James M. Tepper - US grants

This application for a First Award seeks research support for five years to study the afferent control of substantia nigra dopaminergic neuron activity. Many lines of evidence point towards a dopaminergic component in schizophrenia, but there is actually no evidence that the dopaminergic neurons themselves are dysfunctional, and the possibility exists that the problem is one of altered afferent modulation of dopaminergic neuron activity. Previous attempts to study the anatomy and physiology of afferents nigral dopaminergic neurons have been hindered by the complex cytoarchitectonic organization of the substantia nigra, the lack of identification of the neuron being studied as dopaminergic, and uncertainty as to the site of origin of evoked electrophysiological responses or synapses contacting nigral neurons. This application proposes to study dopaminergic afferents by electrophysiological- neuroanatomical techniques in which dopaminergic neurons will be electrophysiologically identified in vivo and recorded intracellularly with horseradish peroxidase (HRP)-containing electrodes. Intrinsic properties and responses to stimulation of suspected afferents will be observed and measured, and then the neuron will be intracellularly injected with HRP and processed for sequential light and electron microscopic analyses. HRP-stained neurons will be examined, drawn and photographed with a light microscope, and representative regions of the entire dendritic extent of identified and individually labelled dopaminergic neuron will be trimmed out, re-sectioned on an ultramicrotome, and examined in the electron microscope, where a qualitative and quantitative analyses of afferent synapses will be conducted. Immunocytochemical labelling of synaptic terminals made onto these neurons will be used to correlate morphologies of synaptic endings with specific neurotransmitters and neurotransmitter-related substances known to exist in substantia nigra, for example, glutamic acid decarboxylase,'substance P, choline acetyltransferase, enkephalin, dopamine beta-hydroxylase and serotonin. The sites of origin of afferents to elec- trophysiologically identified, HRP-filled nigral dopaminergic neurons will be studied by electron microscopic anterograde tracing experiments using Phaseolus vulgaris leucoagglutinin. Lesion degeneration studies will be performed as well in order to identify the sources of both physiologically observed responses to afferent stimulation as well as to correlate physiological responses with the ultrastructural morphology of synapses onto identified dopaminergic neurons in substantia nigra.

These proposed experiments are designed to investigate postnatal changes in the neurophysiology, neuroanatomy and neuropharmacology of neostriatal and substantia nigra dopaminergic neurons in the developing rat. There are many reasons for needing to understand the postnatal ontogeny of neuronal function in the basal ganglia. One is the recent finding that surviving neurons in grafts of fetal neurons to adult hosts, currently used as both an experimental tool as well as a novel clinical approach to treatment of neurodegenerative disorders such as Parkinson's, Alzheimer's and Huntington's disease, exhibit characteristics much more similar to early neonatal neurons in situ than their corresponding adult counterparts. In order to understand the scope and limitations of neuronal grafting as a clinical tool, it is necessary to first understand the neurophysiological properties of immature basal ganglia neurons that occur during the postnatal period. A second important implication of this work is in trying to understand the physiological bases for certain developmental disorders of learning and behavior, for example, attention deficit disorder with hyperactivity (ADDH), and their pharmacological management with stimulants. The experiments utilize extracellular and intracellular single unit recording in vivo to chart the postnatal development of spontaneous and evoked activity of neostriatal and substantia nigra dopamine neurons. Intracellular recordings will be performed with microelectrodes containing biocytin which will be injected into each neuron at the end of the physiological experiments to reveal the entire structure of the neuron, thus allowing a correlation of the postnatal development of neuronal physiology with morphology. Other experiments will use PHA-L to label afferents to substantia nigra and neostriatal neurons, some of which will be subsequently intracellularly labeled with biocytin. Inspection of the tissue at the light and electron microscopic levels will allow us to determine how the pattern of innervation of these important afferents develops in normal postnatal brain, as well as to determine the effect of the nigrostriatal dopamine input on the development of cortical and thalamic synaptic inputs to neostriatum by using 6-OHDA to destroy the dopaminergic inputs. The last major focus of these studies is to use in vitro intracellular recordings to determine the sites and mechanisms of action whereby amphetamine and related stimulants produce a paradoxical excitatory effect on the firing of nigral dopaminergic neurons in early postnatal rats instead of the inhibition that is seen in adults. This phenomenon may have special significance to the mechanism of the paradoxical behavioral effects of amphetamine-like compounds in the treatment of ADDH, as well as to the phenomenon of marked differences in the effects of stimulant drugs on mature versus immature nervous systems.

