DataSheet1.docx

<p>Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. Here we combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. We show that the activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and that DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). We confirmed our previous observation that activating the MB α'β', but not αβ, KCs decreased the SING response, and we identified further MB neurons implicated in SING control, including KCs of the γ lobe and two subtypes of MB output neurons (MBONs). We also observed that co-activating the αβ KCs antagonizes α'β' and γ KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity.</p

Video4.mp4

<p>Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. Here we combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. We show that the activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and that DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). We confirmed our previous observation that activating the MB α'β', but not αβ, KCs decreased the SING response, and we identified further MB neurons implicated in SING control, including KCs of the γ lobe and two subtypes of MB output neurons (MBONs). We also observed that co-activating the αβ KCs antagonizes α'β' and γ KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity.</p

Video3.mp4

<p>Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. Here we combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. We show that the activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and that DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). We confirmed our previous observation that activating the MB α'β', but not αβ, KCs decreased the SING response, and we identified further MB neurons implicated in SING control, including KCs of the γ lobe and two subtypes of MB output neurons (MBONs). We also observed that co-activating the αβ KCs antagonizes α'β' and γ KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity.</p

Video1.mp4

<p>Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. Here we combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. We show that the activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and that DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). We confirmed our previous observation that activating the MB α'β', but not αβ, KCs decreased the SING response, and we identified further MB neurons implicated in SING control, including KCs of the γ lobe and two subtypes of MB output neurons (MBONs). We also observed that co-activating the αβ KCs antagonizes α'β' and γ KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity.</p

Video5.mp4

<p>Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. Here we combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. We show that the activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and that DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). We confirmed our previous observation that activating the MB α'β', but not αβ, KCs decreased the SING response, and we identified further MB neurons implicated in SING control, including KCs of the γ lobe and two subtypes of MB output neurons (MBONs). We also observed that co-activating the αβ KCs antagonizes α'β' and γ KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity.</p

Video2.mp4

<p>Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. Here we combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. We show that the activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and that DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). We confirmed our previous observation that activating the MB α'β', but not αβ, KCs decreased the SING response, and we identified further MB neurons implicated in SING control, including KCs of the γ lobe and two subtypes of MB output neurons (MBONs). We also observed that co-activating the αβ KCs antagonizes α'β' and γ KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity.</p

High-resolution analysis of PN-to-KC synapses within the mushroom body calyx shows increase in T-bar size.

<p>(a–c) Electron micrographs of calyx region of 3d, 30d, and 30d^Spd <i>w</i>^<i>1118</i> animals showing presynaptic specializations in blue (T-bars) at the PN-to-KC synapses. Scale bar: 50 nm. (d) Quantification representing the average T-bar size in 3d, 30d, and 30d^Spd animals (<i>n</i> = 92–100 electromicrographs across four independent animals, with at least 20 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (e–g) STED images of BRP spots reveal ring-shaped structures (arrowheads) within the calyx of 3d, 30d, and 30d^Spd <i>w</i>^<i>1118</i> flies. Scale bar: 500 nm. (h) Comparison of BRP-spot diameter between 3d, 30d, and 30d^Spd flies (total of 94–112 BRP rings across 15 independent animals, with at least 5 BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (i) Electron micrographs of PN bouton within the calyx region of 3d <i>w</i>^<i>1118</i> flies. Scale bar: 200 nm. (j–l) Higher magnification of AZ within PN bouton immunostained for BRP (large gold particles) and RBP (small gold particles) of 3d, 30d, and 30d^Spd <i>w</i>^<i>1118</i> flies. Scale bar: 50 nm. (m) Quantification of BRP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (n) Quantification of RBP-positive gold particles per T-bar (total of 94–108 individual T-bars across three independent animals, with at least 25 T-bars per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, ns = not significant, <i>p</i> ≥ 0.05. Underlying data is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002563#pbio.1002563.s001" target="_blank">S1 Data</a>.</p

Spermidine feeding suppresses age-associated increase in BRP and rim-binding protein (RBP) levels.

