Jay Hirsh - US grants

This proposal is directed at understanding the regulation of a developmentally regulated gene in the fruit fly, Drosophila melanogaster. An additional focus of interest is to study a cluster of genes within which the gene is located. The gene codes for the enzyme dopa decarboxylase (DDC), an essential enzyme involved in metabolic pathways leading to cuticular hardening and neurotransmitter synthesis. The gene is subject to striking developmental regulation, and is hormonally regulated during at least part of the life cycle. We will study the regulation of this gene using a combination of molecular and classical genetic techniques. A high priority is the molecular definition and comparison of Ddc transcripts from different developmental stages and tissues, since the gene is probably expressed via different mechanisms at different developmental stages. We will examine regulatory steps in the expression of Ddc both in vivo and in organ culture systems. Mutant strains will be used to aid in the definition of sequences important in overall gene expression, and in specific developmental expression. Classical ligation experiments will be performed to assay for diffusible substances affecting Ddc expression. These studies are focussed on the study of a gene much like many genes involved in human development. We work on Drosophila because this organism offers many advantages in experimental approach over more complex higher organisms. The results of this study will be basic information on how developmentally regulated genes function, and should generate basic knowledge applicable to many higher organisms.

DESCRIPTION: The broad objectives of this study are to better understand the in vivo functioning of biogenic amines and the factors that modulate these pathways in a simple model system. These pathways are of enormous importance to human biology, movement disorders, and addictive states. We have evidence for a striking conservation in the types of behaviors that can be induced in flies by drugs affecting vertebrate dopamine receptors relative to their functional consequences in vertebrate animals. These behaviors can be induced both in decapitated adult flies, by direct application of drugs to the nerve cord, and also in living flies, by exposure to cocaine, an indirect agonist. Specific aims include (1) genetic selection for genes involved in cocaine responsiveness, selecting for altered cocaine resistance, and resistance to the sensitizing effects of repeated low dose exposure. Isolation of these genes will elucidate the biological pathways involved in these processes; (2) characterization of the mechnisms by which the amine pathways are modulated as a function of sex, time of subjective day, and cocaine sensitization. These studies will provide the basis for understanding how the mutants isolated in aim 1 lead to altered aminergic function; (3) identification of brain specific cis-regulatory elements of the Ddc gene. Using these elements, it will be possible to determine the behavioral roles of specific aminergic cell clusters.

DESCRIPTION (provided by applicant): This proposal uses the fruit fly, Drosophila melanogaster, as a model to study the pathways regulating responses to free base "crack about cocaine. This model organism shows conservation with vertebrate animal models both in the types of cocaine induced behaviors and in the biochemical and signaling pathways underlying these behaviors, it also shows sensitization to repeated cocaine exposures, a process that is likely to represent a component of the addictive process in humans. The current proposal focusses on the neural adaptations and signaling pathways leading to sensitization. The first two aims are directed at understanding two novel pathways required for sensitization in Drosophila that were identified in the previous funding period: In the first aim, the mechanisms by which the trace amine tyramine and the gene inactive are required for cocaine sensitization will be determined. In the second aim, the mechanisms by which the circadian gene products function in cocaine sensitization will be determined. The third aim will focus on the biogenic amine pathways and the direct targets of cocaine, the dopamine and serotonin transporters. For each of the above aims, potential genetic screens are proposed that could detect novel genes interacting with the given pathways. In the fourth aim, global changes in brain gene expression as a function of cocaine exposure and sensitization will be examined in order to identify new genes candidates involved in pathways modulating cocaine responses.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Abuse of addictive drugs puts an enormous burden on society, yet many basic questions regarding drug craving pathways remain poorly understood. Current animal models of drug-self-administration are limited by technical difficulties associated with the invasive procedures required for small animals, particularly in the genetically tractable mouse. Here we describe the development of a non-invasive procedure for drug self-administration that is particularly suited for use in mouse. Preliminary studies conducted in the laboratory of Dr. David Stephens have shown that mice can be trained to self-administer an aqueous solution of cocaine delivered intranasally via an aerosol spray. Here we propose to replicate these results and further develop and apply these methods. Specific aims include: [unreadable] [unreadable] 1. Replication of the initial data generated in Stephens' laboratory, using newly constructed behavioral apparati in our own laboratory. [unreadable] [unreadable] 2. Controls to further validate the self-administration paradigm: these controls include use of related but nonaddicting substances such as lidocaine and measurement of additional physiological correlates of cocaine exposure. The measurements will include determination of serum and brain cocaine levels, and measurement of locomotor and heart-rate stimulation following cocaine exposure. [unreadable] [unreadable] 3. Application of this paradigm to mouse circadian mutants. These studies are based on the observation that mper 1 knockout mice show a lack of conditioned place preference to cocaine. We will study these mice as well as mper 1 and 3 knockouts, and mutants in several other circadian genes. These studies should address the question of whether the defect is specific to cocaine versus defects in more general reward pathways. [unreadable] [unreadable] 4. Screen candidate animals from the Neuromice.org recessive behavioral screen that show alterations in intitial cocaine responses. Initial screening for cocaine responsiveness is showing a large number of 'putants', 36 as of this writing. These mice will be made available to the scientific community even before they are fully characterized. We will obtain these lines when available as homozygotes and test them in our paradigm. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The human serotonin transporter protein SERT is the primary target for antidepressants used by a significant fraction of the population and also is a direct target for several drugs of abuse, yet modes of action are still unclear. This proposal utilizes the genetically tractable model organism, Drosophila melanogaster, to study the behavioral roles of serotonin in Drosophila, primarily via genetic manipulation of the serotonin transporter, dSERT. We take advantage of the unique genetic, behavioral and anatomical tools available in this simple yet relevant model. The proposal is based on the finding that very mild alleles of dSERT show striking abnormalities in circadian behaviors. There are four aims: In the first aim, additional and more severe dSERT alleles will be generated and characterized, and dSERT overexpression and directed under-expression will be studied. In the second aim, the interactions of the dSERT with the circadian gene pathways will be studied. In the third aim, new drivers expressing in subsets of serotonergic neurons will be generated that will elucidate brain regions specific for the various behavioral effects of serotonin. The fourth aim will take advantage of information learned in the previous aims to design forward genetic screens for new genes in the serotonergic and dopaminergic pathways. PUBLIC HEALTH RELEVANCE: The serotonin transporter is the primary target of widely prescribed antidepressants, yet much remains to be discovered regarding the function of the neurotransmitter serotonin and its role in human well-being. The fruit fly, Drosophila melanogaster, contains a highly conserved serotonin system and is an important behavioral model with demonstrated relevance for higher vertebrates. This proposal uses the fruit fly to better understand the behavioral biology of serotonin.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

