Figure 1.
Drosophila habituates to an ethanol-induced olfactory startle.
A) The olfactory startle attenuates with the characteristics of habituation. Ethanol naïve flies exposed to a 30-sec pulse of ethanol vapor showed an olfactory mediated startle response, characterized by a transient increase in locomotor activity. In subsequent pulses of ethanol vapor the olfactory startle increasingly attenuated. To demonstrate habituation (and not sensory adaptation or fatigue) flies were dishabituated (arrow) with a mechanical stimulus before the final pulse of ethanol. B) The frequency distribution of habituation indices (HI) of all strains tested in the genetic screen. The habituation index was calculated as a ratio of the total movement in the fourth pulse (P4) and first pulse (P1) (HI = 1-P4/P1; a HI of 1 indicates complete habituation while a HI of 0 or lower indicate no habituation or sensitization, respectively). A frequency distribution of the habituation indices showed a near normal distribution with a mean of 0.58, median of 0.64 and mode of 0.65. A HI of 0.8 or higher was used as the cut-off for enhanced habituation, while a HI of 0.2 or lower was the cut-off for failure to habituate normally. Strains with low and high HIs were selected for further analysis.
Table 1.
OSH mutants isolated from genetic screen.
Figure 2.
for alleles enhance olfactory startle habituation.
A) for11.247 and for2614 show enhanced OSH. A reduction of distance traveled (compared to Ctrl) was seen in both alleles at pulse 2 (p<0.01), 3 and 4 (p<0.001;(n = 12). B) for11.247 and for2614 have an enhanced HI (indicating more habituation). Significant difference was seen between Ctrl and for11.247 or for2614 (p<0.001; n = 12). C) Compared to Ctrl, the precise excision, forΔ11.247, 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).
Figure 3.
Molecular characterization of for alleles.
A) Schematic of the for transcription unit, with insertion sites of for11.247 and for2614. Blue bars represent translation start/stop sites, grey bars represent region probed for for-T1/T3 and for-T2 transcripts. The 3 major for isoforms, collectively called for-T1/T2/T3 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 for-RA/RH/RI, FOR-T2 is a 894 aa protein encoded by for-RC/RD/RF/RG/RK, and FOR-T3 is a 742 aa protein encoded by for-RB. B) Northern blot of adult fly mRNA using probes specific to for-T1/T3 or for-T2 transcripts. B, top panel) In the control strain (wBerlin) we detected two bands with the for-T1/T3 probe. Based on its size, the upper, more intense, band corresponds to for-T1 transcripts, while the lower, less intense band, to the for-T3 transcript. Compared to wBerlin and forΔ11.247(a precise excision of for11.247) a reduced intensity of for-T1, but not for-T3 transcripts, was seen in for11.247 and for2614. B, middle panel) Using a for-T2 probe we detected no differences in levels of for-T2 transcripts in either for11.247 or for2614. B, bottom panel) A tubulin probe was used to compare total mRNA levels. C) Quantification of Northern Blot showing reduced for-T1, but not for-T2 or for-T3, in for11.247 and for2614. Levels were calculated as a ratio between for and tubulin band intensity. D) 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 for11.247 and for2614 (p<0.001; n = 3).
Figure 4.
Expression pattern of for11.247-GAL4.
A, B) Expression of for11.247-GAL4/+;UAS-GFP/+ flies. A) 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) 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). C) In for11.247-GAL4;UAS-GFP/+ flies strong GFP expression was seen in MB, PIs, LC and sub-oesophageal ganglion (SOG), as well as the ventral lateral neurons (LNvs), the giant dorsal interneuron (DGI), parts of the antennal lobe (AL) and ellipsoid body (EB). D) Higher magnification of for11.247-GAL4;UAS-GFP/+ flies showing partial co-localization with a FOR antibody (red) in the MB and LC, but not in DGI.
Figure 5.
for11.247 -GAL4 flies expressing for-T1 have normal olfactory startle habituation.
A) Habituation profile of functional rescue of for11.247-GAL4 by expressing UAS-for-T1. No significant difference in distances travelled were seen between for11.247-GAL4;UAS-for-T1/+ and either Ctrl or UAS-for-T1/+. At pulse 2, 3 and 4, a significant difference was only seen between for11.247–GAL4;UAS-for-T1/+ and for11.247 (p<0.01; n = 12). B) HI of for11.247 rescue. No significant differences were seen between for11.247-GAL4;UAS-for-T1/+ and either Ctrl or UAS-for-T1/+, but were observed between for11.247-GAL4;UAS-for-T1/+ and for11.247 (p<0.01; n = 12).
Figure 6.
Analysis of neuronal circuitry implicated in olfactory startle habituation.
A) Blocking synaptic activity in for11.247-GAL4 neurons reduces OSH. Heterozygous for11.247-GAL4 flies expressing tetanus toxin (TeTx) had reduced OSH. Significant differences were seen between for11.247-GAL4/+ or UAS-TeTx/+ and for11.247-GAL4/+;UAS-TeTx/+ (p<0.001; n = 9). No significant difference was seen in flies expressing inactive TeTx (TeTxin) with for11.247-GAL4/+ (p>0.05; n = 9). B) Synaptic silencing of ORNs inhibits OSH. Expressing UAS-TeTx with Orco-GAL4 significantly reduced OSH. Differences were observed between Orco-GAL4/+ or UAS-TeTx/+ and Orco-GAL4/+;UAS-TeTx/+ (p<0.01; n = 6). No effect was seen upon expressing UAS-TeTxin with Orco-GAL4/+ (p>0.05; n = 9). C) for-T1 overexpression in ORNs inhibits OSH. Expressing UAS-for-T1 with Orco-GAL4, but not with MB driver OK107-GAL4, reduced OSH. Significant differences were observed between Orco-GAL4/+ or UAS-for-T1/+ and Orco-GAL4/+;UAS-for-T1/+ (p<0.001; n = 14), but not between controls and OK107-GAL4/+;UAS-for-T1/+ (p>0.05; n = 8).