<p>(a–c) Adult brains 3d and 30d <i>w</i>^<i>1118</i> flies, together with 30d^Spd <i>w</i>^<i>1118</i> flies immunostained for Synapsin. Scale bar: 50 μm. (d) Quantification of Synapsin intensity within the central brain region normalized to 3d flies (<i>n</i> = 9–10 independent brains; Kruskal-Wallis test). (e–g) Adult brains of 3d and 30d <i>w</i>^<i>1118</i> flies, together with 30d^Spd <i>w</i>^<i>1118</i> flies immunostained for Synaptotagmin-1 (Syt-1). Scale bar: 50 μm. (h) Quantification of signal intensity of Syt-1 in the central brain region normalized to 3d flies (<i>n</i> = 8–9 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (i–k) Adult brains of 3d, 30d <i>w</i>^<i>1118</i>, and 30d^Spd <i>w</i>^<i>1118</i> flies immunostained for BRP (using Nc82 and N-terminal antibodies) and RBP. Scale bar: 50 μm (l–n) Quantification of BRP (using Nc82 and N-terminal antibodies) and RBP intensities within the central brain region normalized to 3d flies (<i>n</i> = 14–18 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, ns = not significant, <i>p</i> ≥ 0.05. Underlying data is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002563#pbio.1002563.s001" target="_blank">S1 Data</a>.</p

Imaging of SynpH at PN-to-KC synapses to measure odor-evoked SV release.

<p>(a) SynpH expressed in PN boutons and imaged within the calyx neuropil (GH146 > SynpH). Scale bar: 10 μm. (b–c) False color-coded image of the SynpH activity within the presynaptic terminals of PNs in response to 3-Oct and MCH shown in (a). Warm colors indicate high levels, and cold colors indicate low levels or no SynpH activity. The color scale on the right indicates changes in fluorescence (<i>ΔF/</i>F in %). (d) Odor-evoked release of SVs, measured by changes in fluorescence of SynpH of individual flies over time shown as false colors in presynaptic terminals of PN in the calyx region. The left panel is in response to the odorant 3-Oct and the right panel is in response to MCH (<i>n</i> = 6–7 flies). (e) Time course of SynpH activity induced by 3-Oct in the presynaptic terminals of PNs within the calyx neuropil of 3d, 30d, and 30d^Spd animals (SynpH response averaged across three odor exposures from 6–7 flies). (f) Maximum change in SynpH fluorescence (ΔF<i>/F</i> in %) in response to 3-Oct within the presynaptic terminals of PN boutons of 3d, 30d, and 30d^Spd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (g) Time course of SynpH activity induced by MCH in the presynaptic terminals of PNs within the calyx region of 3d, 30d, and 30d^Spd animals (SynpH response averaged across three odor exposures from 6–7 flies) (h) Maximum change in SynpH fluorescence (ΔF<i>/F</i> in %) in response to MCH within the presynaptic terminals of PN boutons of 3d, 30d, and 30d^Spd flies (SynpH response averaged across three odor exposures from 6–7 flies; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). * <i>p</i> < 0.05, ** <i>p</i> < 0.01, ns = not significant, <i>p</i> ≥ 0.05. Underlying data is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002563#pbio.1002563.s001" target="_blank">S1 Data</a>.</p

Homeostasis at PN::KC synapses of aged flies.

<p>(a–c) Mushroom body calyx of 3d and 30d mb247::Dα7^GFP flies and 30d^Spd mb247:: Dα7^GFP flies immunostained for GFP-labeled Dα7 as well as BRP (corresponding single z-planes are shown). Scale bar: 10 μm. Arrows indicate the recurrent presynapses of KCs that remain unopposed to acetylcholine-receptor rings within calycal neuropil; these KCs presynapses are spatially separated from the sites of cholinergic input onto KCs. (d, e) Quantification of signal intensity of Dα7 (using anti-GFP) and BRP (using Nc82) in the calyx region normalized to 3d flies (<i>n</i> = 8–10 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). * <i>p</i> < 0.05, ** <i>p</i> < 0.01, ns = not significant, <i>p</i> ≥ 0.05. (f–i) BRP immunostained within mushroom body calyx from adult brains of 3d and 10d flies expressing UAS-dORK1 ΔC in the KCs compared to age-matched controls. (j) Quantification of signal intensity of BRP (using Nc82) in the calyx region normalized to 3d flies (<i>n</i> = 10–12 independent calyces; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (k) Model showing the age-induced synaptic changes (in red). In the aged brain, the lowering of postsynaptic response with age, due to decrease in membrane excitability or Ca²⁺ homeostasis, might steer retrograde changes in the architecture of AZs. As a result, the AZ characterized by T-bar in flies enlarges in size, leading to higher release of SVs and causing aged synapses to function near the top of their presynaptic plasticity range, leaving little room for additional synaptic strengthening, and possibly impeding further learning. Underlying data is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002563#pbio.1002563.s001" target="_blank">S1 Data</a>.</p