Virginia M. Pickel - US grants

This ia a proposal for continuation of ongoing research examining the synaptic circuitry in the mature (Study I) and developing (Study II) rat striatum. The objectives in Study I are to determine the synaptic relationships between: (1) dopaminergic afferents from the substantia nigra and intrinsic striatal neurons containing gamma-aminobutyric acid (GABA), opioid peptides (Met-and Leu-enkephalin), and acetylcholine; (2) cholinergic neurons and medium spiny neurons containing GABA or opioid peptides; (3) cortical afferents and neurons containing one or more of the three intrinsic transmitters. The major aims in Study II are to determine whether removal of dopaminergic afferents by 6-hydroxdopamine in the early postnatal period alters the regional densities of immunoreactivity for transmitters in intrinsic neurons; the density or distribution of cortical afferents; or the ultrastructural morphology of targets neurons or terminals in the mature striatum. The primary method to be used in these studies is electron microscopic immunocytochemistry with peroxidase, and radio-iodinated markers for identification of one or more antigens in single sections of tissue. The results from the two studies should be complimentary and broaden our knowledge of interactions between three major types of intrinsic striatal neurons and their synaptic relation to dopaminergic and cortical afferents. This information is important for further understanding the etiology and for developing improved therapeutic measures for a number of movement disorders including Parkinson's disease and Huntington's chorea.

Changes in dopaminergic transmission in mesolimbic and mesocortical pathways have been implicated in certain psychiatric disorders and in the mechanism of action of antipsychotic drugs such as haloperidol. The functional activity of these dopaminergic neurons is most likely dependent on their pre- and/or postsynaptic relations with neurons containing other transmitters or modulators. The goals of the presently funded and proposed studies are to determine: (1) the anatomical substrate for functional interactions between the dopaminergic neurons and neurons containing gamma aminobutyric acid (GABA), substance P, or neurotensin, and (2) the time-dependent alterations In the ultrastructural features of neurons containing one or more of the identified transmitters following chronic treatment with neuroleptic drugs. These goals will be achieved by examining the single or dual immunocytochemical localization of tyrosine hydroxylase, an enzyme involved in catecholamine synthesis, GABA, substance P and neurotensin in the nucleus accumbens (Study 1), medial prefrontal cortex (Study 11), and ventral tegmental area (Study 111) of normal adult rats and rats receiving long-term (2-4 mo) treatment with haloperidol. In addition, the anatomical substrate for functional relationships between dopaminergic axons or GABAergic neurons and other afferents from the hippocampal formation or amygdaloid nuclei will be examined in the nucleus accumbens by combining immunoautoradiographic labeling with anterograde degeneration or transport of conjugated horseradish peroxidase. The results will be quantitatively examined by computer-assisted densitometry of immunoautoradiographs and morphometric analysis of peroxidase- labeled profiles by electron microscopy. Further characterization of normal synaptic relationships and morphological changes observed following chronic administration of a neuroleptic drug is relevant to the understanding of the etiology and the implementation of better treatments for psychiatric disorders which Involve mesolimbic and cortical dopaminergic neurons.

