Synaptic activity in serotonergic neurons is required both during pupal development and in adults for flight.

<p><b>A</b>) Flies with Shi^ts expression in serotonergic neurons throughout pupal development (0 h after puparium formation, APF) exhibit a 50% flight deficit in the column test. A lesser but significant deficit is also seen in flies expressing Shi^ts 2 days post eclosion. <b>B</b>) Air-puff stimulated flight response from the DLMs. Control flies, expressing Shi^ts in dopaminergic neurons and maintained at 25°C show rhythmic flight patterns. Animals expressing Shi^ts 2 days post-eclosion can initiate flight (5/15). Remaining flies show wild-type flight patterns. Shi^ts expression throughout pupal development causes complete loss of electrical activity in 8/15 flies. Remaining flies show wild-type flight patterns. <b>C</b>) Quantification of air-puff stimulated spike frequency. The traces are presented as an average of the indicated numbers. Control flies expressing Shi^ts at the permissive temperature (25°C) show a spike frequency of 9 Hz (15 flies). Shi^ts expression at the non-permissive temperature (29°C) during pupal development shows complete loss of spikes in all the intervals in 8/15 flies (group 1), while the remaining flies (group 2) show spike patterns like the controls. When flies expressing Shi^ts are maintained at 29°C from 2-days post eclosion, the spike frequency at initiation remains low (2 Hz) and then diminishes further in 5/15 adults tested. The remaining flies (group 2) show wild-type like frequencies. Spontaneous firing remains unaffected (data not shown).</p

Distribution of serotonergic neurons in second thoracic segment across 10 samples.

<p><b>A</b>) Schematic representation of the serotonergic neurons as seen in T1 and T2 region of the thoracic ganglia, showing the average number of T1 and T2 cells. <b>B</b>) Number of cells marked by anti-GFP in thoracic ganglia. Non-fliers of the genotype <i>TRH/TNT</i> have fewer GFP-positive neurons in T2 as compared with TNTvif controls (what about comparison with fliers also) (*<i>p</i><0.05; Student's <i>t</i> test). <b>C</b>) Number of cells marked by anti-GFP in thoracic ganglia. No significant difference is seen with anti-5-HT staining. <b>D</b>) TNTvif expression in <i>TRHGAL4</i> shows 4 cells, T2a′–d′, in the T2 region. An extra cell, T2e′ is seen in sample S1, which is marked by anti-GFP but not anti-5-HT. In samples S2–S10 equal number of cells were marked by anti-GFP and anti-5-HT staining. <b>E</b>) Fliers of the genotype <i>TRH/TNT</i> show variation in T2c′,d′ cells, but the variation is not significantly different from TNTvif controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046405#pone-0046405-g006" target="_blank">Fig. 6D</a>). <b>F</b>) <i>TRH/TNT</i> non-fliers lack T2c′,d′ in sample S1, S6 and S9. Sample S2 lacks all T2a′–d′ cells.</p

RNAi knock-down of of IP₃R or SOCE components in serotonergic neurons does not affect flight.

<p><b>A</b>) In the cylinder drop test, no flight defect is seen in flies expressing RNAi against IP₃R, STIM or Orai as compared with controls. For each genotype, a total of 100 flies were tested in 5 batches of 20. <b>B</b>) Electrophysiological recordings from the DLMs of tethered flies after delivery of an air puff stimulus (arrows). All flies show rhythmic firing throughout flight. <b>C</b>) Quantification of spontaneous firing. Depletion of IP₃R or SOCE increases spontaneous firing. (*<i>p</i><0.05; Student's <i>t</i> test). <b>D</b>) Representative traces of electrophysiological recordings from the DLMs.</p

Loss of synaptic activity in serotonergic neurons causes flight defects.

