A century later another surprise: A non-visual behavioral function of the white gene

Abstract only availableDiscovery of the white mutation in Drosophila melanogaster has broadly influenced our understanding of the mechanisms of inheritance. We recently discovered a role of the white gene in memory formation. Thus, the white gene continues to provide insight into basic biological functions. We use two conditioning methods to routinely measure learning and memory in D. melanogaster, the heat-box, and classical olfactory conditioning. In the heat box experiments, white mutant flies' learning performance was notably impaired. However, in olfactory conditioning studies the mutant flies performed the same or better than wild-type flies. This differentiates the molecular mechanisms that support these conditioned behaviors. To better understand the regulatory elements that control white expression, we have initiated a molecular characterization of the white genomic locus. We identified the necessary regulatory elements by defining the deletion in the w1118 null allele. Using PCR methods we found that the deletion is about 7 kb long, and includes 5' regions, exon 1, and part of the first intron. Experiments to determine the sufficient set of regulatory elements for conditioned behavior were initiated. Two results argue that existing genomic transgenes do not contain all regulatory elements. First, mutations that affect eye color have molecular lesions outside a 14 kb genomic transgene. Second, attempted behavioral rescue experiments with this transgene fail. We interpret the failure of the 14 kb transgene to rescue as a consequence of incorrect white expression. Thus, we are creating a genomic construct that is 18 kb long that includes genomic DNA up to the next known gene. These approaches should define the regulatory regions necessary and sufficient for behaviorally important white expression.NSF-REU Program in Biological Sciences & Biochemistr

Development of connectivity in a motoneuronal network in Drosophila larvae.

BACKGROUND: Much of our understanding of how neural networks develop is based on studies of sensory systems, revealing often highly stereotyped patterns of connections, particularly as these diverge from the presynaptic terminals of sensory neurons. We know considerably less about the wiring strategies of motor networks, where connections converge onto the dendrites of motoneurons. Here, we investigated patterns of synaptic connections between identified motoneurons with sensory neurons and interneurons in the motor network of the Drosophila larva and how these change as it develops. RESULTS: We find that as animals grow, motoneurons increase the number of synapses with existing presynaptic partners. Different motoneurons form characteristic cell-type-specific patterns of connections. At the same time, there is considerable variability in the number of synapses formed on motoneuron dendrites, which contrasts with the stereotypy reported for presynaptic terminals of sensory neurons. Where two motoneurons of the same cell type contact a common interneuron partner, each postsynaptic cell can arrive at a different connectivity outcome. Experimentally changing the positioning of motoneuron dendrites shows that the geography of dendritic arbors in relation to presynaptic partner terminals is an important determinant in shaping patterns of connectivity. CONCLUSIONS: In the Drosophila larval motor network, the sets of connections that form between identified neurons manifest an unexpected level of variability. Synapse number and the likelihood of forming connections appear to be regulated on a cell-by-cell basis, determined primarily by the postsynaptic dendrites of motoneuron terminals.L.C. was supported by a Fyssen Foundation post-doctoral fellowship. This work was supported by a Biotechnology and Biological Sciences Research Council (UK) grant (BB/I022414/1) to M.L., a Wellcome Trust Programme Grant (WT075934) to Michael Bate and M.L., a Grass Foundation fellowship to A.S.M., and a Sir Isaac Newton Trust grant to A.S.M. and M.L. The work benefited from facilities supported by a Wellcome Trust Equipment Grant (WT079204) and contributions by the Sir Isaac Newton Trust in Cambridge.This paper was originally published in Current Biology (Couton L, Mauss AS, Yunusov T, Diegelmann S, Evers JF, Landgraf M, Current Biology 2015, 25, 568–576, doi:10.1016/j.cub.2014.12.056

Kajian Potensi Energi Arus Laut Sebagai Energi Alternatif Untuk Pembangkit Listrik Di Perarian Selat Lembeh, Sulawesi Utara

