A Genetic Screen for Olfactory Habituation Mutations in <em>Drosophila</em>: Analysis of Novel <em>Foraging</em> Alleles and an Underlying Neural Circuit

<div><p>Habituation is a form of non-associative learning that enables animals to reduce their reaction to repeated harmless stimuli. When exposed to ethanol vapor, <em>Drosophila</em> show an olfactory-mediated startle response characterized by a transient increase in locomotor activity. Upon repeated exposures, this olfactory startle attenuates with the characteristics of habituation. Here we describe the results of a genetic screen to identify olfactory startle habituation (OSH) mutants. One mutation is a transcript specific allele of <em>foraging</em> (<em>for</em>) encoding a cGMP-dependent kinase. We show this allele of <em>for</em> reduces expression of a <em>for-T1</em> isoform expressed in the head and functions normally to inhibit OSH. We localize <em>for-T1</em> function to a limited set of neurons that include olfactory receptor neurons (ORNs) and the mushroom body (MB). Overexpression of <em>for-T1</em> in ORNs inhibits OSH, an effect also seen upon synaptic silencing of the ORNs; <em>for-T1</em> may therefore function in ORNs to decrease synaptic release upon repeated exposure to ethanol vapor. Overall, this work contributes to our understanding of the genes and neurons underlying olfactory habituation in <em>Drosophila</em>.</p> </div

<i>for</i> alleles enhance olfactory startle habituation.

<p><b>A)</b><i>for^11.247</i> and <i>for²⁶¹⁴</i> show enhanced OSH. A reduction of distance traveled (compared to <i>Ctrl</i>) was seen in both alleles at pulse 2 (p<0.01), 3 and 4 (p<0.001;(n = 12). <b>B)</b><i>for^11.247</i> and <i>for²⁶¹⁴</i> have an enhanced HI (indicating more habituation). Significant difference was seen between <i>Ctrl</i> and <i>for^11.247</i> or <i>for²⁶¹⁴</i> (p<0.001; n = 12). <b>C)</b> Compared to <i>Ctrl,</i> the precise excision, <i>for^Δ11.247,</i> had a normal HI (p>0.05; n = 6). Unless indicated, significance was established by a One-Way-ANOVA with post-hoc Newmans-Keuls tests in all figures (*p<0.05, **p<0.01, ***p<0.001).</p

Analysis of neuronal circuitry implicated in olfactory startle habituation.

<p><b>A)</b> Blocking synaptic activity in <i>for^11.247-GAL4</i> neurons reduces OSH. Heterozygous <i>for^11.247-GAL4</i> flies expressing tetanus toxin (<i>TeTx</i>) had reduced OSH. Significant differences were seen between <i>for^11.247-GAL4</i><b><i>/</i></b><i>+</i> or <i>UAS-TeTx/+</i> and <i>for^11.247-GAL4/+;UAS-TeTx/+</i> (p<0.001; n = 9). No significant difference was seen in flies expressing inactive <i>TeTx</i> (<i>TeTxⁱⁿ</i>) with <i>for^11.247-GAL4</i><b><i>/</i></b><i>+</i> (p>0.05; n = 9). <b>B)</b> Synaptic silencing of ORNs inhibits OSH. Expressing <i>UAS-TeTx</i> with <i>Orco-GAL4</i> significantly reduced OSH. Differences were observed between <i>Orco-GAL4/+</i> or <i>UAS-TeTx/+</i> and <i>Orco-GAL4/+;UAS-TeTx/+</i> (p<0.01; n = 6). No effect was seen upon expressing <i>UAS-TeTxⁱⁿ</i> with <i>Orco-GAL4/+</i> (p>0.05; n = 9). <b>C)</b><i>for-T1</i> overexpression in ORNs inhibits OSH. Expressing <i>UAS-for-T1</i> with <i>Orco-GAL4</i>, but not with MB driver <i>OK107-GAL4</i>, reduced OSH. Significant differences were observed between <i>Orco-GAL4/+</i> or <i>UAS-for-T1/+</i> and <i>Orco-GAL4/+</i>;<i>UAS-for-T1/+</i> (p<0.001; n = 14), but not between controls and <i>OK107-GAL4/+;UAS-for-T1/+</i> (p>0.05; n = 8).</p

Expression pattern of <i>for^11.247-GAL4</i>.

