Chloride intracellular channels modulate acute ethanol behaviors in Drosophila, Caenorhabditis elegans and mice.

Identifying genes that influence behavioral responses to alcohol is critical for understanding the molecular basis of alcoholism and ultimately developing therapeutic interventions for the disease. Using an integrated approach that combined the power of the Drosophila, Caenorhabditis elegans and mouse model systems with bioinformatics analyses, we established a novel, conserved role for chloride intracellular channels (CLICs) in alcohol-related behavior. CLIC proteins might have several biochemical functions including intracellular chloride channel activity, modulation of transforming growth factor (TGF)-ß signaling, and regulation of ryanodine receptors and A-kinase anchoring proteins. We initially identified vertebrate Clic4 as a candidate ethanol-responsive gene via bioinformatic analysis of data from published microarray studies of mouse and human ethanol-related genes. We confirmed that Clic4 expression was increased by ethanol treatment in mouse prefrontal cortex and also uncovered a correlation between basal expression of Clic4 in prefrontal cortex and the locomotor activating and sedating properties of ethanol across the BXD mouse genetic reference panel. Furthermore, we found that disruption of the sole Clic Drosophila orthologue significantly blunted sensitivity to alcohol in flies, that mutations in two C. elegans Clic orthologues, exc-4 and exl-1, altered behavioral responses to acute ethanol in worms and that viral-mediated overexpression of Clic4 in mouse brain decreased the sedating properties of ethanol. Together, our studies demonstrate key roles for Clic genes in behavioral responses to acute alcohol in Drosophila, C. elegans and mice.

Alcohol dependence is associated with the ZNF699 gene, a human locus related to Drosophila hangover, in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) sample.

Because tolerance is an important aspect of alcohol dependence (AD) in humans, recent evidence showing that the Drosophila gene hang is critically involved in the development of alcohol tolerance in the fly suggests that variation in related human loci might be important in the etiology of alcohol-related disorders. The orthology of hang in mammals is complex, but a number of human gene products (including ZNF699) with similar levels of amino-acid identity (18-26%) and similarity (30-41%), are consistently identified as the best matches with the translated hang sequence. We tested for association between the dichotomous clinical phenotype of alcohol dependence and seven single nucleotide polymorphisms (SNPs) in ZNF699 in our sample of 565 genetically independent cases and 496 siblings diagnosed with AD, and 609 controls. In analyses of genetically independent cases and controls, four of the seven single markers show strong evidence for association with AD (0.00003

Delayed behavioural aging and altered mortality in Drosophila beta integrin mutants.

The genetic basis for aging is being intensely investigated in a variety of model systems. Much of the focus in Drosophila has been on the molecular-genetic determinants of lifespan, whereas the molecular-genetic basis for age-related functional declines has been less vigorously explored. We evaluated behavioural aging and lifespan in flies harbouring loss-of-function mutations in myospheroid, the gene that encodes betaPS, a beta integrin. Integrins are adhesion molecules that regulate a number of cellular processes and developmental events. Their role in aging, however, has received limited attention. We report here that age-related declines in locomotor activity are ameliorated and that mean lifespan is increased in myospheroid mutants. The delayed functional senescence and altered mortality in myospheroid flies are independent of changes in body size, reproduction or stress resistance. Our data indicate that functional senescence and age-dependent mortality are influenced by beta integrins in Drosophila.

Odor-guided behavior in Drosophila requires calreticulin.

The efficient processing of olfactory information is crucial for many aspects of life in animals, including behavior in insects. While much is known about the organization of the insect olfactory system, comparatively little is understood about the molecules that support its function. To further elucidate the molecular basis of olfaction, we explored the role of the calcium-binding chaperone calreticulin in the behavioral response of Drosophila to aversive odorants. We show that avoidance of naturally aversive odorants is impaired in flies harboring mutations in Calreticulin. Calreticulin mutants have broad defects in odor avoidance without abnormalities in antennal responses to odorants, alterations in central nervous system structure, or deficits in overall locomotor abilities. Interestingly, Calreticulin mutants exhibit defects in behavioral responses to odorants at low strength, whereas responses to higher odorant concentrations are preserved in these animals. Our studies indicate that calreticulin plays a key role in olfactory system function, possibly by establishing its overall sensitivity to odorants.

