebrain of young adult rats at two time-points using sensitive, quantitative immunocytochemical methods. Focus will be on the: (a) dopaminergic system; (b) ascending noradrenergic system; and (c) ascending serotonergic system (especially their innervation of the cerebral cortex and hippocampus), since current experimental evidence indicates that these monoamines are either directly or indirectly affected following MPH administration. Additionally, the basal forebrain cholinergic and cortical glutamatergic systems will be analyzed since both are targeted by monoaminergic afferent systems, play a prominent role in attention and undergo synaptogenesis postnatally. Monoaminergic and cholinergic neurons and their efferent processes will be identified using antibodies to either their synthetic enzymes or transporters (i.e., to label subpopulations that are important in uptake and release), whereas cortical glutamatergic synapses will be identified by antibodies to NMDA receptors. If changes in any of the immunocytochemical markers are seen using these parameters in this model, future experiments would focus on: (1) dosage (e.g., to determine the maximum dose necessary to see these changes); (2) duration (e.g., to determine if the changes diminish with smaller periods of exposure or to identify developmental stages that may be uniquely sensitive to the drug effects); and (3) the age of assessment (e.g., to determine if the changes persist as the brain ages). Determining how therapeutic dosage regimens effect these transmitter systems is critical in understanding the safety of long-term therapeutic doses of MPH administered to children and adolescents with ADHD and other related disorders.

DESCRIPTION (provided by applicant): Drug addiction and relapse to drug taking require associative memory and motivational incentives, processes that critically involve hippocampal formation (HF) output to the mesolimbic reward system. In humans and animals, relapse susceptibility and HF-associated learning behaviors vary by gender, suggesting hormonal as well as sociocultural influences on addiction-linked processes. Separate literatures show that opiate drugs of abuse (e.g., morphine) and ovarian steroids each alter hippocampal function and cognitive performance. Both opiates and ovarian steroids alter HF excitability and long-term potentiation (LTP), the primary model for learning, and neurogenesis that is involved in forms of associative learning. Morphine directly activates mu-opioid receptors (MORs) and affects the availability of delta-opioid receptors (DORs), altering the balance of excitatory and inhibitory circuits. Enkephalins, endogeous HF opioids, activate MORs and DORs and are important in addictive processes. Estrogens and progestins act through both genomic and non-genomic mechanisms to cyclically remodel HF neurons and alter neurotransmission. Understanding interactions of ovarian steroids and opioids/opiates would clarify whether females have unique modulation of drug responses. This renewal application proposes to test the central hypothesis that the hormonal milieu modulates endogenous hippocampal opioid systems and hippocampal responses to exogenous opiates. The proposed studies use electron microscopic immunocytochemistry, autoradiography and in vitro slice electrophysiology in female rats. Aim 1 will determine whether estrogens and progestins: a) affect levels of enkephalins and/or preproenkephalin mRNA in three subregions that critically integrate different afferent information; b) alter the subcellular distribution of MORs and DORs, which are on interneurons that regulate rhythmic output of excitatory projection neurons; and c) interact with opioids to facilitate LTP. Aim 2 will examine whether estrogen and progestin receptors are on: a) enkephalin-containing neurons, suggesting potential direct interactions of these systems; b) hilar mossy cells, major targets of enkephalin terminals and regulators of projection cell output; c) newly generated cells. Aim 2 also will determine if enkephalin-containing neurons, their targets, or newly generated cells contain functional estrogen binding sites. The results will elucidate potential mechanisms and sites where ovarian steroids, by affecting HF opioid systems, may influence hippocampal-dependent learning relevant to drug abuse.

