Yoland Smith - US grants

nervous system; human tissue; Primates; Mammalia;

microscopy; nervous system; human tissue; Primates; Mammalia;

psychology; nervous system; human tissue; Mammalia; Primates; behavioral /social science research tag;

DESCRIPTION (adapted from abstract): The striatum is the major entrance of information to the basal ganglia, a group of interconnected brain structures that play major roles in the control of motor behaviors. The activity of striatal neurons is under the influence of two major excitatory synaptic inputs that arise from the cerebral cortex and the caudal intralaminar thalamic nuclei, namely the centromedian (CM) and parafascicular (PF) nuclei. Despite the fact that the existence of a thalamostriatal pathway has long been established, and that the CM reaches its maximum extent in primates, the role of thalamic influences upon striatal neurons is usually not considered in the functional circuitry of the basal ganglia. Although the functions of the intralaminar nuclei are complex and multifaceted, it is well known that CM plays a role in maintaining a state of high vigilance and attention, two features that might be critical for alerting and preparing striatal neurons for incoming input. Over the past five years, the investigator carried out a series of studies that demonstrate the large extent and high degree of functional and synaptic specificity of the CM afferents to striatal neurons in monkeys. He now proposes to elucidate the sources of synaptic inputs to thalamostriatal neurons to obtain a better understanding of the nature of information that flows through the thalamostriatal pathway. Four hypotheses will be tested in the project: 1) they will test the possibility that the CF/PF nuclear complex is part of functionally distinct circuits with neurons in the basal ganglia, 2) they will verify whether individual thalamostriatal neurons might be the site of convergence of cognitive- and limbic- related information from the basal ganglia, 3) they will test the possibility that thalamostriatal neurons are targets of ascending cerebellar influences and, therefore, represent a major link through which a copy of the cerebellar information is conveyed to striatal neurons and 4) they will better characterize the synaptic relationships and neurotransmitter content of the ascending reticular activating system from the tegmental pedunculopontine nucleus that impinge upon thalamostriatal and thalamocortical neurons in CM. The results of the project, combined with previous data, are prerequisite to future functional studies of the thalamostriatal projection in normal and pathological conditions.

Most of the research conducted in this laboratory was aimed at elucidating various aspects of the connectivity and synaptic chemistry of the basal ganglia in nonhuman primates. The main findings are summarized as follows. (1) The functionally segregated outflow of the basal ganglia remains largely segregated in separate channels at the level of the thalamus, but displays a much higher degree of convergence at the level of the pedunculopontine nucleus in the brainstem. These findings indicate that basal ganglia information processing is likely to be different at thalamic and brainstem levels. This will open new research avenues for understanding better the functional interactions between the pedunculopontine nucleus and the basal ganglia in normal and pathological conditions. (2) NMDA glutamate receptor subunits are much more abundant in dopaminergic neurons of the substantia nigra pars compacts (SNc) than in dopaminergic neurons of the ventral tegmental area (VTA). The se findings provide strong anatomical evidence for the role of excitotoxicity in the death of SNc dopaminergic neurons in Parkinson's disease. (3) The inputs from the intralaminar thalamic nuclei are differentially distributed among subpopulations of striatal interneurons, which suggests a high degree of specificity in the processing of thalamic information by striatal neurons. This should help to understand better the role of the thalamostriatal pathway in the functional circuitry of the basal ganglia. (4) In collaboration with Dr. Michael Kuhar of the Yerkes Center, we analyzed the distribution of a novel family of peptides in the nucleus accumbens and the gut and found that CART is expressed in specific populations of chemically characterized neurons in both the central and peripheral nervous system. The results will help to understand better the role of CART peptides in the centrally mediated phenomenon of addiction to drugs of abuse. (5) In collaboration with Dr. Denis Par[unreadable] in Laval University, Qu[unreadable]bec, Canada, the synaptic organization of intrinsic and extrinsic connections of amygdaloid nuclei in cats was analyzed. The most recent findings indicate that parvalbumin-immunoreactive GABAergic interneurons are a major target of excitatory afferents in the basolateral amygdaloid nuclei. This provides essential anatomical data to elucidate the neuronal mechanisms that underlie the inhibitory network of the amygdala. (6) In collaboration with Dr. Didier Pinault in Laval University, a project was completed on the synaptic organization of the reticular thalamic nucleus in rats. We showed, for the first time, that intrinsic dendrodendritic synapses are the anatomical substrate for neuronal synchronization in the reticular nucleus. These findings are important for understanding the role of the thalamocortical projections in controlling large-scale phenomena such as sleep and wakefulness. Disease Assoc.

DESCRIPTION: GABA and glutamate are the two most abundant inhibitory and excitatory neurotransmitters in the brain. The concept of glutamatergic and GABAergic neurotransmission has changed considerably over the last five years, mainly because of the explosion of molecular data on the structure of glutamatergic and GABAergic receptors. It is now well established that those receptors are highly complex proteins composed of a multitude of subunits, and that the relative abundance of these subunits in the composition of the native receptors underlie many of the functional properties of the receptor channels. Another important series of findings that led to reconsider investigators view of fast excitatory synaptic transmission come from the cloning, and subsequent pharmacological and physiological studies, of a group of G-protein-linked glutamate receptors, called metabotropic receptors. These receptors modulate fast excitatory synaptic transmission and participate in the induction of long term phenomena such as synaptic plasticity and glutamate-induced neurotoxicity. The basal ganglia is a group of brain structures that play a critical role in the control of motor behaviors. A balance in the activity of GABAergic and glutamatergic networks is essential for the normal functioning of these brain structures. For instance, in Parkinson's disease, where midbrain dopaminergic neurones degenerate, the resulting imbalance between the activity of GABAergic and glutamatergic connections underlies many of the clinical manifestations of the disease. Over the last ten years, our laboratory studied in detail the pattern of distribution of GABAergic and glutamatergic terminals on different populations of neurones in the basal ganglia of monkeys. The investigators now, propose to use high resolution electron microscopic techniques to study the subsynaptic distribution of glutamate and GABA receptors associated with those afferents, and to verify the possible alterations in the expression of these receptors in animal models of Parkinson's disease. The results of those studies will provide a basic framework for understanding the complex synaptic interactions that underlie the normal functioning of the basal ganglia circuitry, and will help to identify better targets for the development of drugs that modulate abnormal glutamatergic and GABAergic neurotransmission in Parkinson's disease.

