Model Genetic Organisms for the Study of the Nervous System and Behavior

United States

Model genetic organisms such as the fly, worm, and zebrafish have elucidated the basic principles of development, have identified gene products that regulate excitability in the nervous system, and have lead to discoveries of genes implicated in neurodegenerative disease. The purpose of this satellite symposium is to discuss new developments and opportunities for the analysis of the nervous system and behavior using the powerful tools of fly, worm and zebrafish genetics. The identification of mutations affecting behaviors in these organisms may provide insight into psychiatric disorders and addiction.

Abstracts

Genetic and Pharmacological Approaches to Study Drugs of Abuse in <em>Drosophila

Linus T.-Y. Tsai, Roland J. Bainton, Monica S. Moore, Carol M. Singh, Henrike Scholz, and Ulrike Heberlein
Department of Anatomy, Anesthesia, and Neurology, Programs in Neuroscience and Developmental Biology, University of California at San Francisco, San Francisco, CA 94143-0452

Sensitivity to ethanol and the development of tolerance and dependence are influenced substantially by genotype in humans and rodents. In addition, evidence is emerging from studies in animal and cellular model systems indicating that the effects of ethanol on a variety of cellular functions are mediated by changes in specific proteins. Thus, Drosophila,an organism readily accessible to genetic and molecular analysis should prove useful in establishing missing links between genes and behavior. We have found that adult flies display many of the behaviors observed in humans after both acute or chronic exposure to ethanol vapor. Flies display signs of hyperactivity, incoordination, followed by sedation and hypnosis. In addition, flies develop tolerance after single and multiple ethanol exposures. We are carrying out genetic screens designed to isolate mutations that cause altered sensitivity to ethanol intoxication or that impair or enhance development of tolerance. Sensitivity to intoxication is quantified in an 'inebriometer', a device that measures postural control. Tolerance is defined as a decrease in this sensitivity caused by previous ethanol exposure. The behavioral and molecular characterization of mutations that alter ethanol sensitivity and tolerance will be described.

In mammals, a common property of drugs of abuse is their ability to facilitate dopamine (DA) release in specific brain regions involved in reward and motivation. This increase in DA levels is believed to act as a positive reinforcer and to mediate some of the acute drug responses such as locomotor activation. We have found that DA plays a role in the responses of Drosophila to cocaine, nicotine, and ethanol. We have developed a quantitative behavioral assay, based on negative geotaxis, that measures the effect of psychostimulants on fly behavior. Acute responses to cocaine and nicotine are blunted by pharmacologically-induced reductions in DA levels. Co-administration of cocaine and nicotine shows a high degree of synergy, consistent with an action through convergent pathways. Normal DA levels are also required for ethanol-induced locomotor activation, but not sedation. We conclude that in Drosophila, as in mammals, dopaminergic pathways play a role in modulating behavioral responses to multiple drugs of abuse.

Mutations That Alter Circadian Rhythms of Behavior in Zebrafish

Gregory M. Cahill
Department of Biology and Biochemistry, University of Houston, Houston, TX

Circadian clocks regulate many aspects of behavior, including sleep-wake cycles, sensory function, and reproduction. Genes that code for circadian clock components have been discovered by screening a variety of model organisms for mutations that alter the period or amplitude of circadian rhythms. We have initiated a behavioral screen for clock mutants in zebrafish in an effort to identify new vertebrate circadian clock genes and to gain insight into the functions of known clock genes. Our primary screening assay is the circadian rhythm in spontaneous swimming activity of 9-18 day old larvae, measured by an automated infrared video image analysis system. We are screening for dominant mutations that alter the timing of behavioral rhythms in the progeny of ENU treated males crossed with wild-type females. We recovered two confirmed clock mutants from the first 1275 animals tested. Both of these mutant alleles are incompletely dominant, shortening the freerunning period of behavioral rhythms by 0.5-0.8 h in heterozygotes and 1-1.5 h in homozygotes. One of the mutations maps to Linkage Group 7 and the other maps to LG 20. We have so far confirmed that one of these mutations also shortens the period of the melatonin release rhythms measured from cultured pineal glands, indicating that the mutant gene product affects tissue-level rhythmicity as well as behavior. We are continuing to screen for new mutants in order to identify additional alleles of these genes, as well as mutations in other vertebrate clock genes. Our experience so far indicates that the zebrafish will be a productive model system for genetic analysis of the biological clocks that regulate vertebrate behavior.

Genetic Analysis of Nicotine Adaptation in <em>C. Elegans

William Schafer
Department of Biology and Group in Neuroscience, UCSD, La Jolla, CA 92093-0349

Prolonged exposure to nicotine causes long-lasting and specific inactivation of nicotinic receptor activity in brain neurons, a process though to underlie nicotine addiction. At present, the molecular basis for nicotine adaptation is almost completely unknown. We have begun using a genetic approach to identify molecules required for nicotine adaptation in the nematode C. elegans. Nematode egg-laying behavior undergoes a process of long-term adaptation to nicotine that is similar in many respects to nicotine adaptation in vertebrates. We have found that in C. elegans, prolonged nicotine treatment results in long-term adaptation of nicotinic receptors that control egg-laying. In naive animals, acute exposure to cholinergic agonists led to the efficient stimulation of egg-laying, a response mediated by a nicotinic receptor functionally expressed in both neurons and muscle cells. Overnight exposure to nicotine led to a specific and long-lasting change in egg-laying behavior, which rendered the nicotine-adapted animals insensitive to simulation of egg-laying by nicotinic agonist. Genetic screens conducted in our laboratory have identified a number of adaptation defective mutants, which define several genes that are essential for adaptation to nicotine. For example, mutant animals defective in the gene tpa-1, which encodes a homologue of protein kinase C (PKC), failed to undergo adaptation to nicotine, and remained sensitive to cholinergic agonists even after long nicotine exposure. Long treatments with phorbol esters also inhibited the acute response to cholinergic agonists in a tpa-1 dependent manner. These results suggest that nicotine adaptation may involve long-term inactivation of nicotinic receptors by PKC.

