Nathaniel Heintz - US grants

The major objective of the proposed work is to elucidate the molecular mechanisms governing cell cycle dependent gene expression. The primary focus is to understand the regulation of histone gene expression during the HeLa cell cycle. The proposed studies will be pursued according to the following course. First, additional alleles of the human histone genes will be cloned and fully characterized in order to identify alleles encoding variant core histones, and histone H1 genes, which may be constitutitively expressed during the cell cycle, or, alternatively, expressed in a tissue specific manner. Attention will be given to the gross genomic organization of the human histone genes and to potential relationships between their topological arrangement and function. Second, in vitro studies of both the transcriptional and post-transcriptional mechanisms which operate to regulate individual histone mRNAs (in progress) will be conducted in soluble extracts of synchronized cell populations. This will include the preparation and analysis of mutant templates in order to assess the nucleotide sequences necessary for these processes. Third, in vivo studies of both the rate of synthesis and half life of histone mRNAs under a variety of growth conditions (especially when macromolecular synthesis is blocked using specific inhibitors) will be done to gain insight into these mechanisms. These studies will be extended to other more normal cell types to assess the generality of these processes. Fourth, additional cell cycle regulated genes will be cloned, and their expression studied using all of the above approaches. These studies should provide fundamental insights into the specific mechanisms for regulating cell cycle dependent gene expression at the transcriptional and post-transcriptional levels. It is hoped that they may also provide an avenue towards understanding the mechanisms controlling the mammalian cell cycle.

The major objective of the proposed research is to elucidate the molecular mechanisms governing periodic expression of specific mRNAs during the cell cycle. The primary focus of the studies described herein is to understand the transcriptional and post-transcriptional regulatory events controlling the accumulation of human histone mRNA during the S phase of the HeLa cell cycle. Additional studies, employing the methodologies developed for the analysis of histone gene expression, will be pursued discover those processes controlling the cell cycle specific expression of other cellular genes. The proposed research will be conducted according to the following course. First, the expression of several individual genes encoding each of the histone subtypes will be analysed in vivo to identify those that code for the most abundant cell cycle regulated mRNAs. Second, in vitro studies of both the transcriptional and post-transcriptional mechanisms which control the accumulation of each histone mRNA will be conducted in soluble extracts from synchronized HeLa cells. Current studies of the pHu4A histone H4 gene (see below) provide a useful paradigm for the analysis of other cell cycle regulated histone genes. Thus, these studies will focus on the identification of both nucleotide sequences and protein factors which are involved in the expression of histone genes in vitro. Third, in vivo studies employing either transiently or stably transfected histone genes will be pursued to demonstrate that the nucleotide sequences which are important for histone gene transcription in vitro are also utilized in vivo. This methodology will also be employed to determine whether specific sequences in histone mRNA provide a signal for its rapid and specific degradation after the inhibition of DNA synthesis. Fourth, similar experiments will be conducted to identify those events controlling the cell cycle specific expression of other cellular genes. The initial emphasis in this area will be to gain insight into the dramatic transcriptionsl regulation of the human HSP 70 gene we have observed during the HeLa cell cycle. It seems apparent that studies of this type can generatecrucial information concerning the regulation of specific genes, as well as provide an avenue toward an increased understanding of general processes required for progression through the cell cycle.