The basal ganglia and thalamus are interconnected through a series of loops that process and convey basal ganglia outflow to either frontal cortical regions via the ventral motor nuclei or back to the striatum via the caudal intralaminar group, namely the centre median (CM) and parafascicular (Pf) nuclei. Although the existence of a thalamostriatal system has long been established, the role of these projections in the functional circuitry of the basal ganglia remains enigmatic. For the first four years of this grant, we focused our interest on the sources and chemical nature of basal ganglia and brainstem synaptic inputs that control the activity of thalamostriatal neurons. Both the internal globus pallidus (GPi) and the substantia nigra pays reticulata (SNr) provide GABAergic afferents to specific regions of CM/Pf. In addition, the pedunculopontine tegmental nucleus (PPN) is the source of highly heterogeneous chemical inputs to CM/Pf, some of them co-localize GABA and acetylcholine. In addition, neurons in CM/Pf, as in most thalamic nuclei, are endowed with intrinsic GABAergic influences from the reticular nucleus and local interneurons. Electrophysiological data show that GABA plays a crucial role in regulating thalamic activity. However, the exact mechanisms by which GABA mediates its effects on thalamic neurons are complex and still matter of speculation. In order to further characterize this issue, we propose to use state-of- the-art immunocytochemical procedures at the electron microscopic level to elucidate the subsynaptic and subcellular localization of GABA-A and GABA-B receptors in the basal ganglia-receiving territories of the ventral motor thalamic nuclei and CM/PF in monkeys. Abnormal increased GABAergic outflow from the basal ganglia to the thalamus is a cardinal feature of Parkinson's disease pathophysiology. Such increased activity likely results in downregulation of postsynaptic GABA receptors in basal ganglia receiving thalamic nuclei. In order to elucidate this issue, another goal of this project is to compare the pattern of subsynaptic localization of GABA-A and GABA-B receptors in CM/Pf and ventral motor nuclei of normal monkeys and animal models of Parkinson's disease. This series of studies should provide a comprehensive analysis of GABA receptors localization at specific synaptic sites in basal ganglia-receiving thalamic nuclei in primates. Such information is critical for the interpretation of functional studies and a better understanding of the pathophysiological changes generated at pallidothalamic and nigrothalamic synapses in Parkinson's disease.

DESCRIPTION (provided by applicant): Three major receptor subtypes mediate GABAergic inhibitory effects in the mammalian CNS, the GABA-A and GABA-C receptors that generate fast inhibition, and the metabotropic GABA-B receptors (GBR1, GBR2) which mediate slow inhibitory effects via activation of an intracellular second messengers cascade. Data from our laboratory showed that GBR1 receptors are strongly expressed pre- and postsynaptically throughout the monkey basal ganglia. Interestingly, pre-synaptic GBR1 immunoreactivity is mainly associated with glutamatergic terminals suggesting that GABA-B receptors act as heteroreceptors that modulate glutamate release in these structures. To further elucidate the roles of GABA-B receptors in basal ganglia, we propose a series of anatomical, neurochemical and behavioral studies to characterize various aspects of GABA-B receptor localization and functions in the globus pallidus (GP) and subthalamic nucleus (STN) of normal and parkinsonian monkeys. It is well established that overactivity of glutamatergic pathways from the STN to basal ganglia output structures, namely the internal segment of the globus pallidus (GPi) and the substantia nigra pars reticulata (SNr), is a cardinal feature of the pathophysiology of Parkinson's disease (PD). Our preliminary data raise the interesting possibility that activation of presynaptic GABA-B receptors in GP and STN may reduce transmission at overactive subthalamofugal synapses. In this model, activation of GABA-B receptors would attenuate some of the parkinsonian motor symptoms. In support of this notion, our data also indicate that local administration of GABA-B receptor agonists in GPi and STN reduces glutamate release in primates and that systemic application of these compounds may have beneficial therapeutic effects in parkinsonian monkeys. The following four specific aims are proposed: (1) Characterize and compare the pattern of subcellular and subsynaptic localization of GABA-B Ri immunoreactivity in the GP and STh of normal and parkinsonian monkeys, (2) Determine the exact source of glutamatergic axon terminals that express pre-synaptic GABA-B receptors in GP and STN, (3) Test the possibility that local application of GABA-B agonists decreases glutamate levels in the GP and STN of normal monkeys and (4) Test the potential therapeutic effects of GABA-B receptor agonists in parkinsonian monkeys. These experiments will further delineate the subsynaptic localization and roles of GABA-B receptors in modulating glutamate release and open novel research avenues for the potential use of GABA-B agonists in the pharmacotherapy of PD.