9413198 Jordan This award to Frank Jordan and a group of 11 other faculty will support a multi-disciplinary training program in biophysical aspects of cellular processes at Rutgers University-Newark, an urban campus with significant enrollement of minority students. The faculty group have excellent research programs in three related areas: structure and dynamics of biological macromolecules, analysis of cellular transport mechanisms, and biophysics of excitable cells. The award will support training of students at the undergraduate and graduate levels; funds awarded will provide student stipends and tuition, purchase research instruments and supplies to be used by trainees, seminars by outside experts and student travel. Current training activities will be enriched by three new laboratory courses and additional required rotations in faculty laboratories. ***

Dysfunctioning of the midbrain dopamine system has been suggest as an important etiologic factor in schizophrenia and related psychoses and a number of motor disorders. Although much progress has been made in recent years towards understanding the intrinsic cellular mechanisms that control dopamine neuronal activity and the release of dopamine in the brain, progress towards understanding the afferent control of dopamine neuron activity has been slower in coming. In particular, the inputs that modulate the firing rate and pattern of dopaminergic neurons in vivo remain unclear. That the firing patterns of dopaminergic neurons are controlled principally by afferent input is demonstrated by the fact that in vivo, these neurons exhibit firing patterns that range along a continuum from pacemaker-like firing through random firing to bursty firing, but in vitro, only the pacemaker pattern is observed. Based on neuroanatomical evidence, the most important afferents to substantia nigra neurons are likely to be GABAergic and glutamatergic. The densest and best-studied GABAergic inputs to dopaminergic neurons are thought to originate in the neostriatum and globus pallidus, while the most prominent excitatory inputs arise from the subthalamic and pedunculopontine nuclei, and the frontal cortex. However, it is not possible to elicit bursting activity in dopaminergic neurons by stimulation of any of these sites, and the most common response of dopaminergic neurons to simulation of the subthalamic or pedunculopontine nuclei or frontal cortex is inhibition. The experiments in this application for a competitive renewal are designed to t est various aspects of the general hypothesis that there exists an important GABAergic pathway to dopaminergic neurons originating from the local axon collaterals of non-dopaminergic substantia nigra pars reticulate projection neurons. A corollary of this hypothesis is the idea that many of the most excitatory important inputs to dopaminergic neurons that control the rate and pattern of spontaneous activity are "filtered" through these pars reticulate neurons. These ideas will be tested with a variety of electrophysiological and neuroanatomical methods including in vivo and in vitro intracellular recording and intracellular labeling of substantia nigra dopaminergic and non-dopaminergic neurons in vivo extracellular recording from identified substantia nigra dopaminergic and non- dopaminergic neurons, sequential light and electron microscopy of double labeling of anterograde tracers or intracellular biocytin labeling and dopaminergic neurons, and intracellular labeling coupled with immunocytochemical identification of the postsynaptic targets.

Dopaminergic neurotransmission in the brain modulates a number of important cognitive and behavioral functions. For example, schizophrenia is believed to result from an imbalance in forebrain dopamine systems. Recent advances in molecular biology have led to the identification of 5 different genes coding for dopamine receptors of 2 main families, D1 and D2. Although agonists and antagonists that discriminate between members of the D1 and D2 families exist, drugs do not yet exist that are selective among family members (e.g., D2 vs. D3 vs. D4 or D1 vs. D5). This has resulted in confusion about the sites and mechanisms of action of dopamine in the brain, about precisely which receptor subtypes mediate which cellular effects, and likely also contributes to the mixed therapeutic efficacy and unwanted side effects of antischizophrenic drugs in use today. We have shown that in vivo infusion of specifically designed antisense oligodeoxynucleotides complementary to the mRNA coding for different dopamine receptors produces a highly regional- and receptor-specific pre- or postsynaptic "knockout" of the dopamine D2 or D3 receptors as indexed by receptor autoradiography. In vivo and in vitro electrophysiological techniques will be used to determine the roles that D2 and D3 dopamine receptors play in the modulation of the electrical activity of substantia nigra dopaminergic neurons and neostriatal neurons. Subsequent experiments will utilize similar methods to achieve knockout of the D1 and D4 receptors. The site and receptor subtype of the dopamine receptor that mediates the inhibitory effects of amphetamine on nigral cell firing will be determined. In vivo extracellular and in vitro intracellular recordings will be used to confirm the identity of the nigral somadendritic autoreceptor and determine the role that it plays in the control of the rate and pattern of firing of nigral dopamine neurons. In vivo and in vitro intracellular recordings will be used to define receptor subtype- specific actions of dopamine on neostriatal neurons, and determine the site and subtype of the receptor that mediates dopamine's effect on corticostriatal synaptic transmission. This research is highly relevant to both basic and applied neuroscience. Validating the antisense approach to selective receptor knockout in vivo by electrophysiological means is a necessary first step towards the application of this technique to a variety of research issues. The identification of the particular dopamine receptor subtype(s) that mediates a number of well-characterized physiological responses to dopamine may provide information that will direct future research towards the design of a new generation of antipsychotic drugs that are simultaneously more effective and lack the often devastating side effects of the neuroleptics in use today.