The goal of Project 5 is to analyze the molecular mechanisms which result in cell type and developmental stage specific expression of novel genes that are tightly regulated during cerebellar granule cell differentiation, with particular emphasis on those genes which require specific cell-cell interactions as signals for normal expression. Toward this goal, we will (1) Complete the characterization of a collection of cerebellar cDNAs which we have recently cloned from a stage specific granule cell cDNA library, screening for developmental stage and tissue specific expression. (2) Define the temporal and spatial patterns of expression of regulated clones by in situ hybridization of tissue from normal and neurologic mutant mice (including Lurcher (Lc), weaver (wv), and nervous (nr)) and (3) Isolate both full-length cDNAs and genomic clones of select tightly regulated novel cDNAs as tools for further analysis. To examine the transcriptional regulation of granule cell specific genes, we will carry out transient expression assays in cerebellar slice preparations and analyze critical constructs in transgenic mice. The transcription factors which interact with granule cell specific genes will be examined with particular emphasis on signal transduction pathways mediated by specific cell-cell interactions. Finally, we will examine the functions of selected gene products by (1) in-depth computer analysis of cDNA sequences, (2) immunocytochemical localization of polyclonal antisera against expressed gene products, (3) functional perturbation studies in vitro (see Project 4), and (4) molecular genetic perturbation studies in vivo.

This proposal presents a series of focused studies to further elucidate the molecular basis for S phase specific transcription of histone genes, and to determine whether these mechanisms pertain in the regulation of chromosomal DNA synthesis. The ultimate aim of this work is to gain a molecular understanding of the transition from G1 to S phase, and to identify specific targets for intervention of cell growth at this point in the cell cycle. The experimental emphasis in this proposal is based on fundamental insights into this process gained during the last five years of this grant, and will focus upon six specific aims: 1) further characterization of the histone gene subtype specific cell cycle regulatory proteins OTF1, H1TF2, H1TF1 and H4TF2, 2) identification of enzymes involved in cell cycle regulated modification of OTF1, and demonstration that post- translational modification of OTF1 is functionally important for H2b transcription, 3) exploration of the regulation of H1TF2 (and ultimately H1TF1 and H4TF2) activity during the cell cycle to test whether the mechanisms regulating OTF1 are pleiotropic, 4) biochemical and genetic analysis of S. cerevisiae S phase specific histone gene transcriptional control, 5) analysis of the putative replication initiation factors RIP60 and RIP100 as potential substrates for the S phase regulatory mechanisms involved in transcriptional control, 6) analysis of upstream regulatory molecules in the pathway leading to activation of S phase specific transcription (e.g. protein phosphatase IIa, etc). These studies will result in identification of fundamental cell cycle regulatory mechanisms which may result in opportunities to specifically intervene in cell growth.

The basic principle underlying this work is that identification of strongly developmentally regulated and cell specific genes expressed in the cerebellum provides an opportunity to investigate detailed molecular mechanisms controlling formation of the mammalian CNS. These genes provide the tools both for discovery of transduction pathways critical in the specification and differentiation of cerebellar cell types, and for the exploration of novel biochemical pathways in which their products function. The identification of these mechanisms is critical for understanding both human health and disease. Thus, studies of the transcription factor RU49 and its associated partners will provide insights into cerebellar granule cell growth and differentiation that are directly relevant to medulloblastoma, the most common form of childhood brain tumor. Analysis of brain lipid binding protein (BLBP) and its ligand docosahexanoic acid (DHA) will provide a biochemical basis for understand- ing the utilization of this essential nutrient, which has recently been demonstrated to be required for timely development of the human CNS. And identification of additional molecules involved in critical stages of CNS development will provide tools for investigating other pathways affected in a variety of congenital disorders. To accomplish these goals will require: 1) further characterization of cDNA clones that identify genes involved in development of the cerebellum; 2) identification of the specific functions of RU49 and its associated partners in growth and differentiation of CNS granule cells; 3) investigation of mechanisms regulating RU49 expression using the biolistic transfection procedures developed during the prior period of this project; 4) investigation of the role of brain lipid binding protein (BLBP) and its high affinity ligand docosahexanoic acid (DHA) in CNS development and determine whether DHS is a critical signal for radial glial cell differentiation; 5) identification of the transcription factors controlling expression of BLBP in glial cells in response to neurons and test the idea that these factors include a putative DHA nuclear receptor.

