Identification and distribution of dietary precursors of the Drosophila visual pigment chromophore: analysis of carotenoids in wild type and ninaD mutants by HPLC.

A dietary source of retinoid or carotenoid has been shown to be necessary for the biosynthesis of functional visual pigment in flies. In the present study, the larvae or adults of Drosophila melanogaster were administered specific carotenoid-containing diets and high performance liquid chromatography was used to identify and quantify the carotenoids in extracts of wild type and ninaD visual mutant flies. When beta-carotene was fed to larvae, wild type flies were shown to hydroxylate this molecule and to accumulate zeaxanthin and a small amount of beta-cryptoxanthin. Zeaxanthin content was found to increase throughout development and was a major carotenoid peak detected in the adult fly. Carotenoids were twice as effective at mediating zeaxanthin accumulation when provided to larvae versus adults. In the ninaD mutant, zeaxanthin content was shown to be specifically and significantly altered compared to wild type, and was ineffective at mediating visual pigment synthesis when provided to both larval and adult mutant flies. It is proposed that zeaxanthin is the larval storage form for subsequent visual pigment chromophore biosynthesis during pupation, that zeaxanthin or beta-crytoxanthin is the immediate precursor for light-independent chromophore synthesis in the adult, and that the ninaD mutant is defective in this pathway.

Effect of alpha2-adrenoceptor stimulation on isolated canine Purkinje fiber contraction.

We have recently identified the presence of postjunctional alpha2-adrenoceptors in canine Purkinje fibers. In this study, we examined the effects of alpha2-adrenoceptor stimulation on the contraction strength of isolated Purkinje fibers. Exposure to the alpha2-adrenoceptor specific agonist and antagonist, UK 14,304 (5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) and yohimbine (17-hydroxyyohimban-16-carboxylic acid methyl ester hydrochloride) alone at 0.1 microM respectively, did not produce any significant effect on Purkinje contraction strength. Purkinje contraction strength was augmented by isoproterenol (0.1 microM), forskolin (0.1 microM), or 8-bromo-adenosine cyclic 2',3'-monophosphate (8-bromo-cAMP, 10 microM). UK 14,304 significantly reversed the effects of isoproterenol and forskolin but not those of 8-bromo-cAMP on Purkinje contraction strength. After incubation with pertussis toxin, the positive inotropic effect of forskolin on Purkinje contraction strength remained intact, but the forskolin effect could no longer be reversed by UK 14,304. These results suggest that the postjunctional alpha2-adrenoceptors in canine Purkinje fibers are coupled to a pertussis toxin-sensitive G protein, probably Gi. Stimulation of the alpha2-adrenoceptor antagonizes the effect of beta-adrenoceptor stimulation on Purkinje contraction strength in an accentuated antagonism manner.

Dissociation of photoreceptors from whole heads of the fruit fly, Drosophila melanogaster.

Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple "smash" technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.

On the interpretation of voltage noise in small cells.

Noise analysis is a powerful technique for studying membrane conductance mechanisms, but it can be applied straightforwardly only to current noise recorded under voltage clamp. Many small cells, however, such as some sensory receptors and interneurons, are not suited for voltage clamping. In such cases it is of great interest to be able to interpret recordings of voltage noise. Voltage noise must be corrected for the effects of: (1) changes in membrane potential, (2) the cells' input impedance, and (3) noise contributed by conductances other than the one of interest. This paper describes in detail the factors--including in particular voltage-dependent K+-conductances--that affect voltage-noise recordings, and describes how these factors may be measured and corrected for in practice. A novel method allows membrane impedance to be measured at the same time as voltage noise. An example is given, showing how this approach may be applied to voltage noise from an insect photoreceptor.

An improved microelectrode resistance meter.

A device is described for measuring the resistance of micropipette electrodes. The useful range of electrode resistances that it can measure is 100 k omega to 1 G omega. It is more convenient to use than previously described or commercially available meters, especially for very high-resistance electrodes. Resistance of even the finest microelectrodes can be measured accurately while their tips are inserted by hand into a test solution. This eliminates the need for special holders, speeding and simplifying the screening of large numbers of electrodes. Test solutions are stored in interchangeable reservoirs, making it easy to characterize the resistance of an electrode in solutions of different resistivity. Test solutions can also be capped and removed when not in use to prevent evaporation. To protect very high-resistance electrodes from damage during measurement, the measuring current is low (only 300 pA on the highest resistance range) and the test voltage across the electrode is limited to +/- 1 V.