Integrin-mediated regulation of synaptic morphology, transmission, and plasticity.

Volado, the gene encoding the Drosophila alphaPS3-integrin, is required for normal short-term memory formation (Grotewiel et al., 1998), supporting a role for integrins in synaptic modulation mechanisms. We show that the Volado protein (VOL) is localized to central and peripheral larval Drosophila synapses. VOL is strongly concentrated in a subpopulation of synaptic boutons in the CNS neuropil and to a variable subset of synaptic boutons at neuromuscular junctions (NMJs). Mutant morphological and functional synaptic phenotypes were analyzed at the NMJ. Volado mutant synaptic arbors are structurally enlarged, suggesting VOL negatively regulates developmental synaptic sprouting and growth. Mutant NMJs exhibit abnormally large evoked synaptic currents and reduced Ca(2+) dependence of transmission. Strikingly, multiple forms of Ca(2+)- and activity-dependent synaptic plasticity are reduced or absent. Conditional Volado expression in mutant larvae largely rescues normal transmission and plasticity. Pharmacologicially disrupting integrin function at normal NMJs phenocopies features of mutant transmission and plasticity within 30-60 min, demonstrating that integrins acutely regulate functional transmission. Our results provide direct evidence that Volado regulates functional synaptic plasticity processes and support recent findings implicating integrins in rapid changes in synaptic efficacy and in memory formation.

Differences in agonist-independent activity of 5-Ht2A and 5-HT2c receptors revealed by heterologous expression.

5-Hydroxytryptamine 5-HT2A and 5-HT2C receptors share many properties, including a common ability to stimulate phospholipase C. Traditionally, this activation was thought to be initiated only after agonist binding, in accordance with the ternary complex model of receptor function. Recently, though, the 5-HT2C receptor was shown to deviate from this tenet by spontaneously isomerizing into the active receptor state, thereby activating G proteins in the absence of agonist. To determine if 5-HT2A receptors share this property of constitutive activity, 5-HT2A and 5-HT2C receptor function was evaluated in transiently transfected NIH 3T3 fibroblasts. In 3T3 cells expressing 5-HT2C receptors, agonist-independent phosphatidyl inositol hydrolysis was substantially elevated relative to mock-transfected cells. In contrast, expression of the 5-HT2A receptor at the same density caused only a marginal increase in basal signaling. Control experiments in the current and previous papers establish that basal activity does not reflect contaminating serotonin. In addition, the magnitude of serotonin-induced signaling was the same in cells expressing either receptor, suggesting that the intrinsic ability of the two receptors to couple to G proteins is comparable. These data indicate that the 5-HT2A receptor has a much lower intrinsic ability to spontaneously adopt or maintain the active receptor conformation than does the closely related 5-HT2C receptor.

Integrin-mediated short-term memory in Drosophila.

Volado is a new memory mutant of Drosophila. The locus encodes two isoforms of a new alpha-integrin, a molecule that dynamically mediates cell adhesion and signal transduction. The Volado gene is expressed preferentially in mushroom body cells, which are neurons known to mediate olfactory learning in insects. 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 impaired olfactory memories within 3 min of training, indicating that the integrin is required for short-term memory processes. Conditional expression of a Volado transgene during adulthood rescues the memory impairment. This rescue of memory is reversible, fading over time along with expression of the transgene. Thus the Volado integrin is essential for the physiological processes underlying memory. We propose a model in which integrins act as dynamic regulators of synapse structure or the signalling events underlying short-term memory formation.

DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies.