Kebutuhan akan energi listrik terus mengalami peningkatan dan sumber energi utamanya adalah energi konvensional yang ketersediannya terbatas di alam, untuk itu diperlukan adanya pencarian sumber energi lain yang terbarukan. Selat Lembeh merupakan wilayah perairan sempit yang berada di antara Laut Maluku yang dipengaruhi oleh massa air dari Pasifik dan Laut Sulawesi yang dipengaruhi oleh massa air dari Hindia. Penelitian ini bertujuan untuk mengetahui karakteristik arus laut serta mengetahui potensi arus laut sebagai sumber energi alternatif pembangkit listrik. Pengolahan data terdiri dari analisa data arus dan pasang surut, pemodelan numerik, dan menghitung estimasi rapat daya. Penelitian ini menggunakan metode kuantitatif dan penentuan lokasi dengan sampling area. Berdasarkan hasil penelitian, rapat daya terbesar yang dihasilkan yaitu pada musim barat, sebesar 120,02 kW/m2

Genetic dissociation of acquisition and memory strength in the heat-box spatial learning paradigm in Drosophila

Memories can have different strengths, largely dependent on the intensity of reinforcers encountered. The relationship between reinforcement and memory strength is evident in asymptotic memory curves, with the level of the asymptote related to the intensity of the reinforcer. Although this is likely a fundamental property of memory formation, relatively little is known of how memory strength is determined. Memory performance at different levels in Drosophila can be measured in an operant heat-box conditioning paradigm. In this spatial learning paradigm, flies learn and remember to avoid one-half of a dark chamber associated with a temperature outside of the preferred range. The reinforcement temperature has a strong effect on the level of learning in wild-type flies, with higher temperatures inducing stronger memories. Additionally, two mutations alter memory-acquisition curves, either changing acquisition rate or asymptotic memory level. The rutabaga mutation, affecting a type-1 adenylyl cyclase, decreases the acquisition rate. In contrast, the white mutation, modifying an ABC transporter, limits asymptotic memory. The white mutation does not negatively affect classical olfactory conditioning but actually improves performance at low reinforcement levels. Thus, memory acquisition/memory strength and classical olfactory/operant spatial memories can be genetically dissociated. A conceptual model of operant conditioning and the levels at which rutabaga and white influence conditioning is proposed

Embryonic Origin of Olfactory Circuitry in <em>Drosophila</em>: Contact and Activity-Mediated Interactions Pattern Connectivity in the Antennal Lobe

<div><p>Olfactory neuropiles across different phyla organize into glomerular structures where afferents from a single olfactory receptor class synapse with uniglomerular projecting interneurons. In adult <em>Drosophila</em>, olfactory projection interneurons, partially instructed by the larval olfactory system laid down during embryogenesis, pattern the developing antennal lobe prior to the ingrowth of afferents. In vertebrates it is the afferents that initiate and regulate the development of the first olfactory neuropile. Here we investigate for the first time the embryonic assembly of the <em>Drosophila</em> olfactory network. We use dye injection and genetic labelling to show that during embryogenesis, afferent ingrowth pioneers the development of the olfactory lobe. With a combination of laser ablation experiments and electrophysiological recording from living embryos, we show that olfactory lobe development depends sequentially on contact-mediated and activity-dependent interactions and reveal an unpredicted degree of similarity between the olfactory system development of vertebrates and that of the <em>Drosophila</em> embryo. Our electrophysiological investigation is also the first systematic study of the onset and developmental maturation of normal patterns of spontaneous activity in olfactory sensory neurons, and we uncover some of the mechanisms regulating its dynamics. We find that as development proceeds, activity patterns change, in a way that favours information transfer, and that this change is in part driven by the expression of olfactory receptors. Our findings show an unexpected similarity between the early development of olfactory networks in <em>Drosophila</em> and vertebrates and demonstrate developmental mechanisms that can lead to an improved coding capacity in olfactory neurons.</p> </div

Developmental changes in OSN spontaneous activity patterns depend upon OR function.

