To identify dosage-sensitive genes that influence the response to anesthetics, we previously screened for changes in sensitivity to halothane in lines that were missing one copy of a large block of DNA. We found that the anesthetic phenotypes resulting from deleting two regions, ED1 (75 kb) and ED4065 (540kb), could be attributed to a single gene in each. For ED1, the critical gene was glutathione-S-transferase S1, an antioxidant, implicating redox balance in anesthetic sensitivity. For ED4065, the critical gene was orc4, a key component of the Origin Recognition Complex. During DNA replication, this multi-protein ensemble participates in loading replication origins with key proteins. It is also found in postmitotic neurons, where it participates in microtubule organization and where the human ortholog of another member of the complex, ORC3, has been associated with the severity of symptoms expressed by schizophrenics. While the phenotypes of ED1 and ED4065 result from loss of single copies of single genes, analysis of another deletion, ED2247 (389 kb), is genetically more complex. The anesthesia phenotype of this region could be rescued by a 50 kb transgene containing 7 genes, but it was not possible to recapitulate the phenotype by mutating any single gene. Thus, the screen has reinforced our working model that the study of anesthesia can uncover genes responsible for complex neural functions, but in some cases the observed mutant phenotypes may result from the interaction of multiple genes. Strong effects on anesthesia sensitivity are caused by mutations in the narrow abdomen (na) gene of Drosophila. We earlier showed that the na gene encodes a putative ion channel that is expressed broadly in the central nervous system of all metazoans, and one whose human ortholog has been implicated in susceptibility to bipolar disorder. Our attempts at assessing the anesthetic sensitivity of the NA channel have been thwarted because of the difficulty in reliably replicating published work showing that the vertebrate ortholog functions as a non-selective cation channel. The capriciousness of the channel in heterologous expression systems pushed us to search for accessory subunits and chaperones in addition to those identified in previous collaborations, such as unc79 and unc80. Combining isotopic labeling techniques with mass-spectrographic protein identification, we have identified additional proteins that physically interact with the NA channel. O-18 labeling gave the clearest results, identifying all previously identified interacting proteins, and providing a collection of candidates that are currently being studied. Mutating one of the candidates, l(3)01239, a prefoldin subunit involved in protein trafficking, alters NA protein expression, suggesting a hitherto unsuspected molecular pathway responsible for channel complex formation. Because mutations in na affect anesthesia, which rapidly and reversibly affects consciousness, we have more closely examined the arousal state of na mutants in the absence of anesthetics. Free roaming mutant flies have a tendency to periodically (every 3-10 seconds) lose postural control and enter a quasi-sleep state from which they spontaneously recover. The period of this abrupt shift in postural control correlates with a rhythmic electrical signal that can be recorded from the eyes of mutant flies, suggesting that this behavior reflects a rhythmic change in nervous system excitability. This episodic loss of arousal interfered with behaviors that demand a sustained level of activity, such as tethered flight, male courtship and expanding the wings after emergence from the pupal case. Therefore, in the absence of drug, na mutants appear to dwelling close to the edge of consciousness . We previously reported that sensitivity to halothane strongly correlates with gene dosage of the ryanodine receptor (Ryr), indicating that Ryr is a limiting factor. This effect is mediated by the nervous system, and is halothane selective, with Ryr mutations affecting the response to other anesthetics to a lesser extent. To further challenge our hypothesis that Ryr is a limiting factor for anesthetic sensitivity, we tested a collection of point mutants, and correlated our behavioral results with the sites of mutation as determined by next generation sequencing. Consistent with previous results, alleles that reduced sensitivity to halothane were associated with mutations that eliminated Ryr function. More interestingly, some alleles increased halothane sensitivity. These were associated with missense mutations in conserved regions of the protein, including one with a sequence change identical to that associated with a human genetic disorder that makes the Ryr more likely to open. To establish whether Ryr is a target of halothane, we have used imaging techniques to examine the effects of halothane on intracellular Ca2+. Cultured Sf9 cells stably transfected with Drosophila Ryr and filled with the Ca2+ indicator Fluo-5 show robust, concentration-dependent increases in intracellular Ca2+ in response to halothane. Paralleling the halothane-specificity of the behavioral results, Ryr-transfected cells responded much less strongly to other anesthetics. In addition, the halothane response is blocked by depletion of internal stores, and does not occur in untransfected Sf9 cells. Using genetically-encoded Ca2+ indicators, we have found that central neurons also respond to halothane with elevated internal Ca2+. Because neural Ryr function is a limiting factor for halothane sensitivity, and Ryr responds to halothane by releasing Ca2+ from internal stores, we believe that it is an important anesthetic target. Surgical concentrations of anesthetics cause analgesia and immobility, whereas lower concentrations have more subtle effects on higher functions such as attention and memory. To understand the effects of anesthetics on complex behavior, we have developed a novel assay for social behavior in flies based on their tendency to aggregate when placed in an arena that limits movement to two dimensions. This tendency is independent of the sex or social experience of the flies, but requires ambient light. Different wild-type strains have varying propensities to aggregate, suggesting that there are genetic components of the behavior that can be exploited. Importantly, social aggregation is also blocked by very low concentrations of halothane and enflurane, opening the door to the genetic investigation of the effects of volatile anesthetics on this social behavior. We are also applying our expertise in Drosophila neurobiology to a gene associated with autism via a collaborative study. With Dr. Brian Mozer of NHLBI, David Sandstrom is analyzing the role of Drosophila neuroligin-1 (nlg1) in the development and function of a prototypical synapse, the larval neuromuscular junction. NLG1 protein is known to be localized to the synapse and nlg1 mutations cause reduction in the extent of the synaptic terminal and release of the neurotransmitter glutamate, along with abnormal distribution of glutamate receptors (GluRs) in the muscle. We found that overexpression of NLG1 in the muscle perturbs clustering of GluRs and increases the size of the synaptic terminal, but inhibits glutamate release from the nerve terminal. Terminal growth and inhibition of release are dependent on neurexin, the presumed partner of nlg, but GluR redistribution is not. Paradoxically, mutations and transgenes that increase synaptic terminal area downregulate nlg levels.