After menopause, blood pressure and cardiovascular risk increases. In the periphery, gonadal steroids contribute to cardiovascular regulation by influencing the function of the renin-angiotensin system (RAS) through genomic [via nuclear estrogen, progesterone and androgen receptors (ERs, PRs and ARs)] and non-genomic (via membrane ERs, PRs and ARs) mechanisms. These effects may involve alterations in the number or plasrnalemmal targeting of angiotensin 1 (AT1) receptors, or in AT1 receptor-linked signaling mechanisms, including NAD(P)H oxidase. Similarly, in the CNS, gonadal steroids may influence cardiovascular function by affecting angiotensin II (Ang II) actions in the rostral ventrolateral medulla (RVLM), an area crucial for the control of arterial pressure. Gonadal steroid-RAS interactions in the RVLM may be selective for the C1 adrenergic subset of bulbospinal neurons since these neurons: (a) contain nuclear ER-alpha and AT1 receptors, and (b) are excited by Ang II. It is not known, however, whether extranuclear ERs, PRs and ARs are present and functional on RVLM C1 bulbospinal neurons or their afferents. Our central hypothesis is that estrogens, progestins and androgens differentially modulate central blood pressure regulation, in part by altering the excitability and Ang II responses of RVLM C1 bulbospinal neurons, and that these effects involve both genomic and non-genomic mechanisms. We will examine whether: (1) ERs, PRs and ARs are positioned to have genomic and/or non-genomic effects on neuronal circuits in the RVLM relevant to central cardiovascular regulation; (2) estrogens, progestins and androgens affect the function of C1 bulbospinal neurons including their responses to Ang II and glutamate; and (3) gonadal steroids influence the membrane targeting of AT1 receptors and NAD(P)H oxidase subunits in C1 neurons. These studies will be conducted in normal female rats, in female rats with Ang II-induced hypertension and in ER "knock-out" female mice. Light and electron microscopic immunocytochemistry of C1 neurons, complemented by patch-clamp recording and single-cell reverse transcription polymerase chain reaction, will be used. A better understanding of gender-specific blood pressure regulation will contribute to the design of more effective therapeutic strategies for post-menopausal cardiovascular disorders.

As requested by the review team, the use of quantitative light microscopic (LM) densitometry has been removed from the Neuroanatomy-lmaging Core. In addition, changes have been made that reflect changes in the project proposals and the deletion of Project 4. Specifically, LM and EM autoradiography, DiA anterograde tract-tracing, and TUNEL labeling techniques have been added, and juxtacellular labeling, biocytin anterograde tract-tracing, and LM reconstruction techniques have been deleted. There also have been changes in the antisera that are detailed: descriptions of antisera raised against the progesterone receptor, the alpha2A-adrenergic receptor and p22-phox are now included, while antisera raised against 5HT receptors, c-Fos, nNOS and vasopressin have been deleted. There have also been changes in the percent utilization of the Neuroanatomy-lmaging Core by the projects reflecting the deletion of Project 4. The revised description of the core is provided below. Major changes are indicated by a line in the left margin. Overall, the Budget has been reduced. A detailed account of the budgetary changes has been included in the Budget Justification.

The Neuroanatomv-lmaging Core (Core C) is an integral part of the Program, providing resources and support for structural analysis as required in each of the proposed studies. Core personnel include a Director, a Co- Director, a Technical Director, and a technician. The Director, Dr. Milner, has an internationally recognized reputation in the areas of light (LM) and electron (EM) microscopy, and an extensive number of publications in these areas (see publications (P) for Project 3). The Co-Director, Dr. Pierce, is an experienced quantitative anatomist, having conducted studies involving correlated EM and LM stereological analysis and reconstruction (see Biosketch). The facilities and services provided by this core will support; (a) the selective labeling of neuronal subgroups through tract-tracer injection methodologies; (b) the fixation and processing of rodent tissue for LM, confocal and EM analysis, (c) single and dual preembedding and postembedding immunolabeling of antigens, and antibody characterization, (d) in situ hybridization, (e) reactive oxygen species (ROS) and nitric oxide (NO) imaging and (f) image acquisition for both analysis and presentation. Analytic approaches supported by the Neuroanatomv-lmaging Core will include: plotting neuronal distributions, the stereological determination of neuronal number, EM serial analysis and reconstruction of portions of labeled neurons and their processes, fluorescent densitometry, and the quantification of EM immunolabeling patterns (such as determining the extent to which processes are double-labeled, or immunogold particles are partitioned between different types of membranes, or membranes and cytoplasmic compartments).

