Genetic Neurobiology Of Drosophila

National Institute Of Mental Health

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Abstract

One of the most intriguing genes identified in our laboratorys screens for altered anesthetic sensitivity is called narrow abdomen (na). This gene encodes an ion channel of distinctive structure whose vertebrate ortholog, NALCN, has recently been shown by the lab of Dejian Ren to be the principal source of non-selective cation leak in neurons. We seized upon this advance with the hope of testing whether this leak channel has a strong effect on anesthesia sensitivity because it is normally an important target for action of these drugs. Accordingly, we acquired the expression construct used by the Ren lab and used their protocol to transfect HEK293 cells with NALCN. Despite the presence of significant levels of NALCN protein on the surface of transfected cells, the described voltage-insensitive positive currents were not found. Neuroblastoma cells were also tested, again with no success. After much time spent systematically altering culture and recording conditions, we learned from the original authors that they had lost the ability to achieve functional expression, possibly because of changes in the supply of serum. Thus, the conditions required for expression of active channels are poorly defined and we have moved to other approaches to study the way the physiology of the NA channel is affected by anesthetics.[unreadable] [unreadable] Mutations in the na gene confer a phenotype in the inebriometer, a device that measures postural control,only with certain volatile agents, e.g., halothane but not enflurane. The same agent-specificity was subsequently found in NA channel mutants of C. elegans, an organism whose neuronal circuitry is strikingly different from that of a fly. Rather than invoke two cases of fortuitous wiring, we take these observations to mean that channel-containing neurons and halothane-sensitive neurons are one and the same. In that case, mapping the neurons that need the channel for proper inebriometer performance would identify the neurons that serve as the physiological target for halothane disruption of a behavior. To do such mapping, we start with flies that lack the channel and reintroduce it via expression of a full-length cDNA in selected sets of neurons. This is accomplished by crossing fly lines that contain, in various combinations, transgenes encoding transcriptional drivers, effectors and blockers. Unfortunately, these components have been generated in other labs without regard to genetic background, and we have learned through bitter experience that the effect of drugs on whole animal behavior is polygenic. Accordingly, we spent many months backcrossing a large set of transgenes and subsequently building strains that differ only by domains of channel expression and not by polymorphisms. This effort proved to be vital because the results we got were so unexpected. Specifically, we find that restoration of channel expression in either of two non-overlapping sets of neurons is sufficient to yield normal responses to halothane in the inebriometer. One set of neurons is non-cholinergic and belong to the circadian apparatus of the fly; the other set is cholinergic and non-circadian. We conclude that halothane targets two distinct circuits that redundantly regulate postural control of the fly. [unreadable] [unreadable] It must be acknowledged that not every mutation that influences the sensitivity of an anesthetic endpoint does so in an interesting way. For example, given the fact that na mutant flies are somewhat sluggish, anesthetic effects on climbing ability are hard to interpret. Accordingly, to study this gene more incisively we have resurrected an assay that monitors neural transmission in the brain of a fly by means of electrophysiology. More than ten years ago we reported that anesthetics depress the ability of electrical stimulation of the fly brain to trigger a response in a circuit that normally subserves visually-evoked startle responses. After confirming the basics of our published observations, we have now explored the effect of two independently isolated na mutations. The results are clear: concentrations of halothane that leave the long-latency response intact in over 80% of wild-type flies strongly depress the response of mutant flies. Preliminary results indicate that the same is true for mutations in dunc-79, a gene that encodes an accessory subunit of the channel complex. However, in contrast to results with the inebriometer assay, in the long-latency assay na mutants are similarly hypersensitive to enflurane. Thus the NA channel plays important but significantly different roles in setting the sensitivity of two neural circuits to volatile anesthetics. [unreadable] [unreadable] Although the na gene is clearly a major player in the response to anesthetics, finding other genes that influence arousal is an important goal of our lab. One particularly interesting class of such genes would be those that are dosage-sensitive, i.e, display a phenotype upon reduction of gene copy number from two to one. By definition, the product of such a gene is limiting for proper function of the nervous system, at least under the stress of anesthesia. Moreover, since copy number variation (CNV) is now understood to be a prominent source of genetic diversity in the human population, assessing its impact on a model organism like Drosophila should provide a guide to the influence of this kind of polymorphism on anesthetic potency in the clinic. In work reported earlier, we surveyed almost half of the genome of Drosophila for genes that would affect anesthesia upon reduction of copy number from two to one. Out of 220 strains tested, we have now arrived at a subset of 8 strains that show reproducible shifts in anesthetic potency that range from 25% hypersensitivity to 15% resistance. However, before accepting our survey as providing a guide to the frequency and magnitude of CNV effects, one must consider the possibility that the anesthetic phenotype of a given strain does not reflect haploinsufficiency of a gene in its deleted region but rather is due to an adventitious mutation. Such a mutation might have been introduced during the original construction of the deletion line or might have arisen during its subsequent passages. In one case, to confront this possibility we attempted to reverse the phenotype by putting an extra copy of the deleted region at some other location in the genome. In several other cases, we acquired and tested additional deletions that remove genetic material from the relevant segment of the genome. Although not every strain of interest has been examined, the success of our initial trials with each strategy indicates that adventitious mutations do not seriously confound the conclusion that variation in copy number of selected sets of genes produces significant changes in halothane sensitivity. Moreover, by narrowing the focus within the deleted region, each strategy has provided a stepping-stone to identify the relevant haploinsufficient genes.