DESCRIPTION (provided by applicant): The trafficking and anchoring of neurotransmitter receptors to the neuronal plasma membrane are complex phenomena that involve specific interactions between the receptors and various scaffolding proteins. Over the past few years, the targeting of metabotropic glutamate receptors (mGluRs) has gained particular interest. The group I mGluRs, which comprises mGluR1 and mGluR5, are mostly expressed postsynaptically and produce slow depolarization through coupling with phospholipase C and IP3/Ca2+ receptors. Five years ago, Brakeman et al. (1997) identified a dendritic protein that selectively binds to group I mGluRs. The expression of this protein, which was named HOMER, appeared to be regulated by physiological synaptic activity and likely played a role in group I mGluRs signaling. Since then, 12 Homer cDNAs have been cloned. These cDNAs encode for various proteins with a similar structure named Homer 1a/b/c, Homer 2a/b and Homer 3. Although the exact functions of Homer in the brain remain to be established, various data, largely obtained in cultured cells, suggest that Homer is involved in the trafficking and synaptic targeting of group I mGluRs at specific sites along the neuronal plasma membrane. There is strong evidence that changes in dopamine transmission regulate Homer mRNA expression in the rat CNS. The lack of information on Homer proteins localization and their relationships with group I mGluRs hampers the progress of knowledge on Homer/mGluRs functional interactions in the CNS. During the first five years of this award, our laboratory studied in detail the subsynaptic and subcellular localization of group I mGluRs in the monkey basal ganglia. Two major findings stood out from these studies: (1) Group I mGluRs are expressed at both glutamatergic and non-glutamatergic synapses in various basal ganglia nuclei and (2) a large pool of mGluR5 is expressed intracellularly in basal ganglia output nuclei. These observations raised important questions regarding the functions, synaptic targeting, trafficking and regulation of group I mGluRs in the basal ganglia. In order to further characterize these issues and better understand the potential role(s) of Homer in basal ganglia functions, one of the objectives of this proposal is to elucidate the subcellular and subsynaptic relationships between Homer and group I mGluRs in the striatopallidal complex of monkeys. Another goal of this project is to characterize potential changes in the subcellular and subsynaptic localization of group I mGluRs in normal versus Homer knock out mice with or without lesion of midbrain dopaminergic neurons. Together, findings obtained in these studies will serve as a basic framework to understand Homer/mGluRs interactions in normal and pathological basal ganglia functions.

glutamate receptor; receptor expression; protein structure function; Primates; animal colony; basal ganglia; second messengers;

glutamate receptor; receptor expression; protein structure function; Parkinson's disease; molecular pathology; Primates; animal colony;

neurotoxicology; glutamate receptor; corpus striatum; Primates; animal colony;

The proper functioning of CNS neurons relies on complex and highly specific integration of synaptic inputs along their dendritic trees. The striatum is largely made up of two populations of medium sized GABAergic neurons characterized by densely spiny dendrites and differential expression in dopamine receptors immunoreactivity (D1 versus D2). The cerebral cortex and the thalamus are the two main sources of excitatory glutamatergic afferents to striatal medium spiny neurons (MSN). Together, these two afferents contribute more than 30,000 glutamatergic synapses onto individual striatal projection neurons. Midbrain dopaminergic inputs from the substantia nigra pars compacta (SNc) play a critical role in modulating glutamatergic transmission in the striatum. Consequently, lesion of this system results in significant neurochemical, electrophysiological and morphological changes in striatal MSNs in Parkinson's disease. The vesicular glutamate transporters 1 and 2 (vGluTI and vGluT2) are invaluable tools to label specifically the corticostriatal and thalamostriatal systems, respectively. Although these two striatal inputs have long been established, very little is known about the differential organization of the synaptic microcircuitry of these two pathways in normal and pathological basal ganglia conditions, largely because of the lack of specific markers to differentiate thalamic from cortical afferents in the striatum. For the past 15 years, our laboratory has been at the forefront of research to characterize the organization of synaptic microcircuits in primate basal ganglia. Our work has led to significant advancement in our understanding of the functional anatomy, synaptic connectivity and potential roles of the thalamostriatal system in monkeys. In this project, we propose to use vGluTI and vGluT2 as specific markers of corticostriatal and thalamostriatal terminals to further understand the functional organization and synaptic plasticity of these two pathways in normal monkeys and animal models of Parkinson's disease. Three main hypotheses will be addressed using high resolution electron microscopy immunocytohemistry combined with 3D reconstruction of dendritic spines in normal and MPTP- treated monkeys. Results of these studies will shed light on the substrates the underlie the synaptic mechanisms by which thalamic and cortical glutamatergic afferents control the activity of striatofugal neurons in both normal and pathological basal ganglia conditions.