DESCRIPTION (provided by applicant): Women are more susceptible to several aspects of drug addiction than men, including relapse following stressful events. Addictive processes critically involve hippocampal circuitry that supports spatial and episodic memory acquisition processes. In contrast to the impaired cognition observed in males after chronic stress, females display enhanced spatial memory following chronic stress suggesting ovarian hormone involvement. Within the dorsal hippocampus, it is well established that ovarian steroids, in particular estrogens, can modulate CA1 pyramidal cell activity and long-term potentiation (LTP), the cellular model of learning. During the last grant period we found that ovarian hormones also regulate enkephalins and the mu- and delta-opioid receptors (MORs and DORs, respectively) in a manner that could promote learning processes under certain conditions. Specifically, at proestrus (high estrogen): 1) enkephalin levels are elevated in mossy fibers (MFs), which synapse on the dendrites of CA3 pyramidal cells;2) increased MORs are present on the plasma membrane of parvalbumin GABAergic interneurons, which inhibit CA3 pyramidal cells;3) fewer DORs are present on the plasma membrane of pyramidal cells;and 4) MF stimulation may induce an opioid-dependent potentiation of CA3 field excitatory post- synaptic potentials. Chronic stress, which can trigger the release of opioid peptides, has maladaptive morphological responses in males that are not seen in females, and may also elevate MOR expression in parvalbumin interneurons in females. This renewal application proposes to test the central hypothesis that chronic stress leads to adaptive changes in the opioid system of females to promote CA3 LTP and other plastic processes that support drug-related learning. Approaches using a combination of light and electron microscopic immunocytochemistry, RT-PCR, in situ hybridization, in vitro slice electrophysiology and immobilization stress will test this hypothesis. Aim 1 will determine if chronic stress alters: 1) the levels and/or subcellular distribution of enkephalins within MFs and in lateral perforant path (LPP) afferents to CA3 and 2) opioid-mediated LTP and/or other forms of opioid-mediated plasticity at MF/LPP- CA3 synapses in a manner promoting learning processes in females. Aim 2 will determine if following chronic stress MORs and DORs have altered: 1) expression and cellular distributions;2) trafficking within select cell types;and/or 3) phosphorylated levels or distributions in the CA3 region in a manner promoting excitation and learning processes in females. PUBLIC HEALTH RELEVANCE: The results of these studies will elucidate potential mechanisms through which ovarian steroids, by targeting neurons that release, express or respond to opioids, may influence hippocampal-dependent learning relevant to drug abuse. These studies will improve our understanding of sex differences in the nature and etiology of drug abuse and have implications for tailoring treatment interventions to maximize positive outcomes for females and males.

DESCRIPTION (provided by applicant): Brain derived neurotrophic factor (BDNF) is importantly involved in anxiety, depression and cognitive function. A single nucleotide polymorphism, a valine (Val) to methionine (Met) substitution (Val66Met) in the BDNF gene, results in increased mood and cognitive disorders in both humans and mice. Depression rates rise sharply in women during perimenopause, which is the 7-10 year period before menopause characterized by declining estrogen levels and gradual acyclicity. Evidence from humans and mice that have undergone surgical menopause indicate that estrogen plays a critical role ameliorating anxiety and memory dysfunctions and in modulating the expression of BDNF and its receptor TrkB in the hippocampus, a region critically involved in anxiety and memory. Determining whether disruption of estrogen cyclicity during perimenopause contributes to the greater susceptibility of carriers of Val66Met allele to depression and anxiety disorders and BDNF signaling in the hippocampus has been hampered due to a lack of a rodent model. However, the recent development of a novel model that induces menopause through gradual ovarian cessation now allows for the replication of the perimenopausal period in heterozygote Val66Met mice. Therefore, this proposal will test the central hypothesis that a single copy of Val66Met allele intensifies the mood and memory disorders seen during perimenopause and leads to disruption of BDNF signaling mediated by estrogen in the hippocampus. A multidisciplinary approach combining behavioral measures including open field, object placement and object recognition and quantitative in situ hybridization and light and electron microscopic immunocytochemical methods for the localization of BDNF and TrkB will test this hypothesis. Aim 1 will determine if, after surgical menopause induced by ovariectomy, Val/Met mice experience even greater anxiety and memory problems and less BDNF signaling that Val/Val (control) mice and if these effects can be reversed by estrogen replacement. Aim 2 will determine if during perimenopause disruption of estrogen cyclicity leads to reduced BDNF signaling and increased behavioral instability in anxiety and cognitive tests that are worse in Val/Met than Val/Val mice. Understanding the impact of BDNF genotype and altered estrogen status on mood and cognitive disorders could ultimately lead to clinical treatments that are individualized for sex, genotype and life stage. PUBLIC HEALTH RELEVANCE: In a subpopulation of women, the incidences of anxiety, depression and cognitive dysfunction increase during the transition to menopause (i.e., perimenopause). The proposed studies will use novel rodent models to determine if, during perimenopause, a mutation in the neurotropin brain derived neurotrophic factor (BDNF) gene exacerbates anxiety and cognitive problems and concomitantly alters BDNF communication in the brain. These studies will provide information on the interaction of estrogen and BDNF in affective disorders and could lead to improved hormone replacement therapies to alleviate menopausal symptoms.