Endogenous opiate peptides (enkephalins and dynorphin) within the dorsal (caudate-putamen nuclei, CPN) and ventral (nucleus accumbens septi) striatum have been implicated in motor and sensory responses to peripheral stimuli and in the rewarding properties of several drugs of abuse. These are most likely mediated by interactions between neurons containing endogenous opiates and transmitters in other neurons or afferents in compartmentally specific divisions of the dorsal and ventral striatum. In this competitive renewal, three studies are proposed that continue to investigate the cellular basis for functional interactions involving endogenous striatal opiates. Studies I and II will examine compartmentally specific subregions of dorsal and ventral striatum of normal adult rats using primarily combined immunogold-silver and immunoperoxidase labeling of two antibodies in single sections by electron microscopy (EM). Study I seeks to establish whether there is a cellular basis for functional interactions involving enkephalins and/or dynorphin and gamma-aminobutyric acid (GABA) or acetylcholine, two prominent transmitters in spiny and aspiny neurons. Study II seeks to determine (1) whether cortical or monoaminergic afferents terminate on neurons containing specific opiates, and/or (2) whether the neurons containing these opiates have other types of axonal associations with cortical or monoaminergic afferents that could mediate presynaptic modulation or dual regulation of common target neurons. Study III will use quantitative light microscopic immunocytochemical and in situ hybridization methods and morphometric EM-immunocytochemical analysis. The goal of this study is to determine whether neurons and/or astrocytes containing endogenous opiates, nerve growth factor, or S-100 protein show changes consistent with specific roles in plasticity of adult striatal cholinergic neurons or other afferents following neonatal neurotoxic lesions of dopaminergic neurons. The results from these three studies will provide basic science information relative to understanding how excitatory cortical afferents and monoaminergic afferents to the striatum modulate local circuits between neurons containing endogenous opiates and related transmitters in normal adult animals and in animals showing striatal compensation for neonatal dopamine depletion. The findings have implications for understanding mechanisms important for drug abuse, forebrain analgesia, and several motor and sensory disorders in humans.

electron microscopy; biomedical equipment purchase;

The first order neurons involved in autonomic responses to visceral stimulation are located in the nuclei of the solitary tracts (NTS) and area postrema, a circumventricular organ outside the blood-brain barrier. Integrated autonomic responses to circulating hormones and stress or emotional events are also most likely partially mediated through afferent projections to the NTS. These latter afferents derive respectively (1) from the area postrema, and (2) from forebrain regions such as the prefrontal cortex (PFC) and central nucleus of the amygdala. Pre- and/or postsynaptic interactions between transmitter-specific target and non-target neurons of these afferents are likely to play key roles in modulation of autonomic reflexes. The majority of these interactions have not been established by dual labeling electron microscopic immunocytochemistry. Thus, the goal of proposed Study I is to use these methods to determine in the rat NTS and area postrema whether there is a structural basis for interactions among neurons containing specific transmitters and/or peptides most implicated in autonomic regulation. These include: catecholamines identified by immunoreactivity for the synthesizing enzymes, tyrosine hydroxylase and phenylethanolamine N-methyl transferase; GABA identified by the product or its synthesizing enzyme, glutamic acid decarboxylase; endogenous opiate peptides and neuropeptide Y. The goals of proposed Studies II and III are to determine using tract-tracing and electron microscopic immunocytochemistry (1) whether there is converging afferent input to the NTS from vagal afferents and afferents either from area postrema, PFC or amygdala; and, if so (2) whether these convergences occur on neurons containing one of the putative transmitters identified in Study I. Additionally, in Study II quantitative light microscopic immunocytochemistry and in situ hybridization will be used to determine whether changes in visceral afferents alter the detected levels of neurotransmitter related products or their corresponding mRNA's in the NTS. The findings will further our understanding of the structural and chemical basis for afferent autonomic regulation at the level of the dorsal vagal complex in the rat. More importantly, these studies may provide information relevant to our understanding of the cellular mechanisms underlying autonomic abnormalities in neuropsychiatric illnesses such as schizophrenia as well as in anxiety and panic disorders in human.