DESCRIPTION (provided by applicant): This is a competing renewal of a five-year Institutional NRSA application for the support of outstanding predoctoral students in the interdepartmental Graduate Program in Neuroscience at Emory University. The objective of the Program is to provide graduate students a breadth of knowledge and research skills in modern systems and integrative neuroscience so that they may enter a variety of careers in biomedical research, education, and industry. A fundamental strength of this program is the broad interdisciplinary training we provide in a wide spectrum of neurobiological issues spanning several classical disciplines. We are seeking support for 10 students per year in this renewal. The Neuroscience Program currently has 79 students (+ 17 new recruits for Fall 2005) enrolled and has consistently attracted a large and very high quality applicant pool. The program has been extremely successful in enrolling outstanding underrepresented minority students during the past five years. Fifteen percent of the current pool of students in the program are minorities. An Executive Committee representing the 104 (76 on this grant) faculty from 20 departments and Yerkes Center administers the program. This committee is headed by the Director, who oversees all aspects of program operation, a Director of Graduate Studies, who oversees the monitoring of student progress, and the Director of Admissions, who oversees all aspects of recruitment and admission of students to the program. The laboratory space and core facilities available for neuroscience research at Emory have grown substantially during the past funding period with the opening of two major neuroscience centers. Students in the program receive a broad curriculum of courses in their first two years, including three core courses in Neuroscience, a course in Biochemistry and Cell Biology and a course in Biostatistics. A required written thesis proposal (with oral defense) in the form of an NRSA predoctoral fellowship application teaches valuable grant-writing skills. A wide variety of elective courses ranging from Molecular to Computational Neuroscience and a required research rotation program provides the specialized training necessary for the development and completion of a thesis research project. Students are actively mentored through series of seminars, laboratory meetings and journal clubs, and yearly thesis committee meetings, institutions around the world to visit Emory, present their research, and interact with our students.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The purpose of this study is to elucidate the electrophysiological effects of stimulation of the centromedian nucleus (CM) of the thalamus upon striatal neurons. The rationale supporting this series of experiments is twofolds. First, despite the strong anatomical evidence for the existence of a major glutamatergic CM-striatal projection, very little is known about the physiology of the thalamostriatal pathways. Second, there is empirical evidence that deep brain stimulation of CM alleviates some of the symptoms in Parkinson's disease and reduces dramatically tics in patients who suffer of Tourette syndrome. However, because of our limited knowledge of the thalamostriatal system, these clinical observations remain purely empirical and do not rely on any solid functional basis, which limits considerably their refinement and deeper understanding of their underlying physiological mechanisms. Over the past year, the responses of hundreds of striatal cells were recorded following burst stimulation of CM. The CM stimulation with trains of stimuli at high frequency (100 Hz, 100 pulses/train) had an effect in the majority of recorded cells, which responded with either excitatory or inhibitory responses. Overall, the majority of putamenal projection cells and interneurons responded to thalamic stimulation by complex changes of activity composed of long latency excitatory and inhibitory effects suggesting the involvement of the intrinsic striatal GABaergic and cholinergic microcircuitry. Together, findings of this study provide evidence for complex physiological effects of thalamic inputs upon striatal neurons. These data are highly significant and help to understand the role of the thalamostriatal system in the functional circuitry of the basal ganglia. They also provide solid data to further understand the neural mechanisms that may underlie the beneficial effects of CM deep brain stimulation in PD and/or Tourette syndrome.

DESCRIPTION (provided by applicant): GABAergic transmission is mediated through two major receptor subtypes, GABA-A and GABA-B receptors. GABA-B receptors (GABA-BRs) are composed of GABA-BR1 and GABA-BR2 subunits. During the past funding period, we have described in detail the subsynaptic localization of GABA-BR1 and GABA-BR2 in the monkey basal ganglia. We found that most pallidal GABA-BRs are extrasynaptic in location. Through the use of electrophysiological, pharmacological and neurochemical approaches, we have also studied the effects of GABA-BRs activation on the neuronal activity and neurotransmitter release in the monkey globus pallidus. These studies highlighted not only the effects of GABA-BRs, but have also shown that GABA reuptake mechanisms, utilizing the GABA transporter (GAT), regulate the activation of extrasynaptic GABA- BRs in the primate pallidum. Activation of postsynaptic GABA-BRs in the basal ganglia occurs predominately with intense GABA release, e.g., during burst firing under physiologic conditions. Knowing that synchronized burst firing of GABAergic neurons in the basal ganglia is a key ingredient of the pathophysiology of Parkinson's disease (PD), we hypothesize that GABA-BRs in structures like the external pallidal segment (GPe), which is exposed to increased GABAergic drive from the striatum in parkinsonism, may display significant changes in localization and function in PD. GPe is part of the 'indirect'pathway of the basal ganglia. This pathway originates from the striatum and traverses GPe and the subthalamic nucleus (STN) en route to the basal ganglia output structures, the internal globus pallidus (GPi) and the substantia nigra pars reticulata (SNr). Increased activity along the striatum-GPe pathway in parkinsonism results in compensatory changes in GABA-B receptor distribution. The functional impact of these changes remains to be established. In this project, we propose to use high resolution electron microscopic immunocytochemistry, combined with in vivo and in vitro electrophysiologic techniques to characterize and compare the localization, trafficking and synaptic activation of GABA-BRs and the functional interplay between GAT and GABA-BRs in the basal ganglia of normal and parkinsonian animals. Studies proposed in this application will lay the foundation for a deeper understanding of the functions of GABA-BRs in the basal ganglia, and their role in the alteration of basal ganglia function in PD.