DESCRIPTION (provided by applicant): After menopause, hypertension and cardiovascular risk increases in women. Our preliminary data indicate that a comparable susceptibility to hypertension can be observed following slow pressor angiotensin II (AngII) infusion in a menopausal mouse model. The hypothalamic paraventricular nucleus (PVN) is critical for integrating and coordinating neurohumoral responses involved in cardiovascular regulation. In hypertension, NMDA receptor activation and NADPH oxidase-dependent reactive oxygen species (ROS) production in PVN neurons that project to the spinal cord plays a pivotal role in enhancing the sympathetic drive that underlies the elevation of arterial pressure. A significant number of PVN-spinal neurons contain the estrogen receptor (ER) ¿, suggesting that hormone alterations in menopause could selectively influence excitation in this population. In particular, gonadal steroids could alter excitatory transmission by regulating the expression and/or subcellular distribution of the NMDA receptor, voltage-gated Ca2+ channel currents and/or the generation of NADPH oxidase derived ROS. Such changes could ultimately contribute to the development of hypertension observed in menopause. Therefore, this proposal will test the central hypothesis that changes in postsynaptic NMDA receptors and associated signaling pathways within ER¿ PVN neurons during menopause predisposes these neurons to increase excitability in response to hypertensive challenges. Two aims will examine mouse models of menopause to determine (1) whether menopause increases the susceptibility to slow pressor AngII hypertension through mechanisms involving post-synaptic NMDA receptors in the PVN; and (2) if NMDA-mediated responses in ER¿-containing PVN neurons show adaptations consistent with the potentiation of excitatory transmission during the development of hypertension. These studies will be conducted in the well-established intact aging model and the new VCD model of menopause including some ER¿-GFP transgenic mice, and will use slow pressor AngII- infusion as the hypertensive challenge. These studies will be achieved using a multidisciplinary approach including high resolution electron microscopic immunolabeling to identify the subcellular distribution of essential NMDA NR1 receptors and related signaling pathway components in ER¿-GFP labeled PVN neurons, spatial-temporal deletion of the NR1 gene, quantitative RT-PCR, patch-clamp recording, ROS imaging, and telemetric measurement of blood pressure.

? DESCRIPTION (provided by applicant): Women are more susceptible than men to several aspects of drug addiction, including relapse due to stressful events. Addictive mechanisms require associative memory processes that critically involve hippocampal circuits, including the opioid system. Our light and electron microscopic (EM) studies have revealed notable sex and estrous cycle differences in the hippocampal opioid system: enkephalins, mu opioid receptors (MORs) and delta opioid receptors (DORs) are subcellularly positioned in neurons in the mossy fiber circuitry to enhance excitability and learning processes in females with elevated estrogen states. Moreover, unlike males, females in high estrogen states exhibit a long-term potentiation (LTP) evoked by low frequency stimulation of the mossy fibers that is regulated by DORs. Progress during the last grant period showed that chronic immobilization stress (CIS) in males, which results in CA3 neuron dendritic retraction and parvalbumin interneuron loss, essentially shuts off the hippocampal opioid system. Conversely, CIS in females, regardless of estrogen state, primes the opioid system for greater excitation of CA3 pyramidal cells. Specifically, afte CIS mossy fiber enkephalin levels are elevated and the distribution of MORs and DORs in hippocampal neurons resembles that seen in unstressed females at high estrogen states. Moreover, our EM studies revealed in females, but not males, the involvement of a second mechanism for enhancing hippocampal excitation following CIS: DORs traffic to the near plasmalemma of hilar GABAergic interneurons that are known to project to granule cells dendrites where they converge with entorhinal afferents. Notably one-hour after a single injection of oxycodone (3mg/kg, i.p.) the DORs in these GABAergic interneurons have moved to the plasmalemma where additional exposure to ligand would disinhibit these neurons and thus could promote LTP at entorhinal-granule cell synapses. To date, however, no studies have explored the mechanisms by which chronic stress-induced changes in the opioid system in females could advance associative memory processes important for drug addiction, particularly to oxycodone. Thus, this renewal application proposes to test the central hypothesis that the alterations in the hippocampal opioid system induced by chronic stress predispose females to enhanced sensitivity to oxycodone and promote drug-related associative learning. Aim 1 will first compare the effects of CIS on the acquisition of oxycodone-induced conditioned place preference (CPP) in females and males. Second, MORs and DORs will be selectively antagonized at critical hippocampal sites, to determine their role in oxycodone CPP in unstressed and CIS female and male rats. Aim 2 will determine how oxycodone CPP alters specific molecular (as assessed by targeted PCR gene array) and anatomic (as assessed by light and EM immunocytochemistry and in situ hybridization) profile of the hippocampal mossy fiber opioid system in female and male rats under basal conditions and after CIS.

