Molecular mechanisms underlying sleep in Drosophila

Mentor: Agosto, Jose Luis Off-site Collaborator(s): Leslie Griffith (Brandeis), Axelrod (UT Austin)

Evidence from human sleep disorders and sleep pharmacotherapies indicate that the initiation and maintenance of sleep are differentially regulated. However, the mechanism underlying this regulation remains unknown. Recently, using a pharmacogenetic approach, Dr. Agosto and colleagues showed that altering the desensitization kinetics of a Drosophila GABAA receptor subunit called Resistant to diledrin (RDL) specifically affect sleep initiation without altering sleep maintenance. In addition, they found that carbamazepine (CBZ), a widely used drug in the treatment of multiple neurological disorders, blocks sleep initiation by increasing the kinetics of RDL desensitization but inhibits sleep maintenance by an unknown mechanism. Their main hypothesis is that sleep initiation and sleep maintenance are controlled by different but overlapping circuits and that CBZ interacts with both of these circuits through different mechanisms. The goals of the laboratory are: 1) to perform genome-wide behavioral screen for mutants resistant to CBZ actions on sleep maintenance; 2) to develop a strategy to genetically manipulate CBZ sensitivity in subsets of RDL neurons an: 3) to validate the use of long-term CBZ administration to flies as a model for chronic insomnia and its associated deficits in daytime functioning. These studies not only could bring insights into the genetic bases of sleep regulation but also may provide the basis for the development of more specific drugs for the treatment of sleep disorders such as sleep onset and sleep maintenance insomnia. There are several lines of evidence suggesting that nicotinic acetylcholine receptors (nAChRs) are potential candidates for CBZ actions on sleep maintenance. To test this hypothesis, a summer student could examine if nAChR mutants have defective sleep/wake pattern and/or CBZ sensitivity.

Cellular and Molecular Substrates of Anabolic Steroids Behavioral Effects

Mentor: Barreto, Jennifer

Reproductive-related behaviors, anxiety, cognition and addiction are known to be modulated by androgens. Besides hormone regulation of behavior, it is well known that neurotransmitters and neuropeptides are molecules underlying behavioral characteristics that are also under the influence of peripheral hormones. The long-term goal of the research program is focused in understanding the neurochemical substrates responsible for behavioral changes after exposure to synthetic androgens. Given that anabolic androgenic steroids (AAS) misuse is associated with multiple psychiatric symptoms and endocrine disruption, this study will provides critical data of the biochemical aspects of behavioral changes after androgen exposure, particularly in adolescents, where an increase misuse has been reported. We used pubertal rodents as animal models to determine the effect of chronic exposure to AAS in neurotransmitter and neuropeptide modulation in brain regions involved in anxiety, emotional learning, reproductive-related behaviors, and addiction and dependence. The brain regions curently under study are: nucleus accumbens, medial pre-optic area (mPOA), arcuate nucleus, ventromedial nucleus of the hypothalamus (VMH) and the amygdala. Preliminary data using real-time PCR and in vivo microdyalisis show that anabolic steroids modulate neuropeptide Y (NPY) receptors in the hypothalamus and glutamate levels in the amygdala. These molecules might be important substrates regulated by AAS to attain altered sexual behaviors and anxiety. In fact, we have found increased sexual motivation after AAS exposure during puberty in male rats. Other studies will use proteomics technology to determine proteins that are differentially expressed in the nucleus accumbens of AAS versus vehicle- treated animals. Since the nucleus accumbens is a key player in the mesolimbic circuitry of reward, the obtained results will be essential to find out the particular proteins associated with AAS misuse and dependence. Comparison with classical drugs of abuse will be also highlighted.