DESCRIPTION (provided by applicant): The striatum is the main entrance of extrinsic information to the basal ganglia circuitry. It is largely made up of medium sized GABAergic neurons characterized by densely spiny dendritic trees, the so- called "medium spiny neurons (MSNs)". The cerebral cortex and the thalamus are the two main sources of excitatory glutamatergic afferents to MSNs. Midbrain dopaminergic inputs tightly regulate striatal glutamatergic neurotransmission. Lesion of the nigrostriatal dopaminergic system in animal models of Parkinson's disease (PD) results in a dramatic loss of dendritic spines accompanied with altered transmission and plasticity of corticostriatal synapses. During the past funding period, we characterized in detail the synaptic connectivity of striatal glutamatergic afferents in nonhuman primates using the vesicular glutamate transporters 1 and 2 (vGluT1 and vGluT2) as specific markers of corticostriatal and thalamostriatal systems, respectively. Our data also provided further evidence for a major loss of dendritic spines and a relative increase in vGluT1 immunoreactivity in the striatum of MPTP-treated parkinsonian monkeys, thereby extending previous evidence for alterations in corticostriatal transmission in Parkinsonism. During the next funding period, we propose to extend these recent findings and further investigate plastic changes in spine morphology and AMPA glutamate receptor subunits localization that could possibly underlie functional alterations in corticostriatal and thalamostriatal glutamatergic transmission in Parkinsonism. These anatomical studies will be complemented with an in vivo assessment of changes in the physiological responses of striatal MSNs to activation of the corticostriatal system in awake MPTP-treated parkinsonian monkeys. Our proposal aims at achieving the following three goals: (1) To characterize the morphology and ultrastructural features of dendritic spines that receive cortical and thalamic inputs in the striatum of normal and parkinsonian monkeys, (2) To characterize and compare the subcellular and subsynaptic localization of the AMPA GluR1 subunit in spines contacted by cortical or thalamic inputs in normal and MPTP-treated parkinsonian monkeys and (3) To compare the electrophysiological effects of cortical stimulation upon striatal projection neurons activation between normal and parkinsonian monkeys, and determine if dopaminergic antiparkinsonian therapy mediates its beneficial effect through regulation of corticostriatal glutamatergic transmission . Together, these studies will provide highly significant information that will help further understand the physiology, pathophysiology and structural plasticity of the two main glutamatergic systems that regulate striatofugal neurons activity under normal and parkinsonian conditions. PUBLIC HEALTH RELEVANCE: The main goal of this proposal is to characterize morphological, neurochemical and electrophysiological changes induced in the excitatory glutamatergic transmission from the cerebral cortex and the thalamus onto the basal ganglia circuitry in Parkinson's disease. To achieve this endeavor, we will use high resolution anatomical techniques at the electron microscopic level combined with in vivo electrophysiological recording methods in an awake non-human primate model of Parkinson's disease. The results of these studies should further our current understanding of the neural plasticity that underlies functional changes in the basal ganglia circuitry in the parkinsonian state.

Core B is a central component of the Emory Udall Center of Excellence for Parkinson's disease research. The Core will provide neuroanatomical and behavioral expertise that will be essential to the successful completion of all projects proposed in this application. In contrast to traditional service cores, core B will not only provide technical expertise to the projects, but will play an active role in the study design as well as collection, analysis and interpretation of data related to each project. The proposed work that will be achieved by the core for the different projects include : (1) Project 1: In this project, the core will be responsible for immuno-electron microscopic studies of the localization of muscarinic receptor subtypes in the ventral motor thalamic nuclei of normal mice, and mouse models of Parkinson's disease. These findings will complement the physiological data Dr. Jaeger and his colleagues will collect on the role of these receptors in the regulation of firing activity in the parkinsonian thalamus. (2) Project 2: In this project, light and electron microscopy immunostaining methods will be used to characterize plastic changes in the synaptic connectivity and GABA receptor expression in the ventral motor thalamic nuclei of parkinsonian monkeys with lesions of the internal globus pallidus. These findings will be essential for the interpretation of the electrophysiological and behavioral effects of pallidotomies on thalamic activity and parkinsonian behavior. (3) Project 3: The core will determine the effects of TrkB agonists on striatal spine loss in mouse and monkey models of Parkinson's disease and assess the antiparkinsonian efficacy of TrkB agonist in parkinsonian monkeys. Together, findings of these studies will contribute to the characterization of a novel restorative therapy that could be used in patients with Parkinson's disease. (4) Project 4: In this project. Core B will provide a detailed quantitative assessment of the cellular and ultrastructural localization of the Ml and M4 muscarinic receptor immunoreactivity in the subthalamic nucleus and the substantia nigra pars reticulata. Such information will be critical for the interpretation of electrophysiological and behavioral effects induced by the different muscarinic receptor antagonists used in this project.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The interaction between the thalamus and the striatum has been implicated in disturbances of basal ganglia function such as Parkinson's disease. Deep brain stimulation of the centromedian/parafascicular (CM/PF) thalamic nuclei is effective in relieving motor symptoms associated with these conditions;however, its therapeutic effects are hampered by the minimal information known about the function of the thalamostriatal system. This study aims to examine the CM/PF-striatal anatomical and functional relationship in nonhuman primates, in order to understand the involvement of the thalamostriatal system in basal ganglia dysfunction. Our findings show that cholinergic interneurons mainly respond to CM activation by decreased activity, which is mediated by the local injection of GABAergic drugs. In the anatomical studies, immunohistochemistry at the electron microscopic level was double-labeled with neuronal markers for medium spiny neurons (MSNs) and striatal cholinergic interneurons (SCIs), in order to determine what portion of the GABAergic inputs observed onto SCIs are derived from MSNs, the main cell group of the striatum. Our findings demonstrate that SCIs mainly receive symmetric synaptic inputs along their entire somatodendritic domain, where MSN collaterals comprise about one quarter of these synaptic inputs. In conclusion, intrastriatal GABAergic microcircuitry is shown to be in a position to play an important role in modulating the responses of SCIs to the activation of the CM in monkeys.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The use of viral vectors as carriers of therapeutic agents in the CNS is a field of great interest for the treatment of neurodegenerative diseases, most particularly for Parkinson's disease, because of the preferential loss of nigrostriatal dopaminergic neurons, and the fact that the currently used long term dopamine replacement therapy results in major side effects that considerably limit the use of such therapeutic approach in advanced patients. In the present project, Dr Smith has developed a research program supported by Oxford Biomedica in UK to determine the antiparkinsonian efficacy of a lentiviral vector transfected with the necessary enzymes and co-factor for dopamine synthesis (hereby called Prosavin), in the MPTP-treated nonhuman primate model of Parkinson's disease. A group of seven monkeys were rendered parkinsonian following chronic injections of the toxin MPTP before they received five injections of 10 microliters (ie a total of 50 microliters per structure) of the viral vector in the sensorimotor territory of the postcommissural putamen. Following the injections, animals were allowed to survive for a period of 3-6 months during which their behavior was monitored on a weekly basis to assess changes in their parkinsonian symptoms. Data obtained so far have indicated modest behavioral improvement in some, but not all monkeys that received the vector injections. Postmortem studies are in progress to assess the extent of striatal transfection and the resulting degree of striatal dopamine innervation. These findings in nonhuman primates will serve as the basic foundation for clinical trials in humans with Parkinson's disease.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Parkinson's disease is a progressive neurodegenerative disorder that starts many years prior to the appearance of the first motor symptoms. Thus, if one could intervene early in the disease process to slow down or reverse the progressive degeneration of midbrain dopaminergic neurons, it could have a significant impact on the development of the disease. The goal of this project is to identify preclinical biomarkers that would give the opportunity to pre-treat at risk patients with neuroprotective therapeutic drugs, which could significantly delay or even prevent the death of dopaminergic neurons and the development of motor deficits. In collaboration with Dr Jing Zhang at the University of Washington, this project aims at identifying such biomarkers using proteomics approach from the serum, cerebrospinal fluid and brain tissue in MPTP-treated monkeys. Although no data have yet been collected in this project, a total of 10 monkeys have been treated with MPTP until they reach about 30-40% ("asymptomatic group") or 70-80% ("symptomatic group") striatal dopamine loss, as measured using PET scan imaging for dopamine transporter ligands. Another group of animals, used as controls, was injected with vehicle. After they have reached the required level of striatal dopamine denervation, spinal taps and serum draw were performed before the animals were sacrificed and the brain tissue will be used for proteomics measurements. The results of these monkey studies will be compared with those gathered from serum and CSF measurements collected from at risk patients. Together, these findings could provide the first evidence for the characterization of biomarkers that could predict the development of Parkinson's disease in humans.

