Cocaine Responses Are Not a Circadian Output and <i>pdf</i> Mutants Show Wild-Type Cocaine Sensitivity

<div><p>(A) Cocaine responses do not vary with the circadian clock. Control <i>(EP1631)</i> flies were raised under LD conditions and assayed for cocaine phenotypes in the crackometer at the indicated Zeitgeber (ZT) times. One-way ANOVA revealed no significant effect of time of day (<i>F</i> = 0.53, <i>p</i> = 0.82, <i>n</i> = 32).</p> <p>(B) Flies lacking the neuropeptide PDF <i>(pdf⁰¹)</i> display normal cocaine sensitivity. <i>pdf⁰¹</i> homozygotes <i>(pdf⁰¹/pdf⁰¹)</i> and <i>pdf⁰¹</i> hemizygotes <i>(pdf⁰¹/Df)</i> showed wild-type responses to cocaine in the crackometer. Individual pairwise comparisons using Student's <i>t</i>-tests revealed no significant differences between control (+/+ and +/<i>Df</i>) and <i>pdf</i> mutant genotypes (<i>p</i> = 0.69, <i>p</i> = 0.97, <i>n</i> = 6–8 experiments)</p></div

Molecular Structure of the <i>Lmo</i> Locus

<div><p>(A) A genomic map of the <i>Lmo</i> locus. Three different first exons can be utilized, forming the basis for three alternative transcripts. Exon RA-1 is separated from the alternative start sites RB-1 and RC-1 by a large (∼30 kb) intron. <i>EP1306, EP1383,</i> and <i>pdrm</i> carry insertions 25, 73, and 91 bp, respectively, upstream of the exon RA-1 transcriptional start site. Arrows within the EP elements refer to the orientation of the insertion and the expected direction of inducible expression via UAS sites contained within the EP element. <i>Bx</i> alleles are insertions of natural transposons into the 3′ UTR of the <i>Lmo</i> gene that have been shown to stabilize <i>Lmo</i> transcript (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020408#pbio-0020408-Shoresh1" target="_blank">Shoresh et al. 1998</a>). Protein-coding exons are shaded.</p> <p>(B) Expression of the <i>Lmo</i> RA transcript is enriched in <i>Drosophila</i> heads, and is reduced in the <i>EP1306</i> mutant. RNA was isolated from heads and bodies; after cDNA synthesis, quantitative RT-PCR was performed using primers specific to the RA transcript of <i>Lmo</i> in addition to primers to a reference transcript, the ribosomal protein <i>rp49</i>. Relative abundance is expressed as fold increase over control <i>(EP1631)</i> body mRNA. No detectable amplification was seen in RNase-treated controls (data not shown). Error bars represent standard error of the mean. Asterisk denotes significant difference from control (Student's paired <i>t</i>-test assuming equal variance; <i>p <</i> 0.001, <i>n</i> = 3).</p></div

<i>Lmo</i> Loss-of-Function Mutants Show Increased Sensitivity to Cocaine

<div><p>(A) Cocaine phenotypes of various <i>Lmo</i> mutants. Male flies hemizygous for the indicated <i>Lmo</i> alleles (and their appropriate genetic controls) were exposed to 150 μg of cocaine and tested in the crackometer as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020408#s4" target="_blank">Materials and Methods</a>. Compared to their control (Ctl-1), <i>EP1383</i> (<i>p <</i> 0.02) and <i>EP1306</i> (<i>p <</i> 0.001) flies show significantly increased sensitivity to cocaine. Similarly, compared to their respective controls, <i>pdrm</i> and <i>hdp</i> flies are significantly more sensitive to cocaine (<i>p <</i> 0.001). Asterisks denote significant differences from controls (Student's paired <i>t</i>-test assuming equal variance); <i>n</i> = 20 experiments.</p> <p>(B) Cocaine dose–response. <i>EP1306</i> flies (filled squares) and <i>pdrm</i> flies (filled circles) and their respective controls were exposed to the indicated doses of cocaine. At each dose, the responses of <i>EP1306</i> and <i>pdrm</i> flies are significantly higher than their controls (<i>p <</i> 0.001, <i>n</i> = 16–20 experiments).</p> <p>(C) <i>EP1306</i> flies show alterations in cocaine-induced locomotor patterns of activity. Flies were exposed to 0, 75, or 100 μg of cocaine, as indicated, for 1 min. Representative traces shown correspond to 30 s of recorded activity of about ten flies starting 1 min after the end of cocaine exposure (<i>n</i> ≥ 4). Top panels show response of control flies to indicated amounts of cocaine; bottom panels show activity of <i>EP1306</i> flies after cocaine administration. Ctl-1 is <i>EP1631,</i> Ctl-2 is P[GAL4] line <i>8.142,</i> and Ctl-3 is <i>w¹¹¹⁸</i>.</p></div

