Genetic Neurobiology Of Drosophila

National Institute Of Mental Health

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Abstract

In a screen for genes that influence the sensitivity of fruit flies to the volatile anesthetic halothane, we turned up a mutation in the inaF locus. From work of others, this gene was known to have strong effects on visual signaling through its effect on TRP channels, the principal source of light-induced currents in the fly eye. In previous work, we identified the inaF gene product that interacts with TRP, but the mechanism of its effect on anesthesia sensitivity remained obscure. Because of the effect of this gene on retinal physiology, we investigated the possibility that it ifluences the way illumination affects arousal. We tested the wild-type Canton-S strain for sensitivity to the volatile anesthetic halothane either in dim red light, which fruit flies do not perceive, or in ambient room light: there was no significant difference in sensitivity. In contrast, while the sensitivity of flies bearing a mutation in inaF was indistinguishable from that of the congenic control strain when tested under dark conditions, they became significantly less sensitive to halothane when exposed to ambient light. A null mutation in the trp gene itself confered virtually identical behavior, as did an inaF;;trp double mutant. It appears that damage to the TRP pathway of visual signaling reveals the arousing properties of light. On the other hand, we find that mutations in the white gene confer the opposite effect. When dark-reared white-eyed flies bearing either of two of two white mutations were exposed to ambient light, they increased rather than decreased their sensitivity: in these mutants illumination has an anti-arousing or calmative effect. Our data is thus the first to show that the visual stream provides both positive and negative effects on arousal.[unreadable] [unreadable] Our most advanced understanding of the way anesthetics influence neurons of Drosophila comes from the study of the larval life phase. Here, previous work has shown that isoflurane depresses the excitability of motorneurons, resulting in slowed conduction and reduced neurotransmitter release. The quantitative relationship between these two effects had implicated activation of a leak current as the likely effect of isoflurane in this system. To challenge this hypothesis, we made patch clamp recordings from the somata of larval motorneurons and directly measured the effect of added isoflurane. In current clamp, application of 0.4 mM isoflurane caused a significant and reversible hyperpolarization . Under voltage clamp, 0.3-0.4 mM isoflurane produced a reversible and statistically significant reduction in input resistance. Subtraction of the pre-treatment I-V curves from those recorded in isoflurane yielded an isoflurane-activated conductance of 97.5 pS that reversed at -72.7 + 2.2 mV, values consistent with the hyperpolarization observed in current clamp mode and strongly inidicative of an anesthetic-activated leak.[unreadable] [unreadable] Can the larval preparation be used to investigate genetic effects? As proof of principle, we examined larvae that carried hypomorphic mutations in para, the sole Drosophila gene that encodes a voltage-gated sodium channel. Untreated para ts1 axons were not different from controls, but the effect of 0.1 and 0.15 mM isoflurane was significantly larger in the mutants. Axons from the heteroallelic combination parats1/para60 were also hypersensitive to 0.1 mM isoflurane. Although these experiments demonstrated that para mutations act on anesthetic-sensitive neurons, they do not ditinguish whether sodium channels are themselves a critical target of isoflurane or whether para mutations act indirectly, by sensitizing axons to the effects of isoflurane on other conductances. To answer this question, we turned to the computer model previously developed in our lab that simulates the effect of altered conductances on conduction velocity and neurotransmitter release. The model indicated that reduction of NaV channel function (as expected in the para mutants) could indeed enhance the sensitivity of the model axons to other sources of reduced excitability, such as increased leak. These studies thus serve as a guidepost for future work, indicating both the power and the limitations of the genetic approach. Loss-of-function mutations can identify genes that contribute strongly to the anesthetic response and can even identify genes whose products are needed in anesthetic-sensitive neurons. But, genetic identification of anesthetic targets will require mutations with more specific effects than loss-of-function.[unreadable] [unreadable] One of the most intriguing genes identified in our laboratorys screens for altered ansesthetic sensitivity is narrow abdomen (na). This gene encodes an ion channel of distinctive structure whose mouse ortholog has recently been shown by others to be the principal source of non-selective cation leak in neurons. In addition to the dramatic effects of na mutants on responses to halothane and other anesthetics, the range of phenotypes that accompany loss of this depolarizing conductance in Drosophila na mutants include altered circadian locomotor patterns, presence of rythmic brain field potentials, and disjointed walking movements. These defects are also found in strains bearing mutant alleles of the fly ortholog of the C. elegans gene unc-79. This gene, as well as its fly and vertebrate orthologs, is predicted to encode a large (300kDa) protein that, surprisingly, has no recognizable structural motifs or homologies to well-studied proteins. The discovery that mutants of the Drosophila ortholog (dunc79) accumulte little or no na gene product established a link between the two genes but the nature of that link was unknown. Our current work uses anatomical and biochemical approaches to examine their functional relationship. Using a newly developed antibody to DUNC79, we find that the protein is in the neuropil and not in the cellular rind of adult fly brain; this argues against a role for dunc79 in the synthesis or post-translational processing of the channel. Moreover, the anatomical distribution of DUNC79 shows the same pattern as NA: broad expression throughout the central brain and optic lobes with areas of concentration in the lateral triangles of the ellipsoid body and in the distal tips of large monopolar cells of the lamina. The colocalization of the two proteins suggest they interact and, both by immuno-affinity purification and by Blue Native-PAGE, we find that NA and DUNC79 participate in a membrane-bound complex. This complex appears to be important for both partners since, just as NA levels are very low in dunc79 protein nulls, DUNC79 levels are very low in na protein nulls. An indication that NA plays a structural role in the complex is that levels of DUNC79 are normal in strains that make robust amounts of inactive channel. Taken together, our phenotypic, anatomical and biochemical data support the idea that dunc79 is a gene that is devoted solely to na function, perhaps to help the channel interperet cellular signals.