DESCRIPTION (provided by applicant): This is a competing renewal of a five-year Institutional NRSA application for the support of predoctoral students in the interdepartmental Graduate Program in Neuroscience at Emory University. A fundamental strength of this program is the broad interdisciplinary training provided in a wide spectrum of neurobiological issues spanning several basic and clinical neuroscience-related disciplines. Because of the significant growth in highly qualified applicants to this program during the past funding period, the Emory Neuroscience program seeks support for 10 students per year in this renewal. A total of 84 PhD students (+ 20 new recruits for fall 2010) are enrolled in this program, which has consistently attracted a large and very high quality applicant pool. The program has been extremely successful in enrolling outstanding underrepresented minority (URM) students during the past 5 years. Fifteen percent of the current pool of students in the program are from URM ethnic groups. An Executive Committee representing the 115 (100 on this grant) faculty from 23 departments and the Yerkes National Primate Research Center administers the program. This committee is headed by the Director, who oversees all aspects of program operation, two Directors of Graduate Studies, who monitor student progress, and the Director of Admissions, who is in charge of recruitment of new students in the program. The laboratory space and core facilities available for neuroscience research at Emory have grown substantially during the past funding period. Students in the program receive a broad curriculum of molecular, cellular and systems neuroscience courses in their first two years. A required hypothesis design and grant writing course helps students prepare their thesis proposal (with oral defense) in the form of an NRSA predoctoral fellowship application. They are also required to participate in 3 laboratory rotations before they pick their advisor (usually at the beginning of year 2). Almost 40% of students are successful in competing for national awards after their third year of training. A wide variety of elective courses ranging from Basic Mechanisms of Neurological Diseases, Brain imaging and Computational Neuroscience are available to advanced trainees. Finally, students actively participate in various seminar series and receive significant training in teaching, neuroethics and scholar integrity. PUBLIC HEALTH RELEVANCE: This proposal is a competing renewal to support the training of ten pre-doctoral students enrolled in the PhD graduate neuroscience program at Emory University during years 1 or 2 of training. The interdisciplinary Emory neuroscience program comprises a total of 96 training faculty and 84 PhD students involved in neuroscience research that cuts across a wide array of disciplines that range from the understanding of basic neural communication to the treatment of complex neurological and psychiatric disorders. The Emory neuroscience program has reached a high level of success in attracting the most talented students in the country and over the world to apply for admission in this program. The pool of applicants has increased by more than 50% over the past five years, while maintaining very high standards of academic and research excellence. This dramatic growth of the applicant pool combined with the continued raise in the number of well-funded neuroscience faculty serve as the main basis to justify an increase of 4 trainees/year being supported by this training grant during the next funding period.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The three groups of mGluRs are strongly expressed at pre- and post-synaptic sites in the basal ganglia. Rodent studies achieved in Dr Smith laboratory in collaboration with Dr Conn at Vanderbilt University have provided clear evidence that the group III mGluR subtype, mGluR4, represents a highly promising target for non-dopaminergic therapy in Parkinson's disease. Through the use of electron microscopy procedures and in vitro slice electrophysiology, Dr Smith and colleagues have shown that the mGluR4 is located pre-synaptically at two key synapses in the basal ganglia circuits that become overactive in Parkinson's disease, namely the corticostriatal glutamatergic synapse and the striatopallidal GABAergic synapse. Electrophysiological studies in rat brain slices indicate that activation of group III mGluRs reduces synaptic transmission at these synapses and that intracerebral injections of group III agonists provide antiparkinsonian benefits in rat models of Parkinson's disease. Based on this solid and highly promising foundation, ongoing studies in Dr Smith laboratoryl aim at testing the antiparkinsonian efficacy of intrecerebral administration of group III mGluR agonist and mGluR4 allosteric potentiator (prepared in Dr Conn's laboratory) in the MPTP-treated nonhuman primate model of Parkinson's disease. Furthermore, in order to better understand the possible mechanisms underlying the behavioral antiparkinsonian benefits of these compounds in primates, the physiological activity of pallidal neurons is being recorded in response to the administration of mGluR4 agonists and potentiators.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Current pharmacological treatments for Parkinson's Disease (PD) act on the dopamine neurotransmitter system and cause debilitating motor side effects. This project seeks to study new potential antiparkinsonian drugs (and their mechanisms of action) that act on other neurotransmitter systems and are therefore not likely to cause motor side effects. Antagonists of metabotropic glutamate receptor 5 (mGluR5) and adenosine A2A receptor (A2AR) are the focus of this study. These drugs will be injected systemically or locally into various nuclei of the basal ganglia of MPTP-treated parkinsonian monkeys (the gold-standard model of PD), and their motor behavior will be monitored. These drugs will be administered both alone and together. We have found that when injected systemically, the mGluR5 antagonist MTEP provides a modest anti-akinetic benefit to MPTP-treated monkeys. After trying several different A2AR antagonists, we have been unable to measure any behavioral antiparkinsonian effect in our monkeys, whether the drugs were administered systemically or locally in the basal ganglia, either given alone or in combination with mGluR5 antagonist. We were unable to obtain the only A2AR drug published to have antiparkinsonian effects in monkeys at the time (KW-6002), due to the proprietary nature of the drug. Since then, one more A2AR drug (preladenant) has been published to have antiparkinsonian effects in monkeys. We have made arrangements to get some of this new compound, with better affinity/selectivity/bioavailability, for testing in our monkeys.