With this award to the Biology department at the University of Virginia a bioluminescence/biofluorescence imaging system will be acquired to study molecular signals associated with daily time keeping mechanisms. The instrument will be used for experiments aimed at gaining a better understanding of the internal circadian clocks that allow animals to anticipate daily changes in their environment and to organize a multitude of bodily functions in an optimized daily schedule. The new imaging system optimally visualizes bioluminescent signals produced by clock-controlled expression of luciferase reporter genes in combination with fluorescent signals produced by additional reporter genes that act as markers for specific cell types or biological activities. With the new technology it will be possible to image cell-type specific circadian gene expression at single cell resolution. The proposed experiments will focus on the cellular and network properties of circadian clock function in fruit fly and rodent model systems. These research activities will strongly stimulate the research programs of the (Co-)PIs as well as help in the recruitment and training of researchers and students to the University of Virginia. The instrument and planned research activities will be prominently featured in several undergraduate courses. In addition, existing affiliations will be used to also incorporate training and outreach at the K-12 level. Participating researchers and students will not only be involved in projects at the cutting edge of chronobiological research and imaging technology, but also receive invaluable training in microscopy and data analysis techniques that will be an asset to them throughout their scientific or professional careers.

Project Summary/Abstract There is great interest in the compensatory mechanisms that may function to delay onset of symptoms in the early/presymptomatic phase of Parkinson?s Disease. Here we use Drosophila and mouse models of dopamine (DA) deficiency to characterize compensatory mechanisms that may be relevant to the human condition. In the previous grant period, we partly localized a genetic element responsible for the ?Dopamine Bypass? phenotype, hereafter referred to as ?DD-Hi?, where ?DD? refers to Dopamine Deficient. DD-Hi flies show near normal levels of locomotor activity despite total deficiency of brain dopamine (DA), compared to the low locomotor activity DA deficient line, DD-Lo. The work proposed for the upcoming grant period will work toward more precise genetic mapping of this trait. Related aims will contribute to this effort, identifying and characterizing a co-transmitter that functions in DA neurons that are devoid of dopamine, and analyzing the transcriptomes of single DA neurons. We will pursue a parallel model in mice, where mice that are made dopamine deficient in specific brain regions provide evidence for a dopamine dependent autoregulatory loop that leads to continued expression of a set of genes required for development and maintenance of DA neurons, particularly in the SNc (substantia nigra pars compacta). Given the high susceptibility of SNc DA neurons in early Parkinsons Disease, confirmation of this regulatory circuit could have both clinical and basic science implications. Our hope is that pursuing analogous models in flies and mice will aid in identification of conserved genes and mechanisms that will inform therapeutic targets and strategies in humans.