Wild-Type <i>Lmo</i> Is Required for Robust Circadian Rhythms of Locomotor Activity

<div><p>Locomotor activity of control (Ctl-1 and Ctl-2) and <i>Lmo</i> mutant (<i>EP1383, EP1306, hdp^R26,</i> and <i>hdp^rev83</i>) flies was recorded in constant darkness as previously described (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020408#pbio-0020408-Nitabach1" target="_blank">Nitabach et al. 2002</a>).</p> <p>(A) Representative actograms of control and <i>Lmo</i> mutants. Control flies show robust circadian rhythms with clear distinctions between activity during the subjective day and inactivity during the subjective night. The pattern of the <i>EP1306</i> and <i>hdp^rev83</i> mutants was more stochastic.</p> <p>(B) Graph showing the proportion of strongly rhythmic (white), weakly rhythmic (gray), and arrhythmic (black) flies for each genotype. Most control flies had strong rhythms (28/30 for Ctl-1 and 27/29 for Ctl-2) while <i>Lmo</i> mutants formed a series with an increasing fraction of the flies arrhythmic. For example, 13/26 <i>hdp^rev83</i> flies were arrhythmic, with the other 13 all having weak rhythms. The power of the rhythm was used to estimate the strength of the activity rhythm, with a power of 300 or more classed as a strong rhythm and a power between 300 and 170 classed as a weak rhythm; arrhythmics were given a power of 170 (for analysis below). Between 24 and 30 flies were assayed for each genotype. There were no major differences in the period length of the rhythmic flies in each genotype (Ctl-1, 23.6 ± 0.3; Ctl-2, 23.5 ± 0.5; <i>EP1383,</i> 23.2 ± 0.4; <i>EP1306,</i> 23.4 ± 0.3; <i>hdp^R26,</i> 24.2 ± 0.5; and <i>hdp^rev83,</i> 23.4 ± 0.3).</p> <p>(C) Quantitation of the average power of the rhythm with error bars showing standard error of the mean. One-way ANOVA revealed significant differences between genotypes (<i>p <</i> 0.0001). Post-hoc <i>t</i>-tests using a Bonferroni correction revealed that the power of the rhythm was significantly different between control flies and the <i>Lmo</i> mutants <i>hdp^R26, hdp^rev83,</i> and <i>EP1306</i> (<i>p <</i> 0.01). <i>EP1383</i> flies had a significantly weaker rhythm than Ctl-2 flies (<i>p <</i> 0.05). Ctl-1, <i>w¹¹¹⁸,</i> is the appropriate genetic control for the <i>hdp</i> alleles (black columns); Ctl-2, <i>EP1631,</i> is the appropriate control for <i>EP1306</i> and <i>EP1383</i> (gray columns). All flies tested are in the same genetic background, that of the <i>w¹¹¹⁸</i> flies.</p> <p>(D) Quantitation of average activity (beam crossings per minute) with error bars showing standard error of the mean. ANOVA did not reveal significant differences between genotypes at the 0.01 level. Ctl-1, <i>w¹¹¹⁸,</i> is the appropriate genetic control for the <i>hdp</i> alleles (black columns); Ctl-2, <i>EP1631,</i> is the appropriate control for <i>EP1306</i> and <i>EP1383</i> (gray columns). All flies tested are in the same genetic background, that of the <i>w¹¹¹⁸</i> flies.</p></div