DESCRIPTION: (Adapted from the applicant's abstract) The experiments in this proposal are designed to describe the anatomy and physiology of afferents that control the activity of substantia nigra dopamine neurons. In particular, various aspects of a GABAergic pathway to dopaminergic neurons originating from the axon collaterals of non-dopaminergic substantia nigra pars reticulata projection neurons will be studied. The central hypothesis is that many of the important inputs to dopaminergic neurons that control the rate of firing and spontaneous activity are filtered through pars reticulata neurons. Six specific aims are proposed. Aim 1. To test the hypothesis that there is a physiologically functional monosynaptic connection between substantia nigra pars reticulata GABAergic projections neurons and pars compacta nigrostriatal dopaminergic neurons with electrophysiological means. Aim 2. To determine the subtype of the GABA receptor on the dopaminergic nigrostriatal neuron that mediates the inhibitory influence of GABAergic afferents arising from neostriatum, globus pallidus and substantia nigra par reticulata with in vivo neuropharmacological studies. Aim 3. To determine the relative influences of GABA-A and GABA-B synaptic inputs on the firing pattern of nigrostriatal neurons in vivo. Aim 4. To look for anatomical evidence of a direct monosynaptic connection between substantia nigra pars reticulata projection neurons and nigrostriatal dopaminergic neurons by light and electron microscopic analysis of the axon collaterals of electrophysiologically characterized pars reticulata neurons that were intracellularly or juxtacellularly labeled with biocytin. Aim 5. To test the hypothesis that activation of GABAergic pars reticulata neuron axon collaterals and/or pallidonigral inputs are responsible for the inhibitory responses seen in pars compacta dopaminergic neurons after stimulation of presumed excitatory inputs. Aim 6. To test the hypothesis that individual striatonigral and pallidonigral afferents synapse with either dopaminergic or non-dopaminergic neurons in substantia nigra by intracellular or juxtacellular labeling and tracing axons to nigra and performing double label electron microscopy.

DESCRIPTION (Adapted from applicant's abstract): The basal ganglia are critically involved in the organization of motor behavior and sensorimotor integration. Malfunctions of this system in humans contribute to the pathophysiology of neuropsychiatric disorders such as schizophrenia and obsessive compulsive disorder, as well as to neurological diseases such as Huntington's and Parkinson's disease. Understanding the information processing within this system in mechanistic terms requires the understanding of the physiological properties and anatomical organization of the microcircuitry of its constituent nuclei. The neostriatum, the largest nucleus of the basal ganglia has a central role in controlling the functioning of the basal ganglia and therefore, an understanding of the intrinsic operations of this nucleus is critical to understanding information processing in the basal ganglia. Recently, it has been increasingly recognized that the quantitatively minor population of GABAergic interneurons of the neostriatum may play a critical role in the organization of the population activity of the principal cells. However, until the recent introduction of visually guided whole cell recording these neurons were not accessible for physiological investigation. In the proposed study, this powerful technology will be used in combination with intracellular staining and light and electron microscopic analysis to analyze the role of these interneurons in the control of the activity of their principal postsynaptic targets, the medium spiny neuron. Simultaneous paired recordings will be obtained from interneurons and medium spiny cells and the neurons will be stained for further anatomical investigation. The following specific questions will be addressed. First, what is the nature of the synaptic interaction between the two major types of inhibitory inteneurons and the medium spiny neuron? Second, what are the physiological and anatomical specializations of the inhibitory inputs to the medium spiny neuron from different types of interneurons? Third, what is the pattern of connectivity among the populations of interneurons and medium spiny neurons (i.e. convergence and divergence)? Finally, what are the physiological properties of electrical coupling between interneurons, a potentially significant mechanism affecting the population activity of these cells, as well as, the medium spiny neurons? This data will help in understanding the contribution of these interneurons to the organization of activity in the neostriatum.