ABSTRACT Hypertension is more prevalent in men than in young women, but as women progress towards menopause this relationship is reversed. Unfortunately, compared to men, menopausal women are less likely to receive optimal diagnostic evaluation and therapeutic intervention. In women, the increased incidence of hypertension begins at ?perimenopause?, a transitional phase preceding and extending through menopause beginning at ~45-54 years of age. The perimenopausal period is accompanied by irregular estrous cycles and erratically fluctuating estrogen levels; it may also be a critical period for the emergence of brain plasticity that may contribute to the development of hypertension. The reversal of hypertension liability in young and older women may be phylogenetically conserved: aged female mice, but not young female mice, show increased sympathetic tone and blood pressure following slow-pressor angiotensin II (AngII) administration. To better understand the role of ovarian hormones in female hypertension, we have utilized a mouse model of accelerated ovarian failure (AOF) that uniquely recapitulates hormone fluctuations seen in human menopause. Using AOF mice, we made the novel finding that the susceptibility to hypertension begins at ?peri-AOF?, which is a stage marked by irregular and extended estrous cycles similar to human perimenopause. In addition, peri-AOF hypertension was associated with a unique profile of N-methyl-D-aspartic acid (NMDA) receptor plasticity in estrogen receptor ? (ER?) containing neurons in the hypothalamic paraventricular nucleus (PVN) not seen in pre- or post-AOF females, or in males. These findings define peri-AOF as a critical period when hypertension and adaptations in neuro-cardiovascular regulatory systems emerge. In the current proposal, we will investigate the role of ER? in the emergence of hypertension and PVN NMDA receptor plasticity during peri-AOF. For this, we will test the central hypothesis that ER? influences the susceptibility to hypertension and NMDA receptor plasticity in select populations of PVN neurons in peri-AOF mice following slow-pressor AngII administration. Two aims will test this hypothesis. Aim 1 will test the sub- hypothesis that ER? in the PVN is critically involved in the susceptibility of peri-AOF females to hypertension and potentiated NMDA receptor-mediated excitatory signaling in CRF-expressing neurons. Aim 2 will test the sub-hypothesis that cyclic administration of ER? agonists during peri-AOF reduces both hypertension susceptibility and NMDA-mediated excitatory signaling selectively in CRF-containing PVN neurons. These studies will be achieved using a multidisciplinary approach including spatio-temporal deletion of the ER? gene, electron microscopic immunocytochemistry, molecular and biochemical assays and neurophysiological methods.