Project Summary ? Project 3 Models of the activity changes in the basal ganglia-thalamocortical circuitry in Parkinson's disease have been instrumental for the development of new surgical and pharmacologic antiparkinsonian therapies. However, it has become clear that these models do not fully explain the complex alterations in the patterns of neuronal activity and abnormal network oscillations in the parkinsonian brain. Especially, our knowledge of the anatomical, functional, and pathological changes in the thalamocortical and corticothalamic systems in Parkinson's disease is very limited. In conjunction with the functional studies of projects 1 and 2, and the technical support of Core B, the proposed studies will help us to gain a detailed understanding of the organization and pathology of the synaptic networks through which basal ganglia, thalamic and cortical neurons interact. Our preliminary studies provide strong evidence that the synaptic microcircuits that mediate the communication between the basal ganglia receiving portion of the thalamus and motor cortices display significant pathology in the non-human primate model of Parkinson's disease. At the cortical level, a loss of dendritic spines on projection neurons and a prominent decrease in the thalamic innervation of deep cortical layers was found in M1 of MPTP-treated parkinsonian monkeys. Both findings are strong indicators of pathologic disturbances of thalamocortical communication in parkinsonism. Interestingly, it appears that these changes are particularly prominent in M1, suggesting that the network dysfunction is regionally specific. Combined with recent electrophysiological data showing that the activity of corticospinal, but not corticostriatal, neurons is disrupted in M1 of parkinsonian monkeys, we hypothesize that pathological changes in thalamocortical synaptic connections may differentially affect corticospinal over corticostriatal neurons. Our preliminary data also suggest that the relationships between the thalamus and motor cortices may be impaired by synaptic changes at the thalamic level in parkinsonian monkeys, such that the synaptic connectivity of cortical afferents with GABAergic interneurons and projection neurons is altered. In light of these preliminary findings, we propose a series of morphological, neuropathological, and ultrastructural studies to further assess and quantify the changes in the architecture of the thalamocortical and corticothalamic synaptic networks in normal and MPTP-treated parkinsonian monkeys, as well as in normal and 6-OHDA-treated mice (for comparisons with the studies in primates). The results of these studies will be of critical importance for our understanding of the pathophysiology of parkinsonism, and will aid the interpretation of the functional data obtained in the other projects.

DESCRIPTION (provided by applicant): The long-term objective of this application is to foster the development of clinician- investigators in neuroscience. The goal of the research education program proposed here is to allow selected Emory neuroclinical trainees (in Neurology, Neurosurgery, Neuropathology, Neuroradiology and Emergency Medicine), during residency and fellowship training, to acquire the necessary research skills and background to enable them to be competitive for mentored career development awards and thus catalyze the continuity of the neuroclinician-investigator species. To this end, the specific aim of this application is to provide an outstanding research education experience to one or more residents each year from Neurology, Neurosurgery, Neuropathology, Neuroradiology or Emergency Medicine, to be complemented by a continuation of the participants' research education during subsequent fellowship training. A key component of the research education will be mentored laboratory or clinical research under the supervision of an experienced mentor. The participants in the present program will also have the benefit of a mentor team comprising 1-2 members in addition to the primary mentor, and their development will be closely monitored by the PIs. The participants in the present program will also be able to take advantage of additional educational resources, including courses in research design and analysis, grant-writing and research ethics.