The development and mature function of the mammalian brain must require the precise regulation and concerted action of thousands genes whose products are restricted to the nervous system. The purpose of this grant is to further develop and utilize bacterial artificial chromosomes (BACs) as tools for the discovery and analysis of genes predominantly expressed in the mammalian CNS. The proposal is organized into three specific aims: 1) To utilize BAC transgenic analysis for the characterization of CNS specific gene expression patterns and for the localization of their encoded protein products [BACexpress]. 2) To utilize CNS specific BAC/EGFP or beta-lactamase expressing mice and fluorescence activated cell sorting (FACS) to prepare cell specific probes for gene expression analysis. To use these probes to interrogate either DNA chips or microarrays to profile gene expression for specific CNS cell types (BAC array). 3) To develop and utilize gene trapping in BAC clones to accelerate discovery of CNS specific genes [BACtrap]. The further development of these techniques and their adoption for high throughput analysis can yield: accurate temporal and cell type specific expression profiles for nearly all CNS specific genes; the size, abundance and subcellular distribution of proteins encoded by each of these genes; a library of defined BAC vectors for genetic manipulation of the great variety of CNS cell types; phenotypes resulting from increased dosage of any of the assayed genes; and a "molecular histology" of the CNS that includes the definition of molecularly marked subtypes of morphologically identified neurons.

The development and mature function of the mammalian brain must require the precise regulation and concerted action of thousands genes whose products are restricted to the nervous system. The purpose of this grant is to further develop and utilize bacterial artificial chromosomes (BACs) as tools for the discovery and analysis of genes predominantly expressed in the mammalian CNS. The proposal is organized into three specific aims: 1) To utilize BAC transgenic analysis for the characterization of CNS specific gene expression patterns and for the localization of their encoded protein products [BACexpress]. 2) To utilize CNS specific BAC/EGFP or beta-lactamase expressing mice and fluorescence activated cell sorting (FACS) to prepare cell specific probes for gene expression analysis. To use these probes to interrogate either DNA chips or microarrays to profile gene expression for specific CNS cell types (BAC array). 3) To develop and utilize gene trapping in BAC clones to accelerate discovery of CNS specific genes [BACtrap]. The further development of these techniques and their adoption for high throughput analysis can yield: accurate temporal and cell type specific expression profiles for nearly all CNS specific genes; the size, abundance and subcellular distribution of proteins encoded by each of these genes; a library of defined BAC vectors for genetic manipulation of the great variety of CNS cell types; phenotypes resulting from increased dosage of any of the assayed genes; and a "molecular histology" of the CNS that includes the definition of molecularly marked subtypes of morphologically identified neurons.

neurogenetics; artificial chromosomes; gene expression; biomedical resource; central nervous system; macromolecule; serial analysis of gene expression; mass spectrometry; genetically modified animals; clinical research;

DESCRIPTION (provided by applicant): The purpose of this grant is to generate a novel series of agents for genetic manipulation of receptors, ion channel, and signaling molecules in vivo. These agents (tethered toxins) are chimeric molecules derived from tethering of naturally occurring peptide neurotoxins to the cell surface via GPI anchors or transmembrane. These studies derive from the discovery of mammalian prototoxin genes (e.g. Lynx 1) which are the evolutionary antecedents of snake venom toxins, and which can function as modulators of nAChRs in their native GPI-anchored form. Preliminary results are that tethered bungarotoxins retain their activity on nAChRs, and that they are not cleaved from the cell surface to inhibit adjacent cells. The existence of many thousands of naturally occurring peptide neurotoxins (e.g. bungarotoxins, conotoxins, conantokins, etc.), their exquisite target specificities, and the ability to target expression of the agents in vivo using BAC transgenic mice, suggests that the development of a generic strategy for harnessing their potency for in vivo use will permit genetic control over a wide variety of neuronal functions. For example, cell specific genetic control of neural activity, neurotransmitter receptor function (e.g. ACh, NMDA, 5-HT3 receptors), and specific GPCR signal transduction cascades would become possible. The specific aims are to: 1) Construct additional tethered toxins, particularly tethered conotoxins, and test their activity in Xenopus oocytes; 2) Produce BAC transgenic mice expressing tethered toxins in specific CNS cell types in vivo. Assess the efficacy of tethered toxin action by evaluating phenotypes that would be expected based on results obtained in Specific Aim 1 and current knowledge of the roles of the targeted receptors and ion channels in vivo; 3) Develop inducible tethered toxins to improve the temporal resolution of this strategy for genetic manipulation of specific cells and signaling pathways. These studies will allow unprecedented precision in the genetic dissection of functions required for CNS development, function and dysfunction in vivo.