Catecholamines and opiates potently modulate baro- and chemosensory reflexes attributed to the release of L-glutamate and/or substance P from visceral afferents that terminate mainly in the medial (m) and commissural (com) nuclei of the solitary tract (NTS). The director of the modulation is dependent on the receptor subtype, location, and association with N-methyl-d-aspartate (NMDA) and non-NMDA type glutamate receptors. In this project, these functional sites will be examined in three interrelated studies using electron microscopic (EM) immunocytochemistry for the localization of antipeptide antisera against catecholamine, opioid, and glutamate receptors in the mNTS and comNTS of the rat brain. Study I will test the hypotheses that in these regions, alpha2A - adrenergic receptors (alphs2A -AR) and D2 -dopaminergic receptors (D-R) are localized to (I) sites involved in storage or release of their respective catecholamines, consistent with involvement in autoregulation, or (ii) baro- or chemosensory afferents or their targets, indicating the most probable sites for direct modulation of cardiorespiratory reflexes. Study II will test the hypotheses that mu-opioid receptors (MOR) and/or sigma-opioid receptors (DOR) in the NTS are localized to (I) pre- or postsynaptic sites on neurons containing endogenous opioid peptides, catecholamines, and/or alpha2A -AR, suggesting sites for opioid autoregulation and for functional interactions with catecholamines, (ii) baro- or chemosensory afferents, or substance P containing terminals, suggesting involvement in the presynaptic release of excitatory neurotransmitters, or (iii) second order sensory neurons, suggesting involvement in postsynaptic responses to peripheral excitation. Study III will test the hypothesis that kainate receptors and NMDA receptors are differentially distributed with respect to each other, and to neurons expressing alpha2A -AR or MOR, suggesting that catecholamine and/or opioid modulation may at least partially depend on the presence of a specific excitatory amino acid receptor. Other specific aims of this study are to determine whether kainate or NMDA receptors are present on (I) baro- or chemosensory afferents, suggesting sites for autoregulation of glutamate release, or (ii) second-order sensory neurons, suggesting involvement in glutamatergic excitation. Together, the results will have direct relevance to understanding the pathophysiology of, and devising new treatments for, hypertension, somato-sympathetic pain, and ischemic brain damage associated with reflex changes in cerebral blood flow.

The Neuroanatomy Core plays a critical role in each of the five proposed projects in the revised grant application. This Core will provide facilities and services to support histological and photographic needs of investigators in all projects within the Program. This includes general services such as ordering supplies, maintenance of equipment, instruction in use of equipment, and application of histological techniques. The effort of Core staff, whose major activities are associated with individual projects, is distributed for the most advantageous use of their expertise. The following techniques and professional skills are available in the Neuroanatomy Core.

The Experimental Neurogenic Hypertension Program is a multidisciplinary research program investigating the neurobiology of central neural systems governing arterial pressure (AP), regulating the cerebral circulation, and protecting against cerebral ischemia. The broad objectives of this revised proposal are: (a) to investigate the organization within the rostral ventrolateral medulla (RVL) of neurons controlling regional cerebral blood flow (rCBF), and to characterize the efferent pathway, subcortical relay, and intracortical mechanisms by which electrical, chemical and hypoxic stimulation of the nucleus elevates rCBF; (b) to determine, by anatomical tracing methods, the anatomy of pathways by which visceral signals arising in the nucleus tractus solitarii (NST) ascend to influence the performance of thalamic and forebrain centers involved in processing emotional behaviors; to examine, using techniques of EM immunocytochemistry, the distribution and synaptic relationships of alpha2-2- adrenergic, mu and delta-opiate, and NMDA receptors in and around the perikarya and terminals of sympathoexcitatory reticulospinal neurons of RVL, particularly with respect to sites of action of clonidine; to establish, by similar methods, the sites of and interactions between these, and also the kappa-opioid receptors in NTS in relationship to vagal afferent fibers; (e) to analyze the cellular and genetic mechanisms by which electrical stimulation of the cerebellar fastigial nucleus (FN) can conditionally reduce the volume of focal ischemic infarctions of rat brain, either by modifying Kplus channels, and/or suppressing expression of pro-inflammatory genes, and/or facilitating expression of anti-inflammatory and neuroprotective genes.

