Aging and the negative geotaxis response (NGR) of adult Drosophila.

<p>The NGR of outcrossed wild-type control male and female flies (<i>w¹¹¹⁸/+</i>) was used to determining changes in average climbing index (CI, distance traveled within 5 seconds) between the ages of 1 and 4-weeks. *** P ≤ 0.001. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132768#pone.0132768.s001" target="_blank">S1 Fig</a></b> for the design of the NGR apparatus and additional details.</p

Aging and Autophagic Function Influences the Progressive Decline of Adult Drosophila Behaviors

<div><p>Multiple neurological disorders are characterized by the abnormal accumulation of protein aggregates and the progressive impairment of complex behaviors. Our Drosophila studies demonstrate that middle-aged wild-type flies (WT, ~4-weeks) exhibit a marked accumulation of neural aggregates that is commensurate with the decline of the autophagy pathway. However, enhancing autophagy via neuronal over-expression of <i>Atg8a</i> (Atg8a-OE) reduces the age-dependent accumulation of aggregates. Here we assess basal locomotor activity profiles for single- and group-housed male and female WT flies and observed that only modest behavioral changes occurred by 4-weeks of age, with the noted exception of group-housed male flies. Male flies in same-sex social groups exhibit a progressive increase in nighttime activity. Infrared videos show aged group-housed males (4-weeks) are engaged in extensive bouts of courtship during periods of darkness, which is partly repressed during lighted conditions. Together, these nighttime courtship behaviors were nearly absent in young WT flies and aged Atg8a-OE flies. Previous studies have indicated a regulatory role for olfaction in male courtship partner choice. Coincidently, the mRNA expression profiles of several olfactory genes decline with age in WT flies; however, they are maintained in age-matched Atg8a-OE flies. Together, these results suggest that middle-aged male flies develop impairments in olfaction, which could contribute to the dysregulation of courtship behaviors during dark time periods. Combined, our results demonstrate that as Drosophila age, they develop early behavior defects that are coordinate with protein aggregate accumulation in the nervous system. In addition, the nighttime activity behavior is preserved when neuronal autophagy is maintained (Atg8a-OE flies). Thus, environmental or genetic factors that modify autophagic capacity could have a positive impact on neuronal aging and complex behaviors.</p></div

Progressive accumulation of neural aggregates in normal aged Drosophila.

<p>Sequential protein extracts were prepared from adult male fly heads at different ages (1-day to 4-weeks) and used for Western blot analyses of Ref(2)P and ubiquitinated proteins. The Triton-X Fraction contains the Triton X-100 soluble proteins, while the SDS Fraction identifies the Triton X-100 insoluble/SDS-soluble proteins that have taken on aggregate-like characteristics.</p

Enhanced neural autophagy rescues male nighttime wakefulness.

<p>1 and 4-week old group-housed <i>Appl-Gal4/+</i>, <i>UAS-GFP-Atg8a/+</i> and <i>Appl-Gal4/ UAS-GFP-Atg8a</i> (Atg8a-OE) transgenic male flies were assayed using standard LD conditions. Yellow and black bars indicate day (8:00am to 8:00pm) and night (8:00pm to 8:00am) time periods, respectively. Activity is presented as an average per individual fly. (<b>A-B</b>) The average activity profiles of group-housed male flies at 1-week. (<b>C-D</b>) The average activity profiles of group-housed male flies at 4-weeks. **P ≤ 0.01 and *** P ≤ 0.001.</p

Sleep and arousal thresholds of aging Drosophila.

<p>Fully LD entrained single-housed female and male flies (<i>w¹¹¹⁸/+</i>) were assayed for 5 days using constant DD conditions. Representative double-plotted actograms of individual (<b>A</b>) female or (<b>B</b>) male flies, at 1 or 4-weeks of age. (<b>C-D</b>) Corresponding hypnograms illustrate average sleep profiles of young (1-week) and middle-aged (4-weeks) flies (sleep or inactivity per 30-min bins). CT0-12 depicts subjective daytime sleep and CT12-24 depicts subjective nighttime sleep patterns. (<b>E-F</b>) Young (1-week) and middle-aged (4-weeks) female and male flies were assayed using standard LD conditions over 3 consecutive days. The percentage of files aroused after receiving stimuli of varying intensities is shown for the dark ZT17-19 time period.</p

Activity profiles of group-housed male files in LL and DD conditions.