The Molecular Genetics of Cocaine Responsiveness and Sensitization in <em>Drosophila</em>

R. Andretic, C. McClung, H. Li, S. Chaney, and J. Hirsh*
Department of Biology, University of Virginia; Charlottesville, VA 22903

We are using the fruit fly, Drosophila melanogaster, as a genetic model system to study responsiveness and sensitization to aerosolized free base cocaine. We show that sensitization can be blocked by ectopic in vivo expression of stimulatory or inhibitory G proteins in the dopamine and serotonin neurons of the CNS. This shows the involvement of these transmitter systems, and implicates altered G protein signaling as an important component of sensitization. The degree of responsiveness to cocaine is correlated with the responsiveness of nerve cord receptor preparations to a D2-like dopamine agonist that is expected to interact directly with postsynaptic receptors.

We show the involvement of the octopamine precusor tyramine in sensitization; the mutant inactive (iav), is deficient in tyramine and the tyramine biosynthetic enzyme tyrosine decarboxylase (TDC). Octopamine is not required for sensitization since a null mutant in tyramine beta-hydroxylase, sensitizes normally. Active involvement of tyramine in sensitization is suggested since TDC is induced by cocaine exposure with a time course matching the time course for development of sensitization.

We also show the involvement of a subset of circadian genes in sensitization. Four of the five known circadian genes affect cocaine sensitization; only the gene timeless sensitizes normally. These genes function upstream of TDC since the induction of TDC that normally occurs following cocaine exposure is absent in these mutants. Mechanisms for cocaine sensitization will be discussed.

This work was supported by NIH/NIDA predoctoral grants 1F31DA05897-01 and 1F31DA05942-01, and NIH grant GM/DA 27318.

Molecular, Neurogenetic and Plastic Components of Food Related Behaviors in the Fruit Fly

Marla B. Sokolowski
University of Toronto

The question of how genes contribute to normal individual differences in behavior has captured our imagination for more than a century. Two fundamental questions come to mind: how do genes and their proteins act in the nervous system in order to cause individual differences in behavior? How do genes and their proteins act in response to the environment to affect normal individual differences in behavior? Understanding the connection between genotypic differences and changes in gene expression requires studies on genes that vary under natural conditions. The foraging (for) gene in Drosophila melanogaster is responsible for naturally occurring rover and sitter food search behavior. Animals homozygous for the sitter allele (for^s) exhibit short foraging trails whereas those with a rover allele (for^R) travel farther in search of food. The for gene encodes one of two cGMP-dependent protein kinases (PKG) in Drosophila. for transcript abundance and PKG activity levels are higher in rover adult heads and larval central nervous systems (CNSs) than in sitter heads and larval CNSs when the animals are well fed. When animals are removed from food for several hours rovers behave more sitter-like indicating that the genetically different rovers and sitters can exhibit plastic responses in their foraging behavior in response to environmental change. We show that changes in for gene expression parallel these plastic behavioral responses. Thus it appears that the PKG encoded by for may function in normal individual differences in behavior that arise from both: 1) differences in gene expression due to natural allelic variation and, 2) differences in gene expression due to environmentally induced changes in the behavioral state of an individual.

Cell-Adhesion Molecules and <em>Drosophila</em> Olfactory Learning

Ronald L. Davis
Department of Molecular and Cellular Biology and Department of Psychiatry and Behavioral Sciences. Baylor College of Medicine. Houston, TX 77030

Two new learning mutants of Drosophila have emphasized the importance of cell-adhesion molecules in the processes underlying learning. The Volado gene encodes two isoforms of a novel alpha-integrin, a type of molecule that dynamically mediates cell adhesion and signal transduction. The gene is expressed preferentially in mushroom body cells, neurons known to mediate olfactory learning in insects, and the Volado proteins are concentrated in the mushroom body neuropil, brain areas that contain mushroom body processes in synaptic contact with other neurons. Volado mutants display an impairment of olfactory memories within 3 minutes after training but conditional expression of a Volado transgene during adulthood rescues the memory impairment. A modified behavioral paradigm has revealed that one of the integrin products serves memory formation, while the other is required for both memory formation and stability. The mutant, fasII, encodes a cell-adhesion molecule of the immunoglobulin superfamily. The gene is highly specific for mushroom bodies and when mutant, produces a learning deficit like Volado mutants. Conditional transgenes provide for rescue only during adulthood, confirming a physiological role for the gene product in learning processes. These observations demonstrate the critical importance of cell-adhesion molecules of the immunoglobulin superfamily and integrin family for mediating the formation of memory.

For additional information contact:

Jonathan D. Pollock, Ph.D.
National Institute on Drug Abuse
jp183r@nih.gov

Hemin Chin, Ph.D.
National Institute of Mental Health
hc7v@nih.gov