High resolution electron microscopic immunolabeling of recently cloned proteins now permits clear identification of sites mediating brain functions that are affected in neurological and neurophychiatric diseases. This unique capability, together with a greater demand for quantitative ultrastructural data, has produced an unprecedented need for the use of a modern transmission electron microscope (TEM) by the faculty within the Departments of Neurology and Neuroscience and Pharmacology at Weill Medical College of Cornell University (WMC-CU). Original studies from this group have provided fundamental information on the subcellular distribution of neurotransmitter transporters and receptors using primarily a 1973 model of a Philips 201 TEM, which has become obsolete and unable to cost-effectively meet the needs of the investigators. This proposal requests funds to purchase a new TEM (Tecnai 12 BioTwin) with a digital camera and capacity for on-line imaging and quantitative analysis. The availability of this technologically advanced system will assure the completion of major biomedical research projects that are funded from several different branches of the NIH. These funds cannot be obtained from the research grants of any one investigator, and are not available except through the NIH Shared Instrument Program. The funded projects of the six principal investigators (P.I.'s) who comprise the major users group in this proposal rely exclusively on a TEM for data analysis. These studies require the use of a TEM for quantitatively determining the normal subcellular distribution and drug- or lesion-induced changes in the targeting of opioid receptors and other functional proteins within the rat central nervous system (CNS). The projects of six other investigators who comprise the minor users group require more limited, but crucial, on-line TEM analysis of CNS antigens, particularly in genetic animal models of neurodegenerative diseases and drug addiction. Together, the results from these studies will have major implications for understanding the control of pain, autonomic functions, motivated behaviors, and memory in humans. To assure the successful long-term use of the new TEM system, the daily operation of the instrument will be managed by an internal advisory committee of all major users, and maintained by an experienced staff with the full support of the Medical College.

The nucleus of the solitary tract (NTS) is the brain region most implicated in chemoreflex-induced blood pressure elevation and hypertension-evoked resetting of baroreceptor reflexes, both of which can be modulated through activation of local angiotensin-1 (AT1) receptors. Project 2 will test the central hypothesis that AT1 receptors in the NTS have subcellular distributions supporting direct involvement in cherno- and/or barosensory reflexes and interactions with both catecholamines and NAD(P)H oxidase, an enzyme implicated in the acute and long-term effects of angiotensin II (Angll). This will be achieved by using (1) high resolution electron microscopic immunocytochemical dual labeling of the relevant receptors and NAD(P)H oxidase subunits, and (2) both ultrastructural analysis and patch-clamp recording in barosensory neurons identified by anterograde transport of DiA in rat NTS. There are 5 Specific Aims. Aims 1 and 2 will test the hypothesis that AT1 and alpha2-adrenergic receptors are co-localized within presynaptic axons or their dendritic targets in the NTS, where their distributions are consistent with opposing actions of their agonists on chemo- or barosensory neurons. Aim 3 will test the hypothesis that the physiological actions of Ang II in NTS barosensory neurons are mediated through opening of voltage-dependent Ca 2+ channels also affected by reactive oxygen species (ROS) generated by NAD(P)H oxidase, whose subunits are present in many of the cells that contain AT1 receptors. The opening of voltage-gated Ca 2+ channels is essential for activation of NMDA and certain types of AMPA receptors that are the major mediators of chemosensory and barosensory transmission, respectively. These receptors, like NAD(P)H oxidase, are composed of multiple subunits showing activity-dependent mobilization to plasma and cytoplasmic membranes. Changes in the subcellular distribution of immunogold labeling for glutamate (Aim 4) or NAD(P)H oxidase (Aim 5) subunits will be used to study the potential role of this plasticity in the blood pressure elevations produced either by chronic intermittent hypoxia in the rat model of sleep apnea, or Angll-induced hypertension. Together, the results will contribute to understanding the brain mechanisms underlying the development and maintenance of hypertension.