Gene Profiling of Nervous Regeneration Processes

Mentor: Garcia-Arraras, Jose E. Off-site collaborators: Andrew Cameron (Caltech)

Dr. Garcia-Arraras has pioneered the use of the echinoderm Holothuria glaberrima to study the process of regeneration and organogenesis. His research focuses on the molecular aspects of nervous system regeneration, specifically on the genes that are important for the regeneration process to occur. His lab is generating an expressed sequence tag (EST) database for H. glaberrima sequences obtained from three cDNA libraries, of normal radial nerve cord, and two from regenerating (5-7-days and 12-14-days after nerve cord transection). Their work is aimed at finding different profiles of gene expression and at determining the function of specific genes during the process of regeneration. Students will be involved in bioinformatics analyses to determine gene sequences, structural domains and gene characterization. In addition, the evolution of particular genes will be targeted. Finally, benchwork experiments using PCR, Northerns and Westerns applied to the nervous system regeneration will be done to fully characterize the gene’s expression profile.

Mechanisms of Plasticity in Honeybee Social Behavior

Mentor: Giray, Tugrul Off-site collaborators: Gene Robinson (Univ. of Illinois Urbana-Champaign)

Honeybees live in a society composed of tens of thousands of almost sterile workers, a reproductive female, the queen, and hundreds of males. Studies on behavioral development of honeybee workers, queens, and males revealed a rich and expanding repertoire under neuroendocrine regulation with evolutionary roots in the solitary reproductive cycle of insects. In addition to developmental plasticity, there also is a short-term plasticity in behavior of honeybee workers under regulation of biogenic amines, under natural conditions. Dr. Giray’s lab uses classic and new learning assays to study honeybee social behavior, both in the field and in the laboratory. With these, they probe the neural and molecular bases of plasticity in a mini brain. Simple questions, such as if colony conditions influence behavioral development as measured by flight muscle protein expression are important within this research program and has been answered by undergraduate research students. More recently, the lab has focused on the expression of candidate genes involved in aversive learning in the honeybee brain combining behavioral, bioinformatics, and molecular techniques. This combination of experimentally accessible social behavior and techniques to probe underlying molecular mechanisms make this research program especially suitable for undergraduate research experience.

Structure-Function Studies of the Nicotinic Receptor

Mentor: Lasalde-Dominicci, Jose. Off-site collaborators: Chris Gomez (Univ. Chicago)

The focus of the research in this Dr. Lasalde-Dominicci’s lab is on ion channel structure, ion-channel lipid interactions and ion-channel related disorders. Their long range goals are focused on: (1) the structure function relationships of nicotinic receptors, (2), the role of lipid-protein interaction on acetylcholine receptor function, (3) the question of how genetically abnormal ion channels give rise to neurodegeneration, (4) the regulation of neuronal nicotinic receptor assembly and oligomerization and (5) studies towards a high-resolution structure of the Torpedo AChR. These efforts might identify potential therapeutic targets, or channel-blocking agents that might benefit a broader range of neurodegenerative disorders. To accomplish these goals a wide array of techniques are employed, including: bioinformatics, protein chemistry, as well as molecular biological and electrophysiological techniques.

Neurobiology of Addiction

Mentor: Maldonado-Vlaar, Carmen

The ongoing work in the lab has three goals. The first goal is aimed at characterizing the behavioral and molecular effects of spatial novelty on the acquisition of intravenous cocaine self-administration (SA). The second goal of the present proposal is to focus on the functional role of CREB phosphorylation within the NAc and limbic-related structures in novelty-elicited acquisition of cocaine SA. This aim includes using antisense oligonucleotide technology to examine the role of newly synthesized CREB in recognition of spatial novelty prior to cocaine SA. Findings from these proposed experiments will provide new data on the involvement of CREB regulation within Nac in eliciting novelty-induced behaviors related to cocaine reward. Finally, the last goal of the present proposal is to characterize the role of the protein kinase C (PKC) within the Nac in the phosphorylation of CREB elicited by spatial novelty effects on cocaine SA in rats. Experimental results from this goal will also contribute to establishing the specific role of protein kinase C (PKC) within the NAc in regulating CREB phosphorylation in novelty-elicited acquisition of cocaine SA. To accomplish these goals, direct brain drug microinfusions will be used in conjunction with cocaine intravenous SA and novelty protocols in rats. Protein analysis of CREB phosphorylation will be conducted in all studies.