A grant has been awarded to Rutgers University under the direction of Dr. Esther Nimchinsky to acquire a two-photon laser scanning microscopy (2PLSM) suite, consisting of two custom-designed microscopes operating off a single laser source. The research projects described below represent some of the first studies in what will undoubtedly be the next phase of synaptic physiology research. They go to the heart of the question of how individual synapses interact with their immediate microenvironment, and how neurons are able to receive so many diverse inputs, respond individually to each, and maintain precisely an appropriate level of functioning for the constantly changing demands of the outside world.

The understanding of how neurons communicate with one another has come largely from studies where large numbers of synapses are sampled at the same time, and inferences are drawn regarding their individual behavior from the population averages. While this approach has yielded a great deal of knowledge, there is no escaping the fact that synapses are individual structures. In fact, one of their fascinating properties is that they can be modified separately-with over 10,000 synapses on each neuron, this is an ability that permits an exquisite degree of fine-tuning. However, their extremely small size makes them very difficult to study. In recent years there have been several important technological advances that greatly improve the ability to study individual synapses and their modulation. 2PLSM is an advanced imaging technique that was developed to permit imaging of structures deep in live tissue in vitro and in vivo for extended periods. It thus permits very high-resolution studies at the level of individual synapses in intact tissue, as well as time-lapse studies, which are critical for the uncovering of time-dependent processes. At the same time genetically encoded fluorophores have been characterized and improved, permitting the labeling of living cells with relatively low toxicity. Dyes sensitive to changes in intracellular calcium have also improved dramatically, and these allow the study of functional aspects of neuronal behavior. The system proposed here would be flexible enough to take full advantage of all these innovations. Using 2PLSM and new fluorescent dyes, individual synapses can, for the first time, be studied optically in intact tissue. Specifically, all these techniques will be combined to study the interactions of astrocytes, the major non-neuronal cell type in the brain, with synapses; the ways in which neurons balance the strengths of their synapses across their branches; and the roles of the neurotransmitters dopamine and serotonin in synaptic function, and their mechanisms of action.

This 2PLSM suite will greatly benefit projects that have a broad relevance in neuroscience, and which will be publicized by publication in major journals and presentation at national and international meetings such as that of the Society for Neuroscience. The acquisition of this flexible system will further the teaching mission of the university. Students and postdoctoral fellows in the participating labs and beyond will learn not only how neurons look and how synapses function, but will also acquire hands-on experience in the fundamentals of optics and microscopy, and learn how to optimize experimental conditions and the instruments themselves to make the most of their preparations. In addition, the faculty themselves will learn to use and exploit this important new technology, and perhaps also further to advance it. Furthermore, the acquisition of this microscopy system at Rutgers University-Newark, a campus where underrepresented minorities comprise a very sizeable proportion (40%) of the student body, will put state-of-the-art technology and a cutting-edge approach within reach of a large number of motivated students who would otherwise be very unlikely to have access to them. Finally, the presence at the campus of these microscopes would enhance the strengths of the CMBN in the field of neuronal plasticity, and help to attract faculty and students that are interested in this rapidly expanding field.

DESCRIPTION (provided by applicant): The basal ganglia, and especially the dopaminergic components of this system, are well known to play a central role in the role in the etiology and pathophysiology of several neurological and psychiatric disorders including Parkinson's disease and schizophrenia. More recently, however, mesotelencephalic dopaminergic systems have also been viewed as integral to certain types of learning and memory, affective responses and perception, and several types of higher cognitive function. In vivo, dopaminergic neurons fire spontaneously at low rates. This activity exists along a continuum of firing pattern from a regular pacemaker-like pattern on one end, to an irregular or random pattern to a slow bursty pattern on the other end. Dopaminergic neurons in vivo typically respond to behaviorally relevant environmental stimuli with an increase in firing rate in the form of a low frequency burst that usually lasts for a few hundred milliseconds. The timing of the dopaminergic signal is crucial for many of the functions ascribed to the dopaminergic system in signaling stimulus characteristics, reward salience or predictive error. Although it is clear that switches to the different patterns of activity are triggered by afferent activity, the afferents responsible and in particular the mechanisms of the burst or burst initiation are not clear. It is the overall goal of this competing renewal to extend observations made in the last Brant cycle by concentrating on GABAergic mechanisms in the afferent control of substantia nigra dopaminergic neurons studied by in vivo and in vitro neurophysiology, light and electron microscopy and in vivo microdialysis. There are 5 specific aims that will test the following hypotheses: (1) GABA-A receptors on dopaminergic neurons re predominantly or exclusively activated by GABAergic inputs in vivo under typical experimental conditions and activation of GABA-B receptors only occurs when the GABA transporter is saturated by excessive or high frequency Input and/or pharmacological blockade, (2) Most postsynaptic GABA-B receptors on substantia nigra dopaminergic neurons are located perisynaptically, (3) Afferent induced alterations in the pattern of activity of DAergic neurons lead to significant changes in extracellular levels of DA in striatum and substantia nigra, (4) Nigral GABAergic interneurons ore a source of afferent input to DAergic neurons, and (5) The difference in sensitivity to GABA-A receptor agonists between DAergic and GABAergic neurons in substantia nigra is due to a differential GABA-A subunit composition and/or a difference in the density of GABA-A receptors. These data should provide answers to several important questions about the afferent control of nigral dopaminergic neurons which are essential for understanding the normal function of the basal ganglia and which may also point the way toward improved pharmacotherapies for disorders involving the dopamine system.