<i>Lmo</i> Gain-of-Function <i>Bx</i> Alleles Show Reduced Sensitivity to Cocaine

<div><p>(A) Cocaine phenotypes of <i>Bx</i> mutants. Male flies hemizygous for <i>Bx</i> alleles <i>Bx¹</i> or <i>Bx^J</i> show significant reductions in sensitivity to cocaine compared to control (Ctl) flies (<i>p <</i> 0.001, <i>n</i> = 12 experiments). Asterisks denote significant differences from control (Student's paired <i>t</i>-test assuming equal variance).</p> <p>(B) Dose–response. <i>Bx^J</i> flies (filled circles) show reduced sensitivity compared to Ctl flies (open circles) at all doses tested (<i>p <</i> 0.001, <i>n</i> = 16–20 for all doses except for 250 μg, where <i>p</i> = 0.0015, <i>n</i> = 8). Two additional <i>Bx</i> alleles (<i>Bx² and Bx³</i>) had similar phenotypes to <i>Bx¹</i> (not shown).</p> <p>(C) <i>Bx^J</i> flies show alterations in cocaine-induced locomotor patterns of activity. Flies were exposed to 0, 100, or 125 μg of cocaine, as indicated, for 1 min. Representative traces shown are 30 s of recorded activity of about ten flies starting 30 or 60 s after the end of cocaine exposure (<i>n</i> ≥ 3). Top panels show response of control flies to indicated amounts of cocaine; bottom panels show activity of <i>Bx^J</i> flies after cocaine administration. Ctl flies are <i>w¹¹¹⁸</i>.</p></div

Silencing or Ablating PDF Cells Induces Resistance to Cocaine

<p>Ablation of PDF cells with <i>pdf-GAL4</i> and <i>UAS-hid</i> reduced sensitivity to cocaine compared to parental lines (<i>pdf-GAL4/+</i> and <i>UAS-hid/+</i>). Electrical <i>(UAS-Kir2.1⁸</i> or <i>UAS-Kir2.1⁷)</i> or synaptic <i>(UAS-TeTx)</i> silencing of PDF cells with <i>pdf</i>-<i>GAL4</i> phenocopied PDF cell ablations. One-way ANOVAs with post hoc planned comparisons revealed a significant effect of genotype for the <i>UAS-hid</i> (<i>p <</i> 0.003, <i>n</i> = 20), <i>UAS-Kir2.1⁷</i> (<i>p <</i> 0.008, <i>n</i> = 32), <i>UAS-Kir2.1⁸</i> (<i>p <</i> 0.002, <i>n</i> = 28), and <i>UAS-TeTx</i> (<i>p <</i> 0.001, <i>n</i> = 28) groups, but not for <i>UAS-TeTxⁱⁿ</i> (<i>p</i> > 0.045, <i>n</i> = 28) (<i>n</i> corresponds to the number of experiments). Critical <i>p</i>-value was adjusted to <i>p</i> = 0.025. Asterisks denote significant differences in both planned comparisons (<i>pdf-GAL4/+</i> and <i>UAS-</i>transgene/+ versus <i>pdf-GAL4/UAS</i>-transgene). Variations in phenotype of <i>pdf-GAL4</i> flies for each set of experiments is caused by day-to-day variability.</p