proteins; autophagy; biomedical resource; macromolecule; brain; clinical research; Mammalia; mass spectrometry;

mass spectrometry

mass spectrometry

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 development of methods for the simple and accurate manipulation of Bacterial Artificial Chromosomes (BACs) in my laboratory has allowed the utilization of an alternative and highly efficient strategy for analysis of CNS specific genes (Heintz 2000). This approach is based on two simple facts: large genomic DNA fragments (100KB) are in most instances expressed independent of the site of integration into the genome of transgenic mice;inclusion of epitope tags and marker proteins into endogenous loci of invertebrate genes has in most cases not altered the patterns of expression of these genes or the localization of their encoded products within the cell. To take advantage of this information, a homologous recombination system was established in E. coli that allows for preparation of BACs with highly precise modifications. Using this system, it is possible to create mutations in BACs that range from single nucleotide changes to deletions of tens of kilobases to insertions of marker genes of several kilobases. One can, therefore, construct BACs that allow very rapid analysis of the expression pattern of the gene of interest, the localization of its encoded product, high-resolution visualization of the morphology of cells expressing the gene, and determination of the projection patterns of these cells. Mice made using these techniques also carry epitope tagged proteins that can be used for affinity purification of complexes carrying the protein of interest. The use of epitope tags for determination of the subcellular distribution of proteins in invertebrates and in cultured mammalian cells is very well established. Because of the precision of homologous recombination in E. coli, it is quite simple to introduce an epitope tag into the protein encoded by the gene of interest in the BAC at the same time that one introduces the marker genes. Since a variety of epitope tags and their cognate antibodies are now available commercially, one has a wide range of options from which to choose. Although the introduction of an epitope tag into the protein can in some cases change its subcellular distribution, this is relatively infrequent and usually can be overcome by changing the location of the tag within the protein. Since preparation of useful antibodies for a protein of interest is often an expensive and long-term project, the ability to detect the epitope tagged protein in vivo offers a very efficient and useful alternative. In trying to interpret CNS expressed gene function, localization of its encoded product, or correlation of its subcellular distribution in different cell types or under different conditions can provide crucial information. Obviously, the spectrum of functions one might consider is significantly different for proteins located in the nucleus than those present at the synapse! Furthermore, the redistribution of the protein in response to a stimulus can also be quite informative. For example, there are many well characterized transcriptional responses that involve regulated release of factors from cytoplasmic complexes and their entry into the nucleus in response to growth factors, cytokines, etc. (unpublished data). The ability to obtain this type of information in an efficient manner using epitope tags presents a significant advantage over the time consuming preparation of sufficiently useful antibodies to the native protein for these studies. The development of peptide tags for affinity purification is also of great utility. We have, for example, inserted the 6XHis tag into the Zipro1 locus in BAC transgenic animals for isolation of Zipro1 containing transcription complexes from cerebellar granule cells. It is now possible to utilize Ni+ chelation affinity chromatography to characterize the Zipro1 complexes using whole brain extracts from the BAC transgenic mice as has been very successfully done for His-tagged transcription factors in cultured mammalian cells. This strategy can be extended for purification of any macromolecular complex from any cell type in the brain using the BAC transgenic approach. Since the results from the animal carrying the epitope tagged protein can be directly compared to control animals, background from the purification procedure can be identified readily. While affinity purification methods are not yet fully developed for this purpose, the use of BAC transgenic animals for this purpose is a major advance over current method for identifying protein complexes that exist in vivo. When combined with the advanced mass spectrometric methods carried out in the Chait Laboratory for protein identification, this approach offers a novel and highly efficient alternative to traditional biochemical techniques. Heintz, N. (2000). "Analysis of mammalian central nervous system gene expression and function using bacterial artificial chromosome-mediated transgenesis." Hum Mol Genet 9(6): 937-43. Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N."A gene expression atlas of the central nervous system based on bacterial artificial chromosomes" Nature 425(2003)917-25