DESCRIPTION (provided by applicant): This proposal is to get support from NIH for travel fellowships to the 10th triennial meeting of the International Basal Ganglia Society (IBAGS) run by Dr. James Tepper and Dr. Elizabeth Abercrombie. The purpose of the IBAGS meeting is to advance the understanding of the structure and function of the normal and diseased basal ganglia, to bring together researchers from countries from all over the world and from multiple scientific disciplines working on various aspects of the basal ganglia, and to inform the general public of results and implications of current research in this area. The meeting generally attracts between 175 and 300 participants (although one of our goals for this meeting is to increase the size of the society and the meeting slightly. There are no concurrent sessions;thus no one needs to decide between two competing sessions. All the sessions are held in one room so there is no need to move around from place to place. PUBLIC HEALTH RELEVANCE: The International Basal Ganglia Society meeting is to advance the understanding of the structure and function of the normal and diseased basal ganglia, to bring together researchers from countries from all over the world and from multiple scientific disciplines working on various aspects of the basal ganglia, and to inform the general public of results and implications of current research in this area.

DESCRIPTION (provided by applicant): Neostriatal cholinergic (ChAT) interneurons play an important role in the selection and acquisition of adaptive behavioral actions by encoding the salience and reinforcement value of external events. This information is represented in the precise temporal structure of synchronous multiphasic population responses given to the presentation of behaviorally significant stimuli. An important problem is to understand how these transient signals of ChAT interneurons are detected and decoded in the neostriatal network. Our hypothesis, formulated on the basis of preliminary experiments, is that ChAT interneurons control the activity of the striatal circuitry using nicotinic receptor mediated mechanisms to activate multiple parallel GABAergic mechanisms that elicit kinetically distinct independent inhibitory responses in the spiny projection neurons (SPNs) and in other striatal neurons. These responses originate in part from direct activation of multiple as yet unidentified types of GABAergic interneurons that are distinct from the parvalbumin (PV) containing fast spiking (FS) and NPY expressing interneurons, possibly also involve presynaptic nicotinic facilitation of GABA release from axon terminals and together provide selective, cell type specific inputs to SPNs, ChAT and other interneurons. Using in vitro optogenetic inhibition we also reproduced the population response of ChAT interneurons most commonly observed in behaving animals consisting of a pause of firing followed by weakly synchronous rebound-excitation and demonstrated that this behaviorally directly relevant pattern of activity also engages the inhibitory mechanisms described above. Since these responses are sufficient to inhibit action potential generation in large populations of SPNs they are likely to exert significant effects on the functioning of the basal ganglia and regulate ongoing behavior. The proposed experiments aim at understanding the functional organization of these GABAergic circuits and mechanisms and to quantitatively characterize the effects of reinforcement related population responses of ChAT interneurons on the activity of SPNs in vitro and in vivo. Optogenetic methods will be used to control the activity of ChAT and other interneurons in double transgenic animals allowing the genetic targeting and identification of specific cell types. The disynaptic circuits of ChAT interneurons will be analyzed by identifying the GABAergic interneurons activated by ChAT interneurons and then determining the contribution of individual presynaptic cell types to the various GABAergic response components in SPNs and other neurons by selective optogenetic activation of populations of specific classes of interneurons and by examining the effects of optogenetic inhibition of various interneuron types on IPSCs elicited disynaptically by ChAT interneurons. Finally, we will quantitatively characterize the effects of optogenetically reproduced pause-excitation responses of ChAT interneurons on SPNs in vivo and in vitro. These experiments will describe the functional organization of a powerful novel circuit mechanism of the neostriatum and therefore will significantly advance the understanding of the functioning of the basal ganglia.

