Figure 2. Cholinergic projection neurons functionally decay with age.

(A) Scheme of in vivo functional imaging preparation. The Ca2+ sensor GCaMP3 is expressed in projection neurons (PNs) under the control of GH146-Gal4 (GH146-Gal4;UAS-GCaMP3 or GH146 > GCaMP). (B_1-3) In vivo calcium imaging in PNs (GH146-Gal4;UAS-GCaMP3.0) at the level of the AL using epifluorescence microscopy. The neural response of 1 week and 7 weeks old flies to increasing concentrations of benzaldehyde, 2,3-butanedione and 3-octanol was compared (n = 8 ± SEM). Graphs represent the quantification of neural peak ΔF responses (in %ΔF/F) in the strongest responding glomeruli to different concentrations of odors for 1 week and 7 weeks old flies (n = 8 ± SEM). All GCaMP3-fluorescence responses were calculated in %ΔF/F. All p-values were calculated via Student’s t-test (ns > 0.05, *p≤0.05, **p≤0.01). (C, C´) GCaMP fluorescence changes are recorded in three different responsive glomeruli (DC2, DM6 and DP1) upon stimulation with 3-octanol (12 mM). (D-D´) GCaMP fluorescence changes were measured in three responsive glomeruli (DC1, DP1 und VC2) upon stimulation with 4-methylcyclohexanol (16 mM) in 1, 4 and 6 weeks old flies. (C, D) Maximum fluorescence changes of GCaMP3 upon odor stimulation in three different glomeruli. Scale bars: 20 µm. (C´, D´) Odor-induced fluorescence change of GCaMP3 is indicated as false color images (top row) for one representative animal. Fluorescence changes over time are shown in the lower row for each different glomerulus. The pink bars represent the time window of odor presentation. n = 9; one-way ANOVA with post hoc Bonferroni tests. ns, not significant (p>0.05). *p<0.05. **p<0.01. ***p<0.001. (E–G) Expression of the Ca2+ sensor GCaMP3 in PNs under the control of GH146-Gal4 visualized in two focal planes in presynaptic boutons of projection neurons in calyces. Scale bars: 20 µm. (E) Representative image of in vivo two-photon imaging of fluorescence of GCaMP3 in PNs (GH146 > GCaMP) at their axonal extensions (boutons) in the mushroom body calyx is shown in the top image. Odor-induced fluorescence changes of GCaMP3 are indicated as false color images (bottom image) for one representative animal. (F) Maximal fluorescence changes of GCaMP3 in individual responsive boutons and (G) number of responsive boutons upon stimulation with 3-octanol (12 mM), 4-methylcyclohexanol (16 mM) or linalool (11 mM) in the two imaged focal planes. n = 9–11; one-way ANOVA with post hoc Bonferroni tests. ns, not significant (p>0.05). *p<0.05. **p<0.01. ***p<0.001. All traces represent mean ±SEM of ΔF/F values. Box plots indicate means, medians, interquartile ranges, and 1–99% ranges.

Figure 2—figure supplement 1. Olfactory receptor neuron are not affected by aging.

(A) Expression of a reporter construct for specific olfactory sensory neurons (OSN) in 1–10 weeks old flies with a transgenic reporter construct (Or42b-Gal4;UAS-mCD8GFP). Scale bars: 15 µm. Box plot shows median and upper/lower quartiles for the number of OR42b neurons in 1–10 weeks old flies. No difference in OSN number was detected between young (1 week) and older flies (10 weeks). P-value was calculated via one-way ANOVA (ns >0.05, *p≤0.05, **p≤0.01, ***p≤0.001). (B) The size of OSN cell bodies did not change during aging (OR42b > mCD8 GFP labeled cell bodies of 1 week vs. 10 weeks old flies; N = 20 flies per group, n = 71 and 72 neurons). P-value was calculated using Standard t-test. (C) Number of all ORs, IRs, and GRs upregulated and downregulated (non-significantly) in 7 weeks old fly antenna during aging. In addition, some receptors remained unchanged between the two conditions. (D) Volcano plot of RNA-sequencing data of selected olfactory receptor genes displaying the receptors that are involved in recognition of the tested odorants. Only genes above the cutoff of –log10 (p-value adjusted (padj)) are considered significantly changed. (E) Scatter plot displaying the correlation of gene expression between samples from 1 and 7 weeks old brains. The high correlation indicates that the majority of genes in the brain remains unchanged, while a smaller number of genes change their expression.

Figure 2—figure supplement 2. Olfactory receptor neurons still respond to odors in aged animals.

(A) Schematic illustration of the electrophysiology setup. (B–H) Neural activity of olfactory sensory neurons (OSNs) in electrophysiological single sensilum recordings (SSR) in response to 10 mM of attractive (2,3-butanedione, hexanoic acid, 1-propanol) and aversive (acetophenone,1-octen-3-ol, Benzaldehyde, CO₂, 3-octanol) odors. The responses were compared between 1 week and 5 weeks old flies (n = 8 ± SEM). Each graph shows responses (spike/sec) on the Y-axis while the X-axis indicates the age (weeks) of the flies. (n = 8). Sample response traces are displayed on the right side of each graph. (J) Neural activity response (spike/sec) of young (1 week) and old (7 weeks) flies that show normal aversion (responders) and no aversion (non-responders) in T-maze assay, to aversive odor 3-octanol. The flies were sorted by behavioral performances before the SSR experiments. Responders were flies that showed the expected young fly behavioural response to an odor, while non-responder flies did not respond to an odor as expected in the olfactory behaviour assay. Y-axis shows neural response (spike/sec) whereas X-axis indicates the concentration of the odor (n = 8). (K) Neural activity response (spike/sec) of young (1 week) and old (7 weeks) flies that show normal attraction (responders) and no attraction (non-responders) in the T-maze assay, to attractive odor 2,3-butanedione. Y-axis shows neural responses (spike/sec), whereas the X-axis indicates the concentration of the odor (n = 8). These data suggest that behavioral changes do not correlate with responses of OSNs to odors.

Figure 2—figure supplement 3. Older flies show strongly improved behavior to higher odor concentrations.

(A-D) Schematic illustration of the T-maze olfactory assays (top, left). Olfactory preference index of the 1 week (orange) and 7 weeks (grey) old wildtype (Canton S) flies to standard 1 mM and increased 10 mM attractive (2,3-butanedione, putrescine) and aversive (benzaldehyde, 3-octanol) odors in the T-maze assay. Y-axis indicates the preference index (P.I.) to odors, while the X-axis denotes concentration of the tested odors. Note that the increase in odor concentration strongly improves the flies’ performance in the test suggesting that flies suffer from decreased sensitivity but not from failure to recognize and evaluate the odor. Box plot show median and upper/lower quartiles (n = 8, 60 flies/trial 30 ♀ and 30 ♂).