The overall objective of this contract is to 1) continue the high-throughput analysis of gene expression patterns in the mouse nervous system during development;2) generate new mouse genetic research tools for the scientific community. Using two complementary and coordinated technologies (standard radiometric in situ hybridization and BAC transgenic reporter mice), the contract screens probes for a large number (e.g., thousands) of gene products on a relatively limited number (10 to 20) of sections of the nervous system. Planes of section are chosen by the NINDS to include the structures of most general interest to the neuroscience community (e.g., neocortex, basal ganglia, hippocampus, cerebellum, spinal cord, etc.). The spatial locations/patterns of gene expression are analyzed at 3 or 4 stages of development (early and late embryonic, and early postnatal) and in adult mice. The resulting images of gene expression are digitized and posted in Web-accessible databases, and all BAC transgenic mice generated by the contract are deposited in an NIH-sponsored mouse repository.

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. Four years ago, in collaboration with Nat Heintz (Rockefeller University), we initiated the study of the protein complement present at excitatory synapses in Purkinje cells. We used the Bacterial Artificial Chromosome (BAC) modification strategy to target the specific in vivo expression of GFP-fused GRID 2 to Purkinje cell's excitatory synapses. We performed dissections of mouse cerebella, and purified synapses bearing GFP-GRID2. Although challenging, our approach proved successful, as we isolated synapses and analyzed low-femtomol levels of proteins. During this last year, we continued our mass spectrometric analyses and identified ~70 synaptic proteins, confirming known excitatory proteins, the absence of inhibitory proteins, and identifying novel signatures of excitatory synapses. We have published a manuscript describing this work (F. Selimi, I. Cristea, E. Heller, B.T. Chait, N. Heintz "Proteomic studies of a single CNS synapse type: the parallel fiber/Purkinje cell synapse" PLoS Biology, 2009 Apr 14;7(4):e83). Using a similar approach to the one described above, we hare currently studying the protein composition of inhibitory synapses by isolating GABA receptors from specific cell populations in the Cortex. A paper describing this work has been prepared for submission. The following is the abstract from this manuscript: Electron microscopic studies of the mammalian brain revealed that there are two major classes of synapses (1). Type 1, excitatory synapses were defined as "asymmetric" based on the electron dense material directly apposed to the post- but not the presynaptic membrane. Biochemical studies of this postsynaptic density (PSD) have established it as a complex signal-processing machine that controls synaptic plasticity (2-5). Type 2, inhibitory synapses were defined as "symmetric" because the PSD is greatly reduced or absent. We report here that symmetric synapses contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of excitatory and inhibitory synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and longer term homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.