Drug addiction is a major health issue worldwide, and the central focus of the NIDA Center at Rockefeller University. All addictive substances enhance dopamine in the mesolimbic reward circuit from the ventral tegmental area (VTA) to the nucleus accumbens shell (Acb-SH), a limbic brain region included with the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) as components of the extended amygdala. These regions are also targeted by many excitatory inputs, whose physiological actions are largely ascribed to activation of glutamate (NMDA and AMP A) receptors. Glutamatergic transmission is potently modulated by dopamine acting at Dl receptors and corticotrophin releasing factor (CRF) peptides active at CRF type-1 (CRF1) receptors that are prevalent in both the central extended and basolateral (BLA) amygdala. The more cortical-like BLA has bidirectional connections with the VTA-and other limbic structures implicated in emotional behavior and learning of drug/reward associations. Glutamate receptor plasticity and associations with the dopamine and/or CRF systems contribute to persistent drugseeking behaviors that are powerfully influenced by stress. The subcellular changes in receptor distributions occurring in neurons with these identified transmitter phenotypes in individual brain regions are largely unknown. To begin addressing these key questions, Project 3 in the renewal application will combine research strategies using electron microscopic immunolabeling and spatial-temporal deletion (knock-out) of the NR1 NMDA receptor subunit in limbic brain regions critical for drug seeking behaviors. The long-range goal is to test the hypotheses that (1) limbic NMDA receptors have subcellular distributions conducive to regionally selective associations with dopamine and CRF systems, and (2) NR1 gene expression in the VTA and/or BLA is essential for the synaptic targeting and cocaine-induced trafficking of both AMP A and dopamine Dl receptors, and for cocaine conditioned place preference (CPP) influenced by stress. The studies will be conducted in wild-type and NR1 "floxed" (flanked by loxP) mice, some of which will receive acute or chronic (14 day) cocaine given in an escalating "binge" pattern mimicking that seen in human addicts. Project 3 reflects a collaborative effort by investigators in existing projects within the NIDA Center and is totally dependent on the core resources and facilities. The results obtained from Project 3, together with those in other projects in the renewal application, will provide important new information that is essential for understanding and treating drug addiction.

The Electron Microscopy (EM) Core is a critical component of the NIDA sponsored addiction center, which provides resources and services contributing to the cellular and subcellular localization of transmitters, receptors and transporters under investigation. These are important for all the basic science research in Projects 1-3, and crucial for the quantitative electron microscopic immunolabeling in Project 3. The core personnel include two faculty members of the Department of Neurology and Neuroscience, Weill Medical College of Cornell University. The Director, Dr. Pickel, has pioneered and established many facets of preembedding EM immunolabeling of a variety of antigens in the central nervous system, and also has substantial expertise in brain anatomy and LM detection of mRNA and proteins under investigation in Projects 1 and 2. The co-director, Dr. Glass has made significant contributions to the use of quantitative morphometric analysis of receptor trafficking under the influence of addictive substances. The resources available in the core include a CM-10 and Techni electron microscopes as well as all smaller equipment seeded for LM and EM structural analysis. The core will provide services and facilities related to (a) characterization of antisera and processing of the tissue for both LM and EM immunolabeling either before (pre-) or after (post-) embedding in resin to for microscopic analysis, (b) in situ hybridization of mRNA on frozen slide-mounted brain sections, (c) image acquisition and quantification of immunolabeling. The Core faculty and resources are located at Weill Medical College of Cornell University, which is across the street from Rockefeller University, and occupies one floor of the Kips Bay building. Access to this facility is easily gained by Rockefeller University associates with the presentation of proper identification. The Goals of the EM Resourse are: 1. To offer state of the art ultrastructural examination of tissues from specific brain region, utilizing standard and novel forms of electron microscopy and other standard imaging techniques, for the Center, including Project 3, possibly Projects 1 and 2, and any pilot projects which require this technology. 2. To develop new technologies for ultrastructural imaging as needed. 3. To train scientists and technical support staff in the various techniques of electron microscopy and related imaging techniques.