<p>(A) Five-minute representative trace recordings overlaid with individual OSN units as identified with Spikepy. Top trace control 15 h AEL, middle trace control first instar larvae, and bottom trace Orco mutant first instar larva. OSNs at early stages (15 h and 16 h AEL) often fire in bursts, unlike OSNs at later stages (18.5 h and first instar larvae). OSNs in Orco mutants show throughout development and even in first instar larva stages bursty activity patterns. (B) Coefficient of variation of the interspike intervals (CV) at different developmental stages in controls (blue) and <i>Orco</i> mutants (red). CV is significantly higher in 16 h AEL embryos and first instar <i>Orco</i> mutants than in controls, which demonstrates that at least part of the reduction in spike train variability as development proceeds is due to OR expression. (C) Firing rate at different developmental stages in controls (blue) and <i>Orco</i> mutants (red). The firing rate at 16 h AEL is similar in <i>Orco</i> mutants and controls, but there is a significant difference between controls and <i>Orco</i> mutants in first instar larvae. (B–C) Error bars represent SEM. A single asterisk (*) indicates <i>p</i>≤0.05. <i>N.S.</i> indicates <i>p</i>>0.05.</p

Recording from OSNs during development.

<p>(A–D) OSN recording technique. (A) Schematic showing the way in which the recordings were performed. Embryos were dissected to expose the DOG, a suction electrode was placed in contact with the DOG, and suction applied until a seal was made and action potentials were visible on the recording. (B) Example recording trace from a first instar larvae expressing ChR2 in OSNs (<i>Orco-Gal4;UAS-CD8GFP/UAS-ChR2</i>). The blue bar at the bottom indicates the period of time when the UV light was turned on to activate ChR2. In this particular recording three different units were identified using Spikepy. (C) Experimental trace from (B) showing the shape of individual spikes. (D) Clusters separated with Spikepy. Average spike shapes are plotted with their standard deviation at the top left. The other three panels are an overlay of a maximum of 250 individual spikes for each cluster, with the average spike shape plotted in black. (E) Peri-stimulus time histogram (PSTH), Raster plots, and heat maps showing the response to UV light stimulation of 16 units recorded in three different larvae in response to five different stimuli presentations in <i>Orco-Gal4;UAS-CD8GFP/UAS-ChR2</i> animals. PSTH (top panel) shows the average firing rate of the 16 units in 200 ms bins. Raster plots (middle panel) show the raw data for each of the 16 units. Heat maps (bottom panel) show for each unit the firing rate in 1 s bins. For each unit the average firing rate in the 10 s before light stimulation was significantly lower (<i>p</i><0.001) than the average firing rate in the 10 s during the stimulation. (F) Firing rate, example trace, and example spike shape of OSN recordings at four different developmental stages.</p

Development of OSNs and PNs between 16 h and 21 h AEL.

<p>(A–I) Development of OSNs between 16 h and 21 h AEL as revealed by dye injection. (A) Schematic of the injection procedure; as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001400#pbio-1001400-g001" target="_blank">Figure 1</a>, the square indicates the area shown in the confocal images. Columns show injections at different developmental times. The two rows (B–E) and (F–I) each represent a different example of OSN injection. All figures are at the same scale. Terminals at 21 h AEL (E and I) appear more compact than at earlier stages. Morphologies found at 18.5 h AEL (two examples in D and H) are especially variable, with some showing long filopodia (H) or broad regions of occupancy (D). (J–N′) Development of PNs between 16 h and 21 h AEL as revealed by dye injection. (J) as (A). (K–N′) The two rows show the AL and the MB and LH regions for a representative PN injection at each developmental stage. Red brackets indicate the PN dendrites.</p

PNs require presynaptic innervation during development for their survival.