A Model for LN_v and LMO Regulation of Cocaine Sensitivity

<div><p>(A) In wild type, LN_vs modulate locomotor responses via electrical activity and synaptic transmission. We propose a model in which cocaine acts to directly increase LN_v activity. Upon cocaine administration, synaptic DA concentrations are increased (via cocaine's inhibition of the plasma membrane DA transporter). Activation of presumed DA receptors on the LN_v (dark arrowheads) stimulates electrical activity and subsequent synaptic output. This activity contributes to the behavioral response of the fly to cocaine.</p> <p>(B) LN_v ablations eliminate LN_v contribution to the cocaine response, reducing cocaine sensitivity.</p> <p>(C) In our model, <i>Lmo</i> loss-of-function mutants <i>(Lmo^LOF),</i> which have increased cocaine sensitivity, have increased activity/output during the cocaine response. This increased activity may be mediated by increases in receptor content on the LN_v or by recruitment of other LN_vs that normally do not participate in the cocaine response.</p> <p>(D) <i>Lmo</i> gain-of-function mutants <i>(Lmo^GOF)</i> mutants have reduced LN_v output and reduced cocaine sensitivity. This could also result from a reduction in receptor density.</p></div

<i>pdrm</i>'s GAL4 Expression Is Sufficient to Drive <i>Lmo</i>-Transgene-Mediated Rescue of Cocaine Sensitivity

<div><p>(A) <i>pdrm</i>'s GAL4 expression pattern. <i>UAS-GFP</i> reveals GAL4 expression pattern of the <i>pdrm</i> enhancer-trap insertion in MB lobes and calyces, ALs, the large cell bodies of the peptidergic LN_vs, and neurons of the pars intercerebralis (PI).</p> <p>(B) <i>Lmo</i> expression restores wild-type cocaine responses. In the absence of a <i>UAS</i> transgene (“no UAS” columns), hemizygous male <i>pdrm</i> flies (hatched bar) are more sensitive than controls (Ctl GAL4 is line <i>8.142,</i> solid bar), as shown before in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020408#pbio-0020408-g001" target="_blank">Figure 1</a>A. Male flies hemizygous for <i>pdrm</i> and heterozygous for either of two <i>UAS-Lmo</i> transgenes (<i>UAS-Lmo¹</i> and <i>UAS-Lmo²;</i> hatched black bars), show normal cocaine sensitivity when compared to either <i>UAS-Lmo</i> transgene alone (white bars) or <i>UAS-Lmo</i> transgenes in the presence of a control GAL4 line (Ctl GAL4, line <i>8.142;</i> black bars). To control for non-specific effects of transgene overexpression, <i>UAS-GFP</i> and <i>UAS-lacZ</i> transgenes were also driven by <i>pdrm</i> GAL4. Male flies were hemizygous for <i>pdrm</i> (or heterozygous for the control GAL4 insertion) and heterozygous for the specific <i>UAS</i> transgene. One-way ANOVA revealed a significant effect of genotype in the <i>UAS-lacZ</i> (<i>F</i> = 17.4, <i>p <</i> 0.001), <i>UAS-GFP</i> (<i>F</i> = 19.47, <i>p <</i> 0.001), or no <i>UAS</i> transgene (<i>F</i> = 4.1, <i>p <</i> 0.001) groups, but not in either of the <i>UAS-Lmo</i> transgene groups (<i>F</i> = 1.58, <i>p</i> = 0.22 and <i>F</i> = 1.21, <i>p</i> = 0.31 for <i>UAS-Lmo¹</i> and <i>UAS-LMO²</i>, respectively); thus, <i>UAS-Lmo</i> expression specifically restores normal cocaine sensitivity to <i>pdrm</i> flies. Post hoc pairwise planned comparisons, with the critical <i>p</i>-value adjusted to 0.025, revealed significant differences between the “non-rescued” <i>pdrm/UAS-GFP</i> flies and the appropriate controls (<i>UAS-GFP/+</i> or <i>8.142/UAS-GFP, p <</i> 0.002); similarly, <i>pdrm/UAS-lacZ</i> flies are significantly different from their controls (<i>UAS-lacZ/+</i> and <i>8.142/UAS-lacZ, p <</i> 0.002). Pairwise comparisons revealed no significant differences between “rescued” <i>pdrm/UAS-Lmo¹</i> flies and their “normal” controls (<i>8.142/UAS-Lmo¹</i> and <i>UAS-Lmo¹/+, p</i> = 0.99 and <i>p</i> = 0.14, respectively) or <i>pdrm/UAS-Lmo²</i> flies and their controls (<i>8.142/UAS-Lmo²/+</i> and <i>UAS-Lmo²/+, p</i> = 0.09 and <i>p</i> = 0.99, respectively), indicating full rescue of <i>pdrm</i> cocaine sensitivity. For all genotypes, <i>n</i> = 16–20 experiments.</p></div