The modulatory neurotransmitters that trigger biochemical cascades underlying olfactory learning in Drosophila mushroom bodies have remained unknown. To identify molecules that may perform this role, putative biogenic amine receptors were cloned using the polymerase chain reaction (PCR) and single-strand conformation polymorphism analysis. One new receptor, DAMB, was identified as a dopamine D1 receptor by sequence analysis and pharmacological characterization. In situ hybridization and immunohistochemical analyses revealed highly enriched expression of DAMB in mushroom bodies, in a pattern coincident with the rutabaga-encoded adenylyl cyclase. The spatial coexpression of DAMB and the cyclase, along with DAMB's capacity to mediate dopamine-induced increases in cAMP make this receptor an attractive candidate for initiating biochemical cascades underlying learning.

m-chlorophenylpiperazine and m-trifluoromethylphenylpiperazine are partial agonists at cloned 5-HT2A receptors expressed in fibroblasts.

Serotonin2A (5-HT2A) and 5-HT2C receptors share numerous pharmacological properties. Two compounds thought to discriminate between these two receptor subtypes are m-chlorophenypiperazine (mCPP) and m-trifluoromethylphenylpiperazine (TFMPP). These two drugs have been classified as antagonists at 5-HT2A receptors but as agonists at 5-HT2C receptors on the basis of phosphoinositide hydrolysis studies in cerebral cortex and choroid plexus, respectively. To determine more fully the properties of mCPP and TFMPP at 5-HT2A receptors, NIH 3T3 fibroblasts transfected with the 5-HT2A receptor complementary DNA (GF6 cells) were used as a model system of receptor function. These cells express approximately 15-fold higher 5-HT2A receptor density than is found in cerebral cortex. In GF6 cells, mCPP and TFMPP dose-dependently stimulated phosphoinositide hydrolysis with maximal effects less than that of 5-HT. This agonist activity was blocked by 5-HT2A receptor antagonists but not by prior treatment with pertussis toxin. Partial inactivation of 5-HT2A receptors with phenoxybenzamine decreased the maximal effects of mCPP and TFMPP but did not eliminate agonist activity. Thus mCPP and TFMPP are partial agonists at 5-HT2A receptors in GF6 cells, and these agonist properties are retained even under conditions where receptor density is comparable to that of cerebral cortex. Although it has not yet been demonstrated that mCPP and TFMPP are agonists at central 5-HT2A receptors, this possibility should be considered when evaluating in vivo effects of these drugs.

Regulation of serotonin2A receptors in heterologous expression systems.

The serotonin2A and serotonin2C receptors are unique among receptors coupled to guanine nucleotide binding proteins in that chronic treatment in vivo with agonists as well as antagonists decreases receptor density. In an attempt to uncover molecular events involved in down-regulation of the serotonin2A receptor, the ability of agonists and antagonists to alter receptor density was examined in three heterologous expression systems, i.e., transfected NIH 3T3, transfected Madin-Darby canine kidney, and transfected AtT-20 cells. All three transfected cell lines exhibited pharmacological properties consistent with that predicted for cells expressing the serotonin2A receptor. However, the three cell lines displayed different receptor regulation properties in response to drugs acting at the serotonin2A receptor. In transfected NIH 3T3 cells, neither agonist nor antagonist treatment altered receptor density. Treatment with agonist as well as antagonist led to up-regulation of the serotonin2A receptor in transfected Madin-Darby canine kidney cells. In transfected AtT-20 cells, treatment with agonist led to receptor down-regulation, whereas antagonist treatment increased receptor density. Thus, the cellular background in which the serotonin2A receptor is expressed appears to determine the regulation properties of the receptor.

The effect of PDGF on the healing of full thickness wounds in hairless guinea pigs.

1. Platelet-derived growth factor (PDGF), administered daily for 5 days and every other day for 9 additional days or the day of wounding and the following day, caused dose dependent (0.2, 1.0 or 5.0 micrograms PDGF/wound) increased granulation tissue. 2. The two day 5.0 micrograms treatment resulted in a 73% increase and the multi-day treatment resulted in a 52% increase of alkaline phosphatase activity three days after wounding. 3. Multi-day treatment resulted in significant increases in protein synthesis (132%), vimentin content (160%) and excised wound weight (76%) three days after wounding. 4. These results indicate that limited administration of PDGF alters wound healing although multiple applications provoke a more dramatic response.