<p><b>A</b>) Flight deficit, assayed by the cylinder drop test, is significantly higher in animals expressing either tetanus toxin (TNTH) or the hyperpolarizing K⁺ ion channel (Kir2.1) as compared with controls (*<i>p</i><0.005; Student's <i>t</i> test). Approximately 100 or more flies were tested for each genotype. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046405#s3" target="_blank">Results</a> are expressed as mean ± SEM. <b>B</b>) Electrophysiological recordings from the DLMs of tethered flies after delivery of an air puff stimulus (arrows). Control flies show rhythmic firing throughout flight. Loss of electrical activity is seen in 13/30 animals expressing <i>TNTH</i>. The remaining animals show wild-type like flight pattern. The duration of flight is reduced to <5 secs in 12/30 flies expressing <i>Kir2.1</i>. Intermittent flight patterns are seen in 9/30 flies. The remaining flies show wild-type like flight pattern. <b>C</b>) Quantification of the spike frequency during flight at a bin interval of 5 secs. Control flies (<i>TRHGAL4/+</i>, control 1 and <i>TRHGAL4/TNTvif</i>, control 2) show a spike frequency of 9 Hz in all the bins. The trace is expressed as an average of 15 flies. <i>TNTH</i> expressing flies show either complete loss of flight or normal flight frequency. <b>D</b>) Control flies (<i>Kir2.1/+</i>) show an average spike frequency of 9 Hz (15 flies). Flies expressing <i>Kir2.1</i> show variable spike frequencies. Expression of either TNTH or Kir2.1 in serotonergic neurons does not affect the frequency of spontaneous firing as recorded from the DLMs. <b>E</b>) Quantification of spontaneous firing. <b>F</b>) Representative traces of electrophysiological recordings from the DLMs.</p

Loss of synaptic activity in serotonergic neurons does not affect the cell population in the larval central nervous system.

<p><b>A</b>) Immunohistochemistry of the larval brain expressing <i>mCD8GFP/TNTvif</i> in <i>TRHGAL4</i> domains (control). All the GFP stained cells also show anti-5-HT staining (merge). <b>B</b>) Immunohistochemistry of the larval brain expressing <i>mCD8GFP/TNTH</i> in <i>TRHGAL4</i> domains. No difference is seen as compared with control. <b>C</b>) Schematic of <i>TRHGAL4/mCD8GFP</i> and 5-HT positive neurons marked in the larval brain. <b>D</b>) Number of cells marked by anti-GFP and anti-5-HT staining does not vary between control and tetanus toxin expressing animals.</p

Loss of synaptic activity in serotonergic neurons does not affect cell numbers in the adult brain.

<p><b>A</b>) Immunohistochemistry of the adult brain expressing <i>mCD8GFP/TNTvif</i> in <i>TRHGAL4</i> domains. All <i>TRHGAL4</i> positive neurons (anti-GFP, green) stain with anti-5-HT (red and green merge), except for 6 medial cells (MD), 1 cell in LP1 (LG1), 2 in LP2 (LG2), 1 in SE1 (SG1) and 1 in SE3 (SE3), which are GFP-positive but 5-HT negative. <b>B</b>) Immunohistochemistry on a <i>TRHGAL4/TNTH</i> brain collected from flies which passed the column flight test (fliers). Medial cells are seen (green). <b>C</b>) Immunohistochemistry on a <i>TRH/TNTH</i> brain collected from non-fliers. No difference is seen as compared with controls in A and B. <b>D</b>) Schematic of the brain showing cells marked by <i>TRHGAL4</i> (anti-GFP, green) and anti-5-HT. Medial cells are not stained with anti-5-HT. <b>E</b>) Number of cells marked by anti-GFP and anti-5-HT staining does not vary between control and tetanus toxin expressing serotonergic neurons among fliers and non-fliers. (Nomenclature based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046405#pone.0046405-Monastirioti1" target="_blank">[26]</a>).</p

<i>Clk</i> inactivation in the s-LNv induces faster ARLI in aging flies.