Neuroimmunology of HIV Asociated Dementia

Mentor: Melendez, Loyda Off-site collaborators: Howard Gendelmann (Univ. Nebraska)

To define the roles of the proteins affected by their interactions in the progression to CI in the presence of HAART, we studied the proteome of blood-derived monocytes obtained from Hispanic women with the most severe form of HIV-associated neurocognitive disorder — HIV-associated dementia (HAD) (Kraft-Terry et al, in revision). These experiments were performed in direct collaboration with members of Dr. Gendelman’s laboratory. Underway are experiments that would employ isobaric tag for relative and absolute quantitation (ITRAQ) for expanding the analysis of the macrophage proteome in the setting of HIV-1 infection. These are also planned in collaboration with Dr. Gendelman’s group. These novel techniques can be learned by undergraduate students and applied to proteomics studies in Puerto Rico for further development of quantitative proteomics in projects related to neuropathogenesis. The student will also learn about HIV cultures, ELISA, Western blots, flow cytometry analyses and 2D- DIGE.

Central Pattern Generators and the Control of Motor Behavior

Mentor: Miller, Mark

Repetitive movements, such as locomotion, feeding, and breathing are controlled by neural circuits known as central pattern generators (CPGs). Dr. Miller’s lab investigates the structure and function of CPG circuits in simpler model systems, where individual neurons and their synaptic interactions can be directly examined. Their studies focus on specific neurotransmitters and modulators, such as dopamine and GABA, that control repetitive motor activity in all nervous systems, including our own. These studies will therefore disclose principles that are broadly applicable to adaptive motor activity and to the dysfunctional conditions associated with presently incurable movement disorders, such as Parkinson’s Disease and Huntington’s Disease.

Alcohol Tolerance via Wnt/ß-catenin impacts BK expression and subsequent ethanol consumption

Mentor: Velázquez-Marrero, Cristina

Alcoholism has devastating effects on individuals and society as a whole. Alcohol tolerance is a key factor leading to increased consumption and subsequent alcohol abuse and alcoholism. Our goal is to understand, at the molecular level, the adaptations that occur in response to alcohol exposure that underlie tolerance and its effects on consumption. In this project we will explore a key regulated process triggered during rapid ethanol exposure underlying persistent changes in neuronal excitability, thus providing important insights into the mechanism of molecular tolerance and its behavioral consequences.

The molecular mechanisms of alcohol dependence in a Drosophila model

Mentor: Alfredo Ghezzi, PhD

My research interests revolve around the molecular basis of neural adaptation. In my laboratory, we strive to resolve how the nervous system utilizes a finite number of genes to carefully modulate its activity and adapt to an ever-changing environment, through the integration of molecular genetics, behavioral analyses, and neurophysiology in a Drosophila model system. A central component of this effort is directed to understanding the mechanisms of transcriptional memory that perpetuate adaptations to drugs, such as alcohol, and how these adaptations result in addictive behaviors.

Exposure to anabolic steroids or to social isolation during adolescence have long-lasting effects on motivational behaviors

Mentor: Annabell C. Segarra, PhD

The research in our laboratory focuses on the neurochemical substrates that participate in mediating sex differences in motivated behaviors.  For the last 30 years, our laboratory has studied the mechanisms by which sex steroids modulate the response to drugs of abuse, such as cocaine and opioids.  Our current studies are focused on adolescents. One of our studies examines the impact of social isolation on the behavioral response to cocaine, and if exercise can mitigate the deleterious effects of social isolation.  Our second project investigates the consequences of exposure to anabolic steroids on the behavioral response to cocaine.  Both projects use male and female subjects to assess if the responses to these manipulation vary with sex.  Our studies build on our expertise studying the interaction between sex steroids and drugs of abuse in adult and adolescent male and female rats, as well as on the technical skills currently available in the laboratory.  In areas where we lack those skills, we have established collaborations with colleagues such as Dr. Katia Gysling in Pontificia Universidad Católica de Chile.