Abstract: Understanding how genetic and environmental factors impact drug use and abuse may be critical for addiction prevention and diagnosis, as well as the development of novel effective addiction therapeutics. This application plans to provide 3 predoctoral training slots (for ~2 yrs, starting in the 2nd yr) in the Weill Cornell Graduate School (WCGS) Neuroscience and Pharmacology Programs aimed at understanding the impact of genes and environment on drug addiction. A unique feature of this training plan is the diversity of faculty expertise in both genetic (e.g. sex, single nucleotide polymorphisms, gene splice variants, dendritic/axonal translation, and epigenetics) and environmental (maternal environment, developmental stage, and stress) factors that are essential for the emergence of addictive disease. Our faculty is also noteworthy for the breadth of the approaches they bring to addiction science; we have expertise in studying how several major abused drugs (i.e. opiates, cocaine, and other psychostimulants) impact neuronal function from the expression and behavior of single molecules to the performance of complex functional systems that regulate the behavior of rodents and humans. In addition to our talented faculty, this training grant will take advantage of the WCGS outstanding research environment, educational resources, and recruiting activities, particularly our history of attracting and training under-represented minorities as basic and clinical scientists. Particular strengths of the training grant include: 1) the experience of the Director and Co-Director in mentoring, teaching and drug abuse research; 2) the broad scope of multidisciplinary research training provided by the faculty; 3) extensive collaborations and co-mentoring between the faculty; and 4) the strong emphasis on ?bench-to- bedside? translational research. Training grant faculty will be divided into three groups: 1) Major Sponsors: graduate student thesis mentors with NIDA mission supported research programs; 2) Training Sponsors: individuals with extensive experience in drug abuse research who will work closely with Major Sponsors and their trainees; and 3) Minor Sponsors: individuals with NIDA- mission interests who will collaborate with Major Sponsors and their trainees. Resource Cores comprised of training grant faculty from all three groups will be established to provide a platform for students to incorporate approaches from multiple laboratories into their PhD thesis work. Beyond the laboratory, a new course entitled ?Addiction and Society? developed by a group of graduate students in consultation with faculty, as well as a Drug Abuse focused retreat and WCGS developed programs in grant-preparation, will provide students with important training experiences in teaching, grantsmanship, and networking that will be essential for their career development. These courses together with the existing coursework and curricula, symposia and lectures, as well as each students individual training plan, will provide a solid foundation for the development of independent basic and clinical scientists in drug abuse research.

Opioid driven exacerbations of neuropathological events and alterations in HIV transcription contributing to HIV associated CNS dysfunction are well-reported. Despite years of continuous suppressive antiretroviral therapy (ART), latent HIV persists and finds sanctuary in many of the same brain regions involved in opioid use disorder (OUD) suggesting interactions between HIV and opioids in brain cells. However, there is a sizeable gap in our knowledge on how OUD impacts cellular responses and viral persistence in HIV-infected brain on ART in humans or relevant model organsims. This proposal seeks to generate topographical data sets and evidence at single cell resolution across the hippocampus and prefrontal cortex (PFC), two brain regions known for predilection for HIV persistence and OUD in non-human primate (NHP) and in post-mortem human brain. These data will provide an unprecedented cellular landscape of multiple modalities that can be harnessed to develop strategies to limit viral persistence and restore and retain optimal brain health in people living with HIV. In our published and preliminary work we have developed innovative single-cell approaches: (A) Single-cell isoform RNA sequencing (ScISOr-Seq), which enables single-cell long-read RNA sequencing of polyadenylated RNAs across thousands of single cells; (B) Slide-isoform sequencing (Sl-ISO-Seq) to spatially locate isoforms in brain slices and (C) a single-cell platform that identifies HIV sequences at single cell level (ScHIV-Seq). In concert these novel sequencing and computational methods, along with scATAC-Seq for chromatin accessibility, will permit the mapping of cellular gene expression, open chromatin regions, isoforms and the detection of HIV across single- cells of hippocampus and PFC. Recent literature supports the presence of HIV in the brain and more specifically in microglia and astrocytes present within the hippocampus and PFC. Importantly, these brain regions are also involved in associative learning processes for OUD. Moreover, our prior studies in rodent hippocampus have laid the groundwork for the proposed studies by establishing the regional and cell-specific distributions of opioid peptides and receptors as well as related signaling molecules, and how these distributions are impacted by sex, stress and opioid-associated learning. In further preliminary studies, we conduct opioid receptor mapping, brain spatial transcriptomics, NHP cognitive behavioral assessment and pharmacological profiling of current ART regimens in tissues. These approaches will provide a comprehensive regional landscape to support our single cell specific phenotypes. We propose an overarching hypothesis that: (i) our new integrated single-cell methods will map single-cell and cell-type specific human and NHP transcriptome and epigenome signatures in the hippocampus and PFC of S/HIV in NHPs and post-mortem human brain; (ii) chronic opioid exposure adds a distinguishable signature to S/HIV infection with long-term ART and defines cell subtypes in which these signatures are rooted; and (iii) these signatures are different from chronic opioid exposure on uninfected brain. These studies further an understanding of molecular mechanisms in HIV and OUD in brain.