General anesthetics are of value in the clinic largely because they preferentially interfere with complex neural processes like memory and consciousness. We have shown that these agents affect the nervous system of Drosophila melanogaster with a similar degree of specificity, leaving intact early steps of visual processing and peripheral motor responses while rendering the insect behaviorally inert. The isolation of mutants of the fruit fly that have altered responses to anesthetics thus provides a powerful method for identifying genes whose products are involved in higher-order processing. We do not know how directly such ?anesthesia mutations? affect sensitivity. One approach to clarifying the picture is to determine the molecular nature of the affected gene and the anatomic distribution of its product. Another useful strategy is to assess the effect of the mutation on simple physiological processes that plausibly underly the behavior of the whole animal. In the past year, we have refined the cellular and sub-cellular distribution of two genes of interest and have developed a new assay for an important cellular function. Our past work has implicated the narrow abdomen (na) gene as having strong effects on a variety of anesthetic endpoints. This gene encodes a putative ion channel, specifically an unusual member of the voltage-gated sodium and calcium channel superfamily. The principal evidence that the fly member of this highly conserved family regulates anesthesia sensitivity was the genetic linkage between a sequence change in the gene and an anesthesia mutation isolated in our lab. In the past year we tightened the connection between na and anesthesia by performing a rescue experiment. We first determined the limits of this gene; our collaborator, Dr.Ravi Allada, then constructed a full-length cDNA. This was cloned downstream of transcriptional control signals so that the gene could be turned on by a transcriptional activator which was itself expressed in a tissue-specific manner. With this construct, we find that the halothane sensitivity of an na mutant is restored to normal when the cDNA is expressed pan-neuronally, thereby providing a definitive demonstration of the gene assignment. This experimental system also offers a glimpse into the tissue requirements of na expression. Anesthesia sensitivity is not restored when the construct is driven pan-neuronally at levels much lower than that obtained from the native promoter or when it is driven in glia. However, anesthesia sensitivity is fully restored when the cDNA is driven in the subset of neurons that express the cholinergic marker, Cha. One way to gain insight into the way the na gene contributes to neural performance is to determine the types of cells that express the gene. Our previous work had used immunohistochemistry to show that it is expressed widely but non-uniformly in the brain of adult Drosophila. The staining was generally quite dense but we did note an isolated row of stained cells in the optic lobe lamina; identifying these cells has been a current focus. First, in situ hybridization established that message for na was expressed in the neuronal but not the glial layer of the distal lamina. We then used antibody markers and reporter constructs to compare the expression of various cell types with that of NA. These experiments confirmed that glia were not the source of NA expression. Interestingly, despite the rescue of anesthesia sensitivity by cholinergic expression, the row of NA staining did not overlap with that of a row of cholinergic terminals in the distal lamina: the channel must be expressed in more than one type of neuron. On the positive side, there was strong overlap with antibodies to Frequenin, a putative regulator of intracellular calcium, and with a reporter for LMCs. The latter are large neurons that are almost exclusively postynaptic in the lamina. Since the na gene does not encode a recognizable receptor domain, we conclude that, at least in the lamina, the channel is involved in modulating the postsynaptic response. Another gene identified as influencing sensitivity to halothane is mushroom body defective (mud). Mutations in this gene are highly pleiotropic, showing defects in fertility and neural development; the latter is presumbly responsible for the anesthesia phenotype. In previous work, we mapped this gene adjacent to but distinct from na and showed that it encoded a large coiled-coil protein. To explore the role of Mud in a well-studied tissue, we focused on the female sterile phenotype. We discovered that mutant females laid eggs with normal external anatomy but, although sperm entry was normal, mutant eggs never developed into cellularized embryos. We tracked the defect to the second meiotic division: spindles connecting the two pairs of haploid products were disattached and misaligned. This phenotype indicates a failure in formation or function of the central spindle pole body, a structure that depends on tubulins and centrosomal proteins. Immunostaining of oocytes with anti-Mud showed that is a prominent component of the meiosis II central spindle pole body, implying a direct and non-redundant role for Mud in the function of this organelle. The protein is also found in other places where microtubules are focused, e.g., at the poles of the meiosis I spindle and at mitotic poles in follicle cells. However these structures and the processes which depend on them are unaffected by strong mutations in mud, implying that the contribution of this protein is often redundant. Nevertheless, our results identify a novel component of microtubule-based structures and point the way to exploring a new connection between microtubule function and neural development. Ion channels are crucial for neural function and behavior; in studying them, the uptake of radioactive ions into membrane-delimited structures has been a useful complement to physiological techniques. Although there are numerous such reports from vertebrate systems, there have been very few studies of ion flux in invertebrate systems and, to our knowledge, no studies involving the fruit fly. We have now worked out the experimental details for such an uptake system, with an initial focus on resting calcium ion flux. Heads from adult flies are homogenized in isotonic buffer and, after two rounds of centrifugation, a fraction enriched for membranes is obtained. TEM of these fractions show the presence of resealed vesicular structures ranging in size from 50nm to 500nm. Using conditions of loading and washing that we modified from typical vertebrate protocols, the movement of calcium ions into these microsacs is traced with radiolabel. As revealed by post-uptake osmotic lysis, some of the uptake is genuine flux into microsacs as opposed to mere binding and/or trapping at their surface. The net flux, which shows time- and concentration-dependence, is comparable in magnitude to that reported for vertebrate systems and is strongly depressed by lanthanides. We have begun to apply our new assay by examining the effect of anesthetics on calcium uptake in sacs from wild-type and mutant flies. Regardless of the outcome, our work will provide the field of Drosophila neurobiology with a new tool for assessing an important physiological process.
NIH ExPorter
NIH ExPorter