DESCRIPTION (provided by applicant): The neostriatum is the main input structure of the basal ganglia, a system that is crucial not only for voluntary motor control, but also for reinforcement-mediated learning and higher cognitive functions. The importance of understanding the functioning of the nesotriatum is dramatically illustrated by the severe disability associated with numerous neurological and neuropsychiatric conditions that affect this brain structure. Developments in transgenic methods that allow visualization and targeting of genetically and functionally distinct types of neurons has recently led to the discovery of an unexpectedly large diversity of GABAergic interneurons in the neostriatum. As a result the striatum is now known to contain at least 7 types of GABAergic interneurons that include, in addition to the previously known fast spiking (FS) and the NPY expressing NPY-PLTS interneurons, 4 distinct classes of tyrosine hydroxylase (TH) containing interneurons and a new class of NPY expressing interneuron. We hypothesize, based on preliminary data and earlier studies, that the newly discovered TH and NPY interneurons are integral and important constituents of a highly organized intrastriatal synaptic circuitry and play essential roles in determining the activity and computational function of the neostriatum. The goal of the proposed studies is to understand the synaptic organization of this circuitry and to assess the functional significance of the newly discovered interneuron classes in determining the activity of other constituent neurons, in particular, the activity of functionally distinct types of projection neuros. The circuit organization of TH and NPY interneurons will be mapped in in vitro optogenetic experiments using a series of double transgenic mice in which expression of Cre-recombinase and EGFP in distinct types of neurons will allow high-throughput bidirectional analysis of the connectivity among TH, NPY, and FS interneurons and projection neurons of the direct and indirect pathways. The functional impact of TH and NPY interneurons will be assessed in in vivo optogenetic recording experiments in mice trained to perform operant tasks. First, we will examine how the firing rate of these interneurons varies in relation to distinct phases of the operant tasks. Next, we will examine how optogenetic manipulation (silencing or activation) of the activity of TH and NPY interneurons affects the firing rate of projection neurons, cholinergic and FS interneurons, how these manipulations affect local field potential oscillations, and how qualitative or quantitative measures of behavioral performance are affected. These experiments are expected to yield important new insights into the functioning of the neostriatum and may help to identify new cellular substrates for therapeutic interventions in a variety of neurological and neuropsychiatric disorders.

Project Summary/Abstract The striatum is the main input structure of the basal ganglia, a system that is crucial not only for voluntary motor control, but also for reinforcement-mediated learning and higher cognitive functions. The importance of the striatum is illustrated by the severe disabilities associated with numerous neurological and neuropsy- chiatric conditions that affect this brain structure. The recent introduction of methods for targeting and manipulating genetically and physiologically defined cell types is currently revolutionizing our understanding of the neostriatum. Our preliminary studies, together with recent research demonstrate that the classically known cell types represent less than a third of the interneuron classes in the striatum, and reveal an intricate and precisely organized circuitry of these neurons. The prosed study will test 4 novel hypotheses which were formulated on the basis of preliminary data and capture functionally important principles of organization of the network of striatal interneurons. First, we will investigate the connectivity of genetically identified interneurons and test the hypotheses that their connectivity is highly cell type specific and structured to form at least 2 separate interneuron sub- networks within the neostriatum. Second, building on our previous research and novel preliminary data we hypothesize that cholinergic interneurons (CINs), which traditionally have been thought of as neuromodulatory elements, participate in a fast bi-directional synaptic network with multiple GABAergic interneurons utilizing nicotinic excitatory connections. Importantly, our preliminary data also suggest the existence of a novel source of nicotinic excitation from the brainstem that appears to selectively target a distinct subset of GABAergic interneurons. Third, we will test the hypothesis that a subset of interneurons are hierarchically organized in the sense that one or more classes of interneurons exist which are specialized to control the activity of other interneurons. These interneuron-specific interneurons are of great interest because their impact may be widely distributed and disproportionally amplified via hierarchical control of other interneurons. Finally, based on preliminary data we hypothesize that a class of novel GABAergic interneurons are essential for normal goal-directed behavior, and we will explore the mechanism of action of these neurons using in vivo electrophysiological and Ca2+-imaging methods. These experiments will yield important new insights into the functioning of the striatum and may help to identify new cellular substrates for therapeutic interventions in a variety of neurological and neuropsychiatric disorders.