DESCRIPTION (provided by applicant): Project Summary/Abstract: Recent studies of addiction have highlighted several regions of the brain that are thought to be involved in goal directed and drug seeking behaviors. The specific neuronal classes involved in the regulation of these behaviors are beginning to be identified, and attempts to profile the molecular changes in these cell types occurring as a consequence of addiction have met with some success. While these studies demonstrate that it is possible to discover changes in cell types that are correlated with, and in some cases contribute to, addiction, our knowledge in this area is fragmentary and incomplete. This is due to technical obstacles that have faced this field for decades, and that have recently been overcome by novel methodologies developed in our laboratories. The objective of this program of research is to identify all of the changes in gene expression and accompanying alterations in epigenetic regulation that contribute to the addictive state in cell types known to be important in the neural circuitry controlling addiction. The approach we will take in this program is to: 1) employ TRAP methodology and bacTRAP transgenic mouse lines to comprehensively profile the translated mRNAs from fifteen mouse CNS cell types that are components of the neural circuitry controlling addiction;2) to collect translational profiles from each of these cell types in mice exposed to cocaine, methylphenidate, and methamphetamine 3) perform in depth comparative analysis of these resulting microarray datasets to identify changes in gene expression that accompany the addictive state in each cell type;4) to concurrently isolate nuclei from each cell type and map cell specific sites of mC and hmC modification to the neuronal genomes;5) to conduct follow up studies on genomic loci identified in the preceding aims to map additional epigenetic regulatory events by ChIP assays using H3K9m2 and H3K27m3 specific antibodies to identify genomic loci associated with suppression of gene expression in euchromatin. This project will provide information and experimental animals that will stimulate addiction research in a broad spectrum of laboratories, and provide materials and a paradigm for comprehensive studies of these same neural circuits under other experimental conditions. As such, this program will have an enormous impact on modern molecular neurobiology, and on the development of novel targets for pharmacological interventions in CNS disorders. PUBLIC HEALTH RELEVANCE: According to the National Institute on Drug Abuse, "Estimates of the total overall costs of substance abuse in the United States ..... exceed half a trillion dollars annually". The goals of the project are to comprehensively profile molecular changes occurring in specific cell types in regions associated with addiction, and to concurrently map epigenetic changes occurring in these cell types. It will stimulate in depth research into cell specific molecular events that are responsible for establishment of the addictive state, and identify novel targets for the development of new therapies for drug addiction.

Hypofunction of cortico-basal ganglia circuits has been hypothesized to underlie some of the debilitating cognitive deficits and negative symptoms that are seen in schizophrenic patients. Antipsychotics are thought to mediate some of their therapeutic effects by normalizing this activity. However, the precise cell populations and the molecular changes involved in this response are still not fully understood. In Project 2 of this Conte center application, we hypothesize that cell-type specific changes in neurons projecting from the frontal cortex to the basal ganglia occur in response to typical and atypical antipsychotic drugs. To test this hypothesis, we will make use of a novel mRNA translational profiling approach. In Aim 1 of this project, we will perform these studies on two distinct cortico-striatal cell populations we have targeted genetically. In Aim 2, we will characterize two newly generated mouse lines that may give us translational profiling access to two additional cortico-striatal cell populations. Finally, in Aim 3, in collaboration with the other projects of this center, we will perform a functional analysis of molecules identified in our translational profiling studies of Aims 1 and 2.

SUMMARY: The Administrative Core for the Center for Molecular and Epigenetic Research of Cell Types Mediating Addictive Behaviors will be house in the Collaborative Research Center within the Laboratory of Molecular Biology at The Rockefeller University. It will maintain all financial records and interactions within the Center, coordinate budgets between the Research Support Cores and Pilot Research Project Support Cores, and assist with preparation of financial documents for Progress Reports. It will also oversee data quality control, and data presentation to the public through the Center Website. A critical second role of the Administrative Core will be to foster and maintain interactions between the Center, Center Investigators and NIDA Program Staff. Accordingly, it will administer the Center Advisory Committee and it will host quarterly videoconferences with the committee. It will also manage interactions with the administration of The Rockefeller University, and provide feedback to queries that come to the Center from interested external investigators.

