Genetic Neurobiology Of Drosophila

National Institute Of Mental Health

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Abstract

In the clinic, volatile agents such as halothane and isoflurane induce a unique state in which elementary neural functions are intact but higher-order neural processes that we associate with arousal and working memory are lost. Our project is based on the rationale that genes which influence anesthesia ought to identify players in these important processes and ought to help to figure out how the drugs produce the anesthetized state. To find such genes, we use a genetic model organism (the fruit fly) and identify mutations that affect its response to volatile anesthetics. Work in the past year has focused on understanding how loss of function mutations in one such gene leads to dramatic changes in the anesthesia response. We have also initiated a screen to identify loci at which modest reduction in function leads to changes in anesthetic sensitivity. [unreadable] [unreadable] In past work we have demonstrated that strong loss-of-function mutations in the na gene, which encodes a novel ion channel, produce a distinctive anesthetic phenotype. In assays that measure the ability of a fly to right itself and climb vigorously following a mechanical insult, na mutants are grossly hypersensitive to all volatile agents tested, including halothane and isoflurane. However, in assays that measure the ability of a fly to remain standing under anesthesia, the mutants distinguish between anesthetics. In the presence of halothane, they remain standing much longer than wild-type flies and appear to be frozen in place. But when enflurane is used in such "inebriometer" assays, the mutants' response is no different from that of wild-type flies. Remarkably, the same halothane/enflurane distinction has been seen in unc-79 mutants of the nematode C. elegans, which are very hypersensitive to the immobilizing effects of halothane but very similar to wild-type in sensitivity to enflurane. To determine whether this pattern is adventitious or fundamental, we joined forces with the lab that first described the unc-79 mutations, identified orthologs of each gene, and determined the mutant phenotype within each species. In Caenorhabditis, mutations that inactivate the na channel orthologs produce an unc-79 phenotype and in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway and biochemical studies show that unc-79 controls channel levels by a post-transcriptional mechanism. This interspecies study not only provides new information about channel regulation but has strong implications for the way the channel influences sensitivity to anesthesia. In particular, the evolutionary conservation of agent-specificity argues against many scenarios for indirect action. For example, one might propose that the channel is not in target neurons but influences anesthetic sensitivity only because neurons which depend on it for optimal performance are in communication with those that are affected by anesthetics. But, because the neuronal circuitry of the nematode is strikingly different from that of the fruit fly, it is hard to imagine that in each organism there are halothane-sensitive/enflurane-insensitive neurons that just by chance are connected to channel-containing neurons. Instead, our observations suggest that, in the circuits that support mobility in nematodes and postural control in fruit flies, there exist neurons that directly depend on the channel for resisting the effects of halothane. In this population the channel could be a direct target for halothane (but not enflurane) or could serve to stabilize neuronal performance against the deleterious effects of halothane on some other component in that cell (one which is insensitive to enflurane). Despite this uncertainty, our observations provide a strong clue that, at least in the circuits subserving these endpoints, the relationship between the channel and halothane action is intimate.[unreadable] [unreadable] If the na-encoded ion channel is intimately involved in the response to halothane, the gene should be needed during the time at which the drug is administered. In earlier work, done in collaboration with the lab of Ravi Allada, we showed that neuronal expression of the channel gene was sufficient to suppress the mutant anesthetic phenotype. Although this result was consistent with a relatively direct role, it left open the possibility that the channel was needed primarily to insure normal neural development. To challenge this alternative, we have now used the TARGET system (recently established in the lab of R. Davis) to limit neuronal expression to various periods in the life cycle of Drosophila. We find that, although the channel is normally expressed at low levels during embryonic and larval life, halothane hypersensitivity in the reactive climbing assay persists when expression is limited to these stages. On the other hand, expression after adult eclosion, when the nervous system is fully formed, is at least partially effective at restoring normal sensitivity. This argues strongly against a purely developmental role for the channel. It must be noted, however, that full rescue requires expression during late pupal life, a time when the channel is normally deposited at high levels in the neuropil. Although this might indicate a role for na in neuronal maturation, it is also consistent with the possibility that proper targeting of the channel requires accessory factors that are normally available only at this time.[unreadable] [unreadable] There are many ways in which a given mutation could influence the sensitivity of an organism to general anesthetics. Of particular interest is the subset of such mutations that affect genes that encode direct targets of anesthetic action. Although biochemical and physiological studies have identified several molecules whose function is altered by clinical concentrations of volatile agents, the significance of these targets for the clinical effects of anesthetics has rarely been established. Thus, there is strong motivation to find a genetic strategy that enriches for mutations in anesthetic target genes. We are investigating haploinsufficiency as one such strategy. Our reasoning is based, first, on the widespread correlation between gene dosage and gene expression and, second, on precedents, exemplified by studies with methotrexate, that the amount of its target influences the potency of a drug. Our approach is to identify genes that affect sensitivity to halothane when their copy number is reduced in half. To survey the Drosophila genome for such loci, we use the collection of DrosDel deficiencies. These have been constructed to have sharply defined endpoints, remove on average 50 genes at a time, and share a common genetic background. We have now screened more than 200 of these deletions by outcrossing each to a standard strain and measuring the potency of halothane in producing loss of reactive climbing. From this survey, covering more than 45% of identified Drosophila genes, we have identified only a few loci that are significantly and reproducibly distinguishable from control. Interestingly, half of these produce not hypersensitivity but relative resistance to halothane, insuring that we are not only dealing with genes that are required for robust health. Our collection of dosage-sensitive loci is a resource for identifying genes, like those that encode anesthetic targets, whose expression is limiting for neuromuscular function under the stress of anesthesia.