<p>(A) Down-regulating <i>Clk</i> expression selectively in all PDF-expressing neurons (<i>pdf>Clk</i>^RNAi) results in a premature locomotor decline. (B) Flies with reduced <i>cyc</i> expression in the PDF neurons (<i>pdf>cyc</i>^RNAi) showed no ARLI defect. (C) Expressing <i>Clk</i>^RNAi in the s-LNv selectively (<i>R6-Gal4</i> driver) had the same effect on climbing abilities as <i>pdf>Clk</i>^RNAi. (D) Ablation of PDF neurons by expressing the pro-apoptic gene <i>hid</i> in these cells did not alter ARLI. Graphs display the means ± SEM from 2 independent experiments.</p

Specific loss of dopaminergic PPL1 neurons in <i>Clk</i>-deficient flies.

<p>TH-IR cells were counted in confocal stacks of brains dissected from flies of the indicated ages and genotypes. In all panels, histograms display the mean ± SEM of TH-IR cell numbers in the PAL, PPL1, PPL2, PPM1/2 and PPM 3 clusters, from 2 independent experiments with 8–10 brains per genotype in each experiment. (A) At 10 days of age, the number of TH-IR neurons in the different dopaminergic neuronal clusters of Canton-S (ctrl), <i>Clk</i>^AR and <i>cyc</i>⁰ flies was similar. (B) <i>Clk</i>^AR mutants exhibited a selective loss of TH-IR cells in the PPL1 neuronal cluster at 31 days post-eclosion when compared to either controls or <i>cyc</i>⁰. (C) Loss of TH-IR cells in the PPL1 neuronal cluster was also observed at 31 days post-eclosion in <i>Clk</i>^AR/+ heterozygote flies. (D) Reducing <i>Clk</i> levels in the PDF neurons specifically decreased the number of PPL1 TH-IR cells. PAL, protocerebral anterior lateral; PPM1-3, protocerebral posterior median 1–3; PPL1-2, protocerebral posterior lateral 1–2.</p

Age-related locomotor impairment (ARLI) in <i>cyc</i>⁰, <i>tim</i>⁰ and <i>Clk</i>^AR arrhythmic mutants.

<p>(A) ARLI of <i>cyc</i>⁰ and <i>tim</i>⁰ mutants is accelerated relative to wild-type flies (ctrl) housed in LD. (B) ARLI of wild-type Canton-S flies (ctrl) is accelerated under constant light (LL) that stops the clock. (C) The effect of <i>Clk</i>^AR mutation on ARLI is stronger and occurs earlier than the effect of <i>cyc</i>⁰, <i>tim</i>⁰ or LL. (D) <i>Clk</i>^AR/+, but not <i>cyc</i>⁰/+, heterozygote mutants also exhibit an accelerated locomotor decline as compared to controls. (E) No effect on ARLI is observed for <i>cyc</i>⁰ and <i>tim</i>⁰ mutants in constant light (LL). (F) In contrast, <i>Clk</i>^AR mutants exhibit an accelerated ARLI under constant light when compared to wild-type flies (ctrl) kept in LL as well. Graphs display the means ± SEM from 2–3 independent experiments.</p

<i>Clk</i> regulates brain ROS levels independently from ARLI.

<p>(A, B) Brain ROS levels are higher in <i>Clk</i>^AR mutants at both 10 and 31 days of age as compared to ctrl and <i>cyc</i>⁰ flies. Scale bars in A: 100μm. (C) Downregulating <i>Clk</i> expression in the PDF neurons, which accelerated ARLI (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006507#pgen.1006507.g002" target="_blank">Fig 2A</a>) had no effect on brain ROS levels as compared to controls. (D) Brain ROS levels of <i>Clk</i>^AR flies in which <i>Clk</i> expression was restored in the PDF neurons only, resulting in largely rescued ARLI (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006507#pgen.1006507.g003" target="_blank">Fig 3A</a>), are unaltered in comparison to respective controls in the same <i>Clk</i>^AR background. Histograms display the mean ± SEM of brain ROS levels from 2–3 independent experiments with 6–8 brains per genotype in each experiment.</p