DESCRIPTION (provided by applicant): The NIDA P30 Center for Molecular and Epigenetic Research of Cell Types Mediating Addictive Behaviors proposed here will provide to NIH investigators state of the art resources, technologies and expertise that will significantly enhance the programs of each of the participating laboratories, and enable development of additional advanced methodologies that are of general utility for the investigation of mechanisms of drug abuse. To meet these objectives, the Center will be organized into four Cores: The Administrative Core will organize and manage all programmatic and fiscal operations of the Center, including outreach to each of the Center investigators, management of quarterly online meetings of the External Advisory Committee, and hosting of Center investigators or their staff during the conduct of on-site experiments. The BAC Recombineering and Transgenic Targeting Core will generate, characterize and distribute specialized transgenic lines for advanced molecular studies of cell types contributing to addiction circuitry; The Molecular and Epigenetic Profiling Core will provide specialized behavioral and experimental services for the conduct of TRAP translational profiling and epigenetic mapping studies in the context of chronic drug treatment, drug self-administration, and drug withdrawal; The Molecular Informatics Core will provide facilities and expertise to help investigators collect, store and analyze the very large amounts of data that will result from their microarray, TRAPseq, epigenome mapping and ChlPseq studies. The Center will also support a small number of Pilot Projects aimed at developing new technologies that will advance discovery of molecular mechanisms that contribute to drug abuse, or extension of existing approaches to mammalian model systems that have been refractory thus far to modern molecular genetic approaches The outstanding scientists participating in the Center share the belief that: it is beyond the capacity of any participating laboratory to generate the large number of specialized transgenic lines required for comprehensive genetic, molecular and epigenetic analysis of addiction circuits; and that a Center in which their laboratories can apply advanced methods for analysis of addiction circuits will significantly advance their research programs.

The importance of new technological approaches for the advance of science cannot be overestimated. Although an impressive array of new technologies for investigation of complex tissues has been developed over the past decade, the nervous system is composed of hundreds (at least!) of distinct cell types and many interiocking circuits that control behavior in normal or pathophysiological situations. The purpose of the Pilot Project Program is to stimulate new and more efficient approaches for the discovery of novel molecular mechanisms associated with drug abuse, and to aide in breaching the experimental barriers that restrict current discovery methodologies to genetically tractable organisms. This program is intended to support less established investigators because more established laboratories normally have sufficient resources to pursue creative ideas as they arise.

SUMMARY: The BAC Recombineering and Transgenic Targeting Core (BRTT) will generate, characterize and distribute specialized transgenic lines requested by the NIDA PSO Center for Molecular and Epigenetic Research of Cell Types Mediating Addictive Behaviors. The BRTT core will be in charge of the following subprojects/tasks: 1) BAC modification of Cre-recombinase and TRAP vectors, 2) Generation of BAC transgenic mice, 3) Histological confirmation of accurate transgene expression, and 4) Archive and distribution of BAC transgenic mice

SUMMARY: The Molecular and Epigenetic Profiling Core (MEPC) will provide specialized behavioral and experimental services for the conduct of TRAP translational profiling and epigenetic mapping studies in the context of chronic drug treatment, drug self-administration, and drug withdrawal. In particular, the MEPC will perform TRAP gene expression analysis and epigenetic mapping of 5hmC and 5mC on cell types labeled by bacTRAP transgenic mice generated in the BAC Recombineering and Transgenic Targeting Core. The MEPC will be a critical component for the success of the NIDA P30 Center in that it will serve as a centralized facility providing the expertise and resources for performing these sensitive techniques, thus increasing the quality and reproducibility of data and streamlining the productivity of collaborating investigators. MECP personnel will characterize all new bacTRAP lines generated by the Center and disseminate these data to other participating laboratories. In addition, the MEPC will perform on-site, comprehensive pilot experiments using the self administration behavioral paradigm to analyze cell type specific molecular and epigenetic changes that occur in animal models of addiction. The MEPC will also train personnel from all participating laboratories on using TRAP translational profiling and epigenetic methods used in this core. In addition, the MEPC will host investigators to carry out more in-depth profiling projects that reach beyond the scope of the pilot experiments. This obviates the need to ship mice to laboratories with only short-term experimental goals or that do not have access to resources needed to carry out these experiments. The data and facilities provided by this research support core will offer immediate high-impact application of the transgenic tools generated by the NIDA PSO Center.

