Repetitive mild head trauma induces activity mediated lifelong brain deficits in a novel Drosophila model

Mild head trauma, including concussion, can lead to chronic brain dysfunction and degeneration but the underlying mechanisms remain poorly understood. Here, we developed a novel head impact system to investigate the long-term effects of mild head trauma on brain structure and function, as well as the underlying mechanisms in Drosophila melanogaster. We find that Drosophila subjected to repetitive head impacts develop long-term deficits, including impaired startle-induced climbing, progressive brain degeneration, and shortened lifespan, all of which are substantially exacerbated in female flies. Interestingly, head impacts elicit an elevation in neuronal activity and its acute suppression abrogates the detrimental effects in female flies. Together, our findings validate Drosophila as a suitable model system for investigating the long-term effects of mild head trauma, suggest an increased vulnerability to brain injury in female flies, and indicate that early altered neuronal excitability may be a key mechanism linking mild brain trauma to chronic degeneration.


Gross Morphology and Acute Survival Following Repetitive Head Impacts (a)
Representative whole-body micrograph of an injured Oregon R male fly showing no signs of gross morphological damage to the head or body following repetitive head impact exposure. Scale bar= 100 μm. (b) Barplot of acute survival (24h) following varying number of iterative successive head impacts delivered 10s apart. Black text indicates (# dead/# at risk).
Acute recovery of climbing deficits following minimally lethal repetitive head impacts is sexually dimorphic. Repetitive head impacts elicit acute climbing deficits in both male and female, seen as a reduction in (a) total climbing distance and (c) total vertical distance traversed during the startle-induced climbing assay. (b&d) Injured female flies show progressive relative behavioral deficits that worsen after the second impact session, while injured male flies show active acute recovery 24h after each session of impact. Plotted values are median (a&c) raw or (b&d) relative (to respective sex) values with 95% confidence intervals as error bars. Mann-Whitney U test between (a&c) injured and non-injured groups, with Holm correction and (b&d) injured female and male performance relative to non-injured, with Bonferroni correction. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=25-35 flies per sex/time/injury group.

Repetitive head impacts result in long-term behavioral climbing deficits.
Repetitive head impacts elicit chronic climbing deficits that are more pronounced in female flies, seen as a reduction in (a) total climbing distance and (b) total vertical climbing distance traversed during the climbing assay. Plotted values are median values with 95% confidence intervals as error bars. Mann-Whitney U test between injured and noninjured groups, with Holm correction: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n≥22 flies per sex/time/injury group except day 56 n≥11 flies.
Repetitive head impacts result in no acute neurodegeneration. Repetitive head impacts elicit no acute (1.5h post-injury) neurodegeneration, neither seen as an (a) increased number of vacuoles and (b) vacuole area per brain. Boxplots contain individually plotted values with whiskers corresponding to the maximum 1.5 interquartile range. Within sex differences between sham and repetitive head impact conditions were analyzed with the Mann-Whitney U test with Bonferroni correction.

Supplemental Figure S6
Suppressing acute injury-induced neuronal activity following repetitive head impacts preferentially benefits females. Blocking activity protects against acute climbing deficits in female flies, specifically (a&b) relative total distance and (c&d) relative vertical distance traversed. Plotted values are relative median values (compared to respective genotype sham) with 95% confidence interval error bars. Differences in relative climbing behavior were analyzed using the Mann-Whitney U test with Holm correction, between injured LexA Only and Shi ts1 -containing flies. *p<0.05, **p<0.01, ***p<0.001.

Supplemental Figure S7
Suppressing acute injury-induced neuronal activity following repetitive head impacts preferentially benefits females. Blocking activity protects against chronic climbing deficits in female flies, specifically (a&b) relative total distance and (c&d) relative vertical distance traversed. Plotted values are relative median values (compared to respective genotype sham) with 95% confidence interval error bars. Differences in relative climbing behavior were analyzed using the Mann-Whitney U test with Holm correction, between injured LexA Only and Shi ts1 -containing flies. *p<0.05, **p<0.01, ***p<0.001.

Supplemental Figure S8
Climbing effect of pan-neuronal expression of the female-specific splicing regulator transformer in male flies following repetitive head impacts. Male flies with (nSyb>Tra) and without (nSyb-GAL4) pan-neuronal transformer expression were subjected two sessions of repetitive head impacts as described in Fig. 2. Their startleinduced climbing behaviors were assessed at various points before and after the repetitive head impacts. (a) Plotted climbing slope of sham and injured male flies with (nSyb-Tra) and without transformer (nSyb-GAL4). (b) Plotted relative climbing slope of injured male flies with (nSyb>Tra) and without transformer (nSyb-GAL4 Only). ( slope= (Injured Slope-Median Sham Slope)/Median Sham Slope). Plotted values are median values with 95% confidence interval error bars. Differences in relative climbing behavior were analyzed using the Mann-Whitney U test with Holm correction, between (a) sham and injured male flies or (b) injured GAL4 Only and Transformer-containing flies. *p<0.05.