<p>(A) Schematic of the experimental approach. OSNs on one side were laser ablated at stage 14, before OSN axons contact PNs. Animals were left to develop and hatch and PN morphology was examined in mid-first instar larvae. (B) Anterior part of a first instar larva in which OSNs have been unilaterally ablated. All sensory neurons are labelled using <i>PO163-Gal4;UAS-CD8GFP</i>, including taste neurons that appear as the most anteriorly located cluster of PO163 positive cells on both sides. OSNs labelled both with PO163 in green and with α-Orco antibody (magenta) are present on the control side (bottom) but not in the ablated side (top). (C) Quantification of the OSN ablation experiments. Number of PNs in the control (blue dots) and OSN ablated side (pink dots) for each animal are linked by a grey line. (D–D″) Z projection of a brain in which OSNs were unilaterally ablated (right side). PNs visible with <i>GH146-QF;QUAS-mTomato</i> on the control side are missing on the ablated side. The AL, visualized with Nc82 antibody staining in the control side (dashed line), is absent on the ablated side. (E–E″) Z projection of a brain in which OSNs were unilaterally ablated (right side). PNs (<i>GH146-QF;QUAS-mTomato</i>) can be seen innervating the AL in the control side. Some PN of the ablated side are missing, while surviving ones are found innervating the subesophageal ganglion (SOG). The AL, visualized in the control side as a gap with DAPI staining, is missing on the ablated side (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001400#pbio.1001400.s002" target="_blank">Figure S2</a>).</p

Development of OSNs and PNs between 10 h and 14 h AEL.

<p>(A–F) OSN and PN development as followed with <i>acj6-Gal4:UAS-CD8GFP</i> between 10 h and 12 h AEL, before any contact is made. In the schematics (A–C, G, and K) OSNs are yellow and PNs green. The black square in the schematics indicates the region that is being visualized in the confocal images. In the confocal stacks (E–F, J, L, and M) OSN axons are indicated with an arrow and PN cell bodies with an asterisk. (A–B and D–E) OSNs are born in direct contact with the brain (dashed line) and send short axonal projections into the brain from the very early stages (arrow in E). (F) At early stage 15 (11.2–12 h AEL) OSNs and PNs have not yet contacted each other, although they are within filopodial reach. (G–J) OSN dye injections at 13 h AEL. (G) Schematic showing the injection procedure and marking the region shown in the confocal stacks (H–J) with a black square. (H) Injection of two OSNs with different dyes showing growth cones in both OSNs. (I) Schematic showing the position of OSN axons and PNs. Both components are in red, as in the confocal pictures, showing GFP staining expressed on the acj6-Gal4 pattern. One OSN is green showing filopodia, which represents a single injected OSN as in (J and J′). OSN axons enter the brain in a ventral position respective to PN cell bodies. PN axons run ventrally towards the place of OSN terminals and then turn dorsally towards higher brain centres, making thus a U shape. Different PNs turn their axons at different dorso-ventral depths, and OSN axonal growth cones contact even the most dorsally turning PN axons. The line in (I) marked with the letter J′ represents the confocal z stack shown in (J′). (J) is a confocal z projection of what is represented in (I), while (J′) is a single z stack at the position indicated in (I). From here onwards letters with apostrophe (‘) indicate images taken from the same animal. (K–M) PN dye injections at 14 h AEL. (K) Schematic showing the injection procedure and marking the regions (AL, and MB&LH) shown in the confocal stacks (L–M). (L–M) In each example a single PN was injected (green), and all OSNs and PNs are labelled in red with <i>acj6-Gal4;UAS-CD8GFP</i>. The AL and the MB and LH regions are shown for each injected PN. At 14 h AEL PNs have already extended axons towards higher brain centres, but they have not yet sprouted dendrites at the AL. The axons in the MB and LH are still immature with growth cones. Arrows indicate OSN axons, and brackets indicate the region where the AL is forming and therefore the region where PNs will sprout dendrites.</p