SUMMARY: The Molecular Informatics Core will be a state-of-the-art facility that will support all of the biostatistical and bioinformatics needs of the NIDA P30 Center for Molecular and Epigenetic Research of Cell Types Mediating Addictive Behaviors. This Core will be organized into two components. The first component will create an annotated high-resolution image database of all of the new transgenic strains generated by the BAC Recombineering and Transgenic Targeting Core and will include 256 unanalyzed Cre driver lines generated by the GENSAT project. This database will provide a web-based section-by-section description of cell types labeled in each new transgenic line and their axonal projections based on software written for the GENSAT project. Annotations will be done by an expert neuroanatomist that worked with the GENSAT project for eight years. This will provide a valuable resource for NIDA investigators to identify novel cell types and transgenic tools available in order to expand their individual research programs. Second, the Molecular Informatics Core will provide comprehensive statistical and bioinformatics support to all NIDA Center investigators for the analysis of all molecular profiling and epigenetic data. This includes genomic alignment of sequencing reads, determination of significantly expressed or altered genes/genomic locations, and an extensive selection of data mining tools to extract meaningful biological information from these large data sets. A full-time research support specialist, with an advanced degree in biostatistics/bioinformatics, will provide expertise, consultation, and training for all applications supported in this core. Users will also have access to high performance computer workstations with licenses for software needed for data analysis and a high capacity server for data storage. The Molecular Informatics Core will provide an innovative facility crucial for navigating, correlating, and interpreting anatomical and profiling information.

This INSPIRE award brings together research areas traditionally supported in the Division of Integrative Organismal Systems in the Directorate for Biological Sciences, in the Division of Chemical, Bioengineering,Environmental, and Transport Systems in the Directorate for Engineering, and in the Division of Behavior and Cognitive Sciences in the Directorate for Social, Behavioral and Economic Sciences. Cognition arises from the activity within complex brain circuits. These brain circuits are laid out according to a species' genetic blueprint. Past and current successes in uncovering the biological basis of cognition include the discovery of areas in the human brain supporting specific cognitive functions, the determination of the roles that specific cell types play in the behavior of animals like the mouse, and the increasingly detailed understanding of the impact specific genes and their alterations can have on cognitive functions in health and disease. The goal of this interdisciplinary project, conducted at The Rockefeller University, is to directly determine the genetic specificity of brain circuit elements that are critically important for high-level cognitive function. This project will be significant by 1) elucidating the complexity of biological organization from the level of genes, through cell types, brain areas, and neural circuits to behavior, 2) developing new technology that will allow researchers to dissect brain circuits underlying cognition with the precision and specificity of model organisms, and 3) improving the understanding of how genetic alterations impact cognition. The interdisciplinary project at the interface of cognitive neuroscience, neural systems, and neurotechnology, is expected to have broader impacts on society by providing insights into some of the deepest questions about the human mind and by offering unique educational and outreach opportunities to improve public understanding of the organization and function of the brain.

The project will investigate the genetic specificity of a multi-node brain circuit that supports cognitive function. The circuit will be localized with functional and structural magnetic brain imaging. Genetic expression patterns of projection neurons within multiple circuit nodes will then be determined using cutting edge molecular techniques. The functions of the projection neurons linking the nodes will then be determined through advanced and custom-designed optogenetic and electrophysiological techniques. The same optogenetic approach will then be used for causal interrogation of projection neurons in cognitive and emotional behaviors. Combining the gene expression and functional data, predictions for how specific polymorphisms in human genes may alter cognitive-emotional abilities will be generated. These predictions will be tested through functional brain imaging and behavioral testing of genotyped subjects. Together, these investigations will provide deep insights into the brain circuits and genetic underpinnings that make possible the cognitive functions of the human mind.