PROJECT 2 (Pickel): Hypothalamic plasticity enabling slow pressor hypertension Neurohumoral output neurons in the hypothalamic paraventricular nucleus (PVN) are activated by glutamateand Angll-containing neuronal inputs from the subfornical organ (SFO), a brain structure responsive to circulating angiotensin 11 (Angll). These inputs target PVN output neurons that increase hormonal release from the pituitary and sympathetic activity through monosynaptic projections to the thoracic spinal cord. Slow pressor hypertension can be induced by chronic systemic infusion of low doses of Angll (600 ng/kg/min) or by chronic exposure to intermittent hypoxia (CIH). The CIH-induced sympathetic activation and elevation in arterial pressure is dependent on plasticity in the carotid body, but also on changes in glutamate NMDA receptor-dependent transmission in the brain. Chronic exposure to Angll or CIH may result in NMDA receptor-dependent long-term facilitation of glutamatergic transmission in the PVN spinal projection neurons, which is enabled in part by suppression of opposing inhibitory neurons. NADPH oxidase generated reactive oxygen species (ROS) are important modulators of NMDA receptor mediated synaptic plasticity, and are also mediators of the intracellular signaling for Angll, a neuropeptide present in the glutamatergic SFO inputs to the PVN and active mainly through the Angll type-1 (ATi) receptor. Project 2 will test the central hvpothes\s that plasticitv in pre-svmpathetic output and inhibitorv neurons of the PVN enables the development of slow pressor hvpertension through mechanisms that are dependent on postsvnaptic NMDA receptors and influenced bv both Angll and ROS. Aim 1 will examine the basal distribution and function of NMDA and ATi receptors in PVN neurons identified as projecting to the thoracic spinal cord by retrograde transport. Aim 2 will determine whether changes in the surface/synaptic availability of the essential NMDA NR1 subunit and NMDA currents are concomitants of Angll or CIH hypertension, both of which are attenuated by a spatial-temporal deletion of postsynaptic NR1 in the PVN. Aim 3 will determine whether the development of Angll and/or CIH hypertension is linked to NADPH oxidase generated ROS in the PVN. This research will be conducted in male mouse models using in vivo measurement of arterial pressure (tail-cuff or radiotelemetry), high resolution electron microscopic immunolabeling, patch-clamp recording, and ROS imaging. Project 2 is interdependent with each of the other projects and reliant on all core facilities of this PPG.

The escalation in recreational use of marijuana by today?s teenagers is a major health concern, because of the increased risk for psychiatric disorders in individuals who abuse marijuana during adolescence. The psychiatric symptoms include abnormalities in cognitive functions that are mediated largely through the prefrontal cortex (PFC) and associated limbic brain regions. Both pyramidal cells and interneurons in the PFC express cannabinoid-1 receptors (CB1Rs) that are activated by endocannabinoids and by ?9-tetrahydro- cannabinol (?9-THC), the major psychoactive compound in marijuana. Chronic activation of these receptors by ?9-THC downregulates the endocannabinoid system that is a key regulator of experience-dependent learning in the still immature PFC of adolescence. This learning is triggered by calcium influx through glutamatergic NMDA receptors comprised of subunits that are physically and functionally coupled to dopamine D1-like receptors (D1Rs). Pyramidal cells expressing D1Rs are among the PFC neurons most implicated in controlling subcortical brain networks that drive cognitive, social, and attentional functions that are often dysfunctional in psychiatric patients. These neurons are activated not only through Gs-coupled D1Rs and NMDA receptors, but also through Gq/ii-coupled M1 muscarinic acetylcholine receptors (M1Rs) that provoke calcium release from IP3-operated intracellular stores and also mediate endocannabinoid-dependent inhibition. However, there is a major gap in knowledge of the extent to which adolescent abuse of marijuana produces changes in the availability and functionality of these receptors in PFC neurons, which culminate in cognitive or social dysfunctions in adulthood. The proposed studies will test the Central Hypothesis that adult behavioral dysfunctions resulting from chronic adolescent administration of ?9-THC are maintained by persistent suppression of NMDA/D1R and M1R/IP3 receptor systems that mediate the influx and intracellular release of calcium in dopamine-regulated prefrontal cortical neurons. The research will be conducted in male and female C57BL/6J mice that are available in wild-type and mutant forms that can be used for determining potentially critical sex-specific differences in the deleterious effects of adolescent marijuana, which are not directly assessable in humans. The first two Specific Aims will assess the potentially deleterious impact of chronic adolescent administration of ?9-THC on the functional expression of ionotropic (NMDA) glutamate receptors and muscarinic (M1) acetylcholine receptors in D1R-containing output neurons within the adult PFC. Specific Aim 3 will assess the functional relevance of observed THC-induced changes in these receptor systems by examining whether they are accompanied by (1) depression of intracellular Ca2+ and (2) behavioral dysfunctions recapitulated by genetic or pharmacological disruption of NMDA/D1 or M1R/IP3 signaling in the prefrontal cortex. Together, this research will identify molecular targets useful for treating and/or preventing the adverse neurobehavioral abnormalities resulting from marijuana abuse during adolescence.