<p>Male flies (<i>w¹¹¹⁸/+</i>) were entrained in LD conditions before being assayed in constant light (<b>LL</b>) or dark (<b>DD</b>) conditions for 48 hours, at 1 and 4-weeks of age. The average activity profiles of male flies assayed in constant (<b>A-B</b>) LL and (<b>C-D</b>) DD conditions. *** P ≤ 0.001.</p

Aging and the negative geotaxis response (NGR) of adult Drosophila.

<p>The NGR of outcrossed wild-type control male and female flies (<i>w¹¹¹⁸/+</i>) was used to determining changes in average climbing index (CI, distance traveled within 5 seconds) between the ages of 1 and 4-weeks. *** P ≤ 0.001. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132768#pone.0132768.s001" target="_blank">S1 Fig</a></b> for the design of the NGR apparatus and additional details.</p

Age-dependent increase in male courtship behaviors.

<p>Still images, representing individual courtship behaviors of group-housed male flies (4-weeks) were made from infrared video recordings taken during periods of darkness (midnight to 1:00am). Recordings and images of 4-week old WT males show extensive male courtship, which include (<b>A</b>) multi-male courtship bouts or chaining behaviors, (<b>B</b>) wing extension or male courtship singing (arrows) and (<b>C</b>) attempts to copulation (arrows). (<b>D</b>) The average number of male courtship events that occurred during light (noon) and dark (midnight) time periods were determined for 1 and 4-week control (<i>w¹¹¹⁸/+</i>) and Atg8a-OE (4 Wk Rescue) male flies. (<b>E</b>) RNA was isolated from adult fly heads and used to examine the expression profiles of the <i>Obp56g</i>, <i>Obp57a</i>, <i>Obp57c and Obp99b</i> genes in young (1-week) and middle-aged (4-week) WT (<i>w¹¹¹⁸/+</i>) and Atg8a-OE male flies. * P ≤ 0.05 and *** P ≤ 0.001.</p

The LD activity profiles of single and group-housed male and female flies.

<p>Male and female control flies (<i>w¹¹¹⁸/+</i>) were aged, entrained and assayed between the ages of 1 to 4-weeks. Flies were assayed singly or in large group activity monitors (10 per vial) for 48 hours under standard 12-hr lights on, 12-hr lights off (<b>LD</b>) conditions. Yellow and black bars indicate day (8:00am to 8:00pm) and night (8:00pm to 8:00am) time periods, respectively. Activity is presented as an average per individual fly. (<b>A-B</b>) The average activity or actogram profiles of single-housed WT (<i>w¹¹¹⁸/+</i>) male and female flies, between 1 to 4-weeks of age. (<b>C-D</b>) The average activity profiles of group-housed male and female from 1 to 4-weeks of age. (<b>E-F</b>) The average activity levels of group-housed male and female flies (per fly) during day (ZT0-12), night (ZT12-24) and mid-dark (ZT15-21) time periods. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132768#pone.0132768.s007" target="_blank">S2 Fig</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132768#pone.0132768.s007" target="_blank">S2 Table</a></b> for additional information.</p

Enhanced neural autophagy rescues male nighttime wakefulness.

<p>1 and 4-week old group-housed <i>Appl-Gal4/+</i>, <i>UAS-GFP-Atg8a/+</i> and <i>Appl-Gal4/ UAS-GFP-Atg8a</i> (Atg8a-OE) transgenic male flies were assayed using standard LD conditions. Yellow and black bars indicate day (8:00am to 8:00pm) and night (8:00pm to 8:00am) time periods, respectively. Activity is presented as an average per individual fly. (<b>A-B</b>) The average activity profiles of group-housed male flies at 1-week. (<b>C-D</b>) The average activity profiles of group-housed male flies at 4-weeks. **P ≤ 0.01 and *** P ≤ 0.001.</p