<i>Lmo</i> Expression in PDF Neurons Regulates Cocaine Responses

<div><p>(A) <i>pdf-GAL4</i>-driven expression of <i>Lmo</i> using the <i>EP1306</i> element rescues the <i>EP1306</i> insertional phenotype. <i>Lmo</i> mutants <i>EP1306, hdp,</i> and <i>pdrm,</i> as well as a control EP line (Ctl-1 <i>= EP1413</i>), were tested in the absence (−<i>pdf-GAL4;</i> white bars) and presence (<b><i>+</i></b><i>pdf-GAL4;</i> black bars) of <i>pdf-GAL4</i>. All male flies are hemizygous for the <i>Lmo</i> mutation (or Ctl-1) and heterozygous for <i>pdf-GAL4</i> (when carrying the transgene). One-way ANOVAs with post hoc planned comparisons (critical <i>p-</i>value adjusted to 0.0125) confirmed that <i>EP1306, pdrm,</i> and <i>hdp</i> flies (in the absence of <i>pdf-GAL4</i>) had significantly increased sensitivity to cocaine compared to control flies (Ctl-1 <i>= EP1413</i>) (<i>p</i> ≤ 0.003, <i>n</i> = 15–26 experiments). One-way ANOVA with post hoc planned comparisons (critical <i>p-</i>value adjusted to 0.01) revealed a significant difference between <i>pdrm/pdf-GAL4</i> and <i>hdp/pdf-GAL4</i> flies and their controls (<i>EP1413/pdf-GAL4, pdf-GAL4/+,</i> or <i>EP1413/+, p <</i> 0.003, <i>n</i> = 24–27 experiments), showing that the presence of <i>pdf-GAL4</i> does not rescue the cocaine sensitivity of <i>pdrm</i> or <i>hdp</i> flies. In contrast, similar comparisons for “rescued” <i>EP1306/pdf-GAL4</i> flies revealed no significant differences from their “normal” controls (<i>p</i> ≥ 0.026, <i>n</i> = 27–36). Furthermore, within-group comparisons (<b>+</b>/− <i>pdf-GAL4</i>) using <i>t</i>-tests indicate a significant difference only in the <i>EP1306</i> group (<i>p</i> = 0.002). Asterisk denotes significant difference between −<i>pdf</i>-GAL4 and <b>+</b><i>pdf</i>-GAL4 phenotype.</p> <p>(B) Flies overexpressing <i>Lmo</i> in PDF cells show decreased sensitivity to cocaine. Flies heterozygous for both <i>pdf-GAL4</i> and either one of two <i>UAS-Lmo</i> transgenes (black bars) were compared to flies carrying <i>UAS-Lmo</i> (white bars) or <i>pdf-GAL4</i> (gray bar) alone. One-way ANOVA revealed a significant effect of genotype for both <i>UAS-Lmo</i> transgene groups. Post-test planned comparisons, with the critical <i>p</i>-value adjusted to 0.025, showed significant differences between the <i>pdf-GAL4/UAS-Lmo</i> flies and either <i>pdf-GAL4/+</i> (<i>p <</i> 0.02) or <i>UAS-Lmo/+</i> controls (<i>p <</i> 0.005). Asterisks denote significant differences<i>, n</i> = 16 experiments.</p> <p>(C) Confocal images demonstrate overlap between <i>pdrm</i> and PDF expression in the LN_vs. In the left panel, <i>UAS-mCD8GFP</i> reveals the <i>pdrm</i>-driven GAL4 expression pattern (green) in the adult brain, and α-PAP staining (magenta) reveals PDF-expressing LN_vs. Right panels are close-ups of the cell bodies of the LN_vs; white areas correspond to regions of overlap between GFP (green) and PAP (magenta) expression.</p></div