<p><b>A, B)</b> Expression of <i>for^11.247-GAL4/+;UAS-GFP/+</i> flies. <b>A)</b> In the antenna, GFP (green) was expressed in the arista (AR) and a sub-population of olfactory receptor neurons (ORNs) in the third antennal segment. <b>B)</b> In the CNS, GFP was expressed in specific glomeruli in the antennal lobe (AL), par intercrebalis (PI) neurons, and weakly in the mushroom body (MB) and lateral cells (LC). <b>C)</b> In <i>for^11.247-GAL4;UAS-GFP/+</i> flies strong GFP expression was seen in MB, PIs, LC and sub-oesophageal ganglion (SOG), as well as the ventral lateral neurons (LN_vs), the giant dorsal interneuron (DGI), parts of the antennal lobe (AL) and ellipsoid body (EB). <b>D)</b> Higher magnification of <i>for^11.247-GAL4;UAS-GFP/+</i> flies showing partial co-localization with a FOR antibody (red) in the MB and LC, but not in DGI.</p

Molecular characterization of <i>for</i> alleles.

<p><b>A)</b> Schematic of the <i>for</i> transcription unit, with insertion sites of <i>for^11.247</i> and <i>for²⁶¹⁴</i>. Blue bars represent translation start/stop sites, grey bars represent region probed for <i>for-T1/T3</i> and <i>for-T2</i> transcripts. The 3 major <i>for</i> isoforms, collectively called <i>for-T1/T2/T3</i> have a total of nine splice forms, all encoding a common kinase domain at the 3′ end. FOR-T1 is a 1088 amino acid (aa) protein encoded by <i>for-RA/RH</i>/<i>RI</i>, FOR-T2 is a 894 aa protein encoded by <i>for-RC/RD/RF/RG/RK</i>, and FOR-T3 is a 742 aa protein encoded by <i>for-RB</i>. <b>B)</b> Northern blot of adult fly mRNA using probes specific to <i>for-T1/T3</i> or <i>for-T2</i> transcripts. <b>B, top panel)</b> In the control strain (<i>wBerlin</i>) we detected two bands with the <i>for-T1/T3</i> probe. Based on its size, the upper, more intense, band corresponds to <i>for-T1</i> transcripts, while the lower, less intense band, to the <i>for-T3</i> transcript. Compared to <i>wBerlin</i> and <i>for^Δ11.247</i>(a precise excision of <i>for^11.247</i>) a reduced intensity of <i>for-T1</i>, but not <i>for-T3</i> transcripts, was seen in <i>for^11.247</i> and <i>for²⁶¹⁴</i>. <b>B, middle panel)</b> Using a <i>for-T2</i> probe we detected no differences in levels of <i>for-T2</i> transcripts in either <i>for^11.247</i> or <i>for²⁶¹⁴</i>. <b>B, bottom panel)</b> A <i>tubulin</i> probe was used to compare total mRNA levels. <b>C)</b> Quantification of Northern Blot showing reduced <i>for-T1,</i> but not <i>for-T2</i> or <i>for-T3,</i> in <i>for^11.247</i> and <i>for²⁶¹⁴</i>. Levels were calculated as a ratio between <i>for</i> and <i>tubulin</i> band intensity. <b>D)</b> Quantification and representative Western blot of extracts from adult heads analyzed with an antibody that recognizes FOR-T1. Compared to controls, we saw a reduction of FOR-T1 in both <i>for^11.247</i> and <i>for²⁶¹⁴</i> (p<0.001; n = 3).</p

OSH mutants isolated from genetic screen.

<p>Initial startle: distance moved per fly during first 30-second startle. Arrows represent direction of the P element. Molecular classes: cell signalling (CS), DNA binding (DB), RNA binding (RB), cell adhesion (CA), cytoskeleton (CY), metabolism (M), proteases (PP), annotated genes unknown molecular function without homology (CG), and those annotated genes with conserved structural domains (CGd). Information current to FlyBase release: FB2012_05, Sept 7th, 2012.</p

<i>for^11.247 -GAL4</i> flies expressing <i>for-T1</i> have normal olfactory startle habituation.

<p><b>A)</b> Habituation profile of functional rescue of <i>for^11.247-GAL4</i> by expressing <i>UAS-for-T1</i>. No significant difference in distances travelled were seen between <i>for^11.247-GAL4</i>;<i>UAS-for-T1/+</i> and either <i>Ctrl</i> or <i>UAS-for-T1/+.</i> At pulse 2, 3 and 4, a significant difference was only seen between <i>for^11.247–GAL4</i>;<i>UAS-for-T1/+</i> and <i>for^11.247</i> (p<0.01; n = 12). <b>B)</b> HI of <i>for</i>^11.247 rescue. No significant differences were seen between <i>for^11.247-GAL4</i>;<i>UAS-for-T1/+</i> and either <i>Ctrl</i> or <i>UAS-for-T1/+</i>, but were observed between <i>for^11.247-GAL4</i>;<i>UAS-for-T1/+</i> and <i>for^11.247</i> (p<0.01; n = 12).</p