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

Increase in levels of core AZ proteins BRP and RBP leads to early memory impairment.

<p>(a–d) Adult brains of 3d and 30d flies expressing 4xBRP together with age-matched controls <i>brp</i> (2xBRP), immunostained for BRP (using Nc82 and BRP N-terminal antibody) and RBP. Scale bar: 50 μm. (e, f) Quantification of BRP (using N-terminal antibody) as well as RBP intensity within the central brain region normalized to 3d flies (<i>n</i> = 12–13 independent brains; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (g–j) STED images of BRP label within the calyx region of 3d and 30d flies expressing 4xBRP as well as 2xBRP. Ring-shaped structures are indicated (arrowheads). Scale bar: 500 nm. (k) Quantification of BRP ring diameter in 3d and 30d 4xBRP flies along with age-matched 2xBRP flies (total of 47–68 BRP rings across eight independent animals, with at least six BRP rings per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (l) Aversive associative memory performance 3 min after training (short-term memory; STM) markedly reduced in 3d 4xBRP flies in comparison to 3d wild-type 2xBRP flies (<i>n</i> = 10–16; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (m) Aversive associative memory performance at 3 h after training (intermediate-term memory; ITM), anesthesia-resistant memory (ARM), and anesthesia-sensitive memory (ASM) of 3d and 30d 4xBRP flies compared to age-matched control (2xBRP) flies (<i>n</i> = 7 independent experiments; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (n) Aversive olfactory memory performance 3 min after training (STM) higher in appl-gal4 > histone deacetylase-6 (HDAC6) RNAi in comparison to age-matched controls (<i>n</i> = 13–21; 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

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

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

Ultrastructural analysis of PN-to-KC synapses within the mushroom body calyx.

<p>(a) Overview of the calyx neuropil, obtained by amalgamation of several images over a whole calyx cross-section of a 3d <i>w</i>^<i>1118</i> fly. Scale bar: 10 μm. (b–d) Higher magnification of PN boutons and dendritic claws of KCs within the calyx of 3d, 30d, and 30d^Spd <i>w</i>^<i>1118</i> flies. Scale bar: 2 μm. The asterisk indicates the PN bouton, and the arrowhead indicates the dendritic claws of KCs. (e) Quantification of AZs normalized to bouton area (1/pm²) (total of 95–103 boutons across three independent animals, with at least 25 boutons per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (f) Quantification of total SVs within a shell of 150 nm surrounding the AZ scaffold (total of 92–100 electron-micrographs across four independent animals, with at least 20 electron-micrographs per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (g) Quantification of SVs touching the presynaptic plasma membrane (total of 92–100 electron-micrographs across four independent animals, with at least 20 electron micrographs per animal; Kruskal-Wallis test with Dunn’s multiple comparison test, p-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

Imaging of Homer-GCamp3.0 within the dendritic claws of KCs to measure odor-evoked Ca²⁺ activity.

<p>(a) Expression of Homer-GCamp3.0 in the dendritic claws of KCs and imaged within the calyx region (mb247 > Homer-GCamp3.0). Scale bar: 10 μm. (b–c) False color-coded image of Homer-GCamp3.0 activity within the postsynaptic terminals of KCs in response to 3-Oct and MCH shown in (a). Warm colors indicate high activity and cold colors indicate low or no Ca²⁺ activity. The numbers indicate changes in fluorescence (<i>ΔF/</i>F in %). (d) Odor-evoked postsynaptic Ca²⁺ activity, measured by changes in fluorescence of Homer-Gamp3.0, of an individual fly over time, shown as false colors in dendritic claws of KCs 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> = 10 flies). (e) Time course of Ca²⁺ activity induced by 3-Oct in the dendritic terminals of KCs within the calyx region of 3d, 30d, and 30d^Spd animals (GCamp3.0 response averaged across three odor exposures from ten flies). (f) Maximum change in GCamp3.0 fluorescence (ΔF<i>/F</i> in %) in response to 3-Oct within dendritic claws of KCs of 3d, 30d, and 30d^Spd flies (GCamp3.0 response averaged across three odor exposures from ten flies; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). (g) Time course of Ca²⁺ activity induced by MCH in the dendritic terminals of KCs of 3d, 30d, and 30d^Spd animals (GCamp3.0 response averaged across three odor exposures from ten flies). (h) Maximum change in GCamp3.0 fluorescence (ΔF<i>/F</i> in %) in response to MCH within dendritic claws of KCs of 3d, 30d, and 30d^Spd flies (GCamp3.0 response averaged across three odor exposures from ten flies; Kruskal-Wallis test with Dunn’s multiple comparison test, <i>p</i>-values were subject to Bonferroni correction). The grey bars indicate the duration of the odor stimuli. * <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