Abstract Neurodegenerative diseases that involve the basal ganglia commonly lead to cognitive impairments. In this proposal, we hypothesize that the massive projections from the thalamic parafascicular (PF) and centromedian (CM) nuclei to the caudate nucleus and the putamen, respectively, are key pathways that regulate cognitive processing, and that lesion of these thalamostriatal systems produce selective attention-related cognitive impairments in behavioral flexibility, habit learning, and possibly other forms of cognition. The CM and PF provide functionally organized glutamatergic projections that target striatal projection neurons and interneurons (particularly cholinergic interneurons). Functional imaging data from human and monkey studies have shown that CM and PF neurons respond strongly to behaviorally significant sensory events. It was also shown that CM/PF inputs to the striatum regulate responses of striatal tonically active neurons (TANs; likely corresponding to cholinergic interneurons) to salient reward-related stimuli, and that inactivation of Pf disrupts performance in attention tasks. Because the CM/PF complex profoundly degenerates early in the course of Parkinson?s disease (PD) and Huntington?s disease (HD), a detailed knowledge of the role of the CM- and PF-striatal projections will help us to understand the importance of the degeneration of these nuclei in the development of cognitive impairments in PD and HD. In this pilot project, we will evaluate the behavioral consequences of selectively lesioning the CM- or Pf-striatal projections in monkeys, using an immunotoxin lesioning approach. These preliminary studies will set the foundation for the development of a future R01 proposal that will assess the contribution of the primate thalamostriatal system to cognition. A better knowledge of the role of this projection system in normal basal ganglia function is essential to gauge the importance of its degeneration in neurodegenerative diseases.

SUMMARY According to current models, the involvement of the basal ganglia in motor and non-motor functions is explained in the context of information processing in segregated basal ganglia-thalamocortical loops. These models predict that striatal dopamine loss in Parkinson?s disease (PD) eventually leads to abnormal processing in the ?motor? thalamocortical network, and the antiparkinsonian effects of deep brain stimulation (DBS) of the sensorimotor internal globus pallidus (GPi) is explained as a release of movement-related thalamic neurons from overactive inhibitory GPi inputs. However, recent evidence suggests that descending basal ganglia output, specifically the massive projection of GPi to the pedunculopontine nucleus (PPN), may also be relevant for normal behavior and parkinsonism. Thus, manipulations of the PPN influence limb movements and postural adjustments, PPN activation has antiparkinsonian effects in monkeys, and DBS of the PPN ameliorates gait disturbances in some PD patients. The PPN is a highly heterogeneous brain region that gives rise to widespread ascending and descending projections. Our lack of knowledge of the anatomical targets of GPi projections to the PPN, and the effects of activation of the GPi-PPN pathway on PPN activity limits our understanding of the normal role of the GPi-PPN interaction and its role in the pathophysiology of PD, particularly in primates. The proposed studies aim therefore to examine the functional connectivity between the GPi and the PPN (aims 1 and 2), determine whether the anatomy and physiology of these networks are altered in the parkinsonian state (aims 2 and 3), and how GPi-DBS alters firing rates and patterns of GPi-receiving PPN neurons, as well as local field potential activity in the PPN (aim 3). These studies will be done in normal and MPTP-treated parkinsonian monkeys, using a combination of state-of-the-art optogenetic, anatomical and electron microscopy procedures. The knowledge gained from these studies is needed to develop or refine antiparkinsonian therapies that target the PPN or its projections for treatment of PD or other basal ganglia disorders.

ABST RACT In current schemes of the pathophysiology of Parkinson?s disease (PD), neuronal activity changes in the sen- sorimotor region of the subthalamic nucleus (STN) play a central role in the development of parkinsonism. Until recently, the changes in STN activity were thought to result solely from reduced inhibition from the external globus pallidus (GPe). However, recent findings from animal models of advanced parkinsonism have suggested that a profound loss of glutamatergic cortico-subthalamic terminals and an increased strength of GABAergic pallidosubthalamic synapses may contribute to activity changes in the STN and to the development of parkin- sonism. Our preliminary data demonstrate that a loss of cortico-subthalamic terminals is also present in the sensorimotor STN territory of people with advanced PD. It remains unclear, however, how these anatomical and physiologic changes relate to the degree of nigrostriatal dopamine loss and to the expression of parkinsonism. Further, it is unknown if these changes also affect non-motor regions of the STN, perhaps contributing to cogni- tive or affective PD symptoms. We will examine these issues with neuropathological and electrophysiological studies in monkeys with different degrees of MPTP-induced dopamine loss (Aim 1), and with longitudinal 7T ultra-high field MRI studies in people with early PD (Aim 2). In Aim 1, we will record responses of STN neurons to optogenetic activation of cortical and pallidal inputs in monkeys that remained either asymptomatic after ex- posure to small dose of the dopamine-depleting neurotoxin MPTP or became parkinsonian after exposure to (larger doses of) MPTP. We will also assess changes in local field potentials (LFPs) and abnormal spiking activity in STN, and in the coherence between STN LFPs and motor cortical electrocorticograms. In postmortem studies of the same animals, we will use high resolution microscopic immunohistochemical studies and 3D-EM reconstructions to assess whether the number, localization, and morphology of glutamatergic and GABAergic synapses in the STN changes as a function of dopamine loss. We will also compare the number of cortico- subthalamic terminals and examine changes in GABAergic markers in STN tissue from patients with PD and age-matched controls. In Aim 2, we will use state-of-the-art diffusion and resting state functional MRI to test whether humans with early stage PD exhibit significant changes in the volume and microstructural organization of the STN and its cortical and pallidal afferents, and determine if these changes are related to the expression and progression of motor and non-motor impairments. The same patients will be studied at enrollment and 30 months later to examine changes in the MRI measures. The results of this project will increase our understanding of the temporal evolution of parkinsonism-associated plastic changes in the STN, and determine their potential relationships to the development and severity of motor and non-motor signs and symptoms of the disease. These studies may lead to novel interventions to control or prevent abnormal firing patterns in STN and may contribute to the development of imaging biomarkers to identify early stages of PD and predictors of disease progression.