(A) Effects of Mtss1L knockdown on the spine density of exercise-TRAPed cells were assessed by stereotaxically injecting a control shScramble-GFP lentivirus into the left hemisphere and a shMtss1L-GFP lentivirus in the right hemisphere of Fos-TRAP mice. After 21 days, dentate granule cells were exercise-TRAPed and analyzed 3 days later as shown in the schematic. (B) The left panel shows representative OML dendritic segments of exercise-TRAPed cells (red), exercise-TRAPed cells co-expressing shRNA (orange) next to several dendrites expressing only shRNA (green). The middle panel shows the dendritic field isolated in the red channel to allow comparison of dendritic spines in exercise-TRAPed cells at 3 days post exercise with (top, middle panel) and without (bottom, middle panel) co-expression of the Mtss1L shRNA. Mtss1L shRNA, but not shScramble, blocked the exercise–induced increase in dendritic spines in exercise-TRAPed cells. Summary graph at right shows dendritic spine density in the dentate OML for each condition (Homecage TRAPed: 0.8 ± 0.1, n = 4, Exercise TRAPed: 1.2 ± 0.2, n = 5, Exercise TRAPed/shScramble: 1.17 ± 0.1, n = 4, Exercise-TRAPed/shMtss1L: 0.87 ± 0.06, n = 5, one-way ANOVA, multiple comparisons Dunnett’s test, p<0.01). (C) Schematic of simultaneous whole-cell recordings from control (black) and exercise-TRAPed cells co-expressing shRNA-Mtss1L (orange) to assess functional synaptic activity. Lateral perforant path axons were stimulated in the OML. (D) Superimposed EPSCs from a representative control cell (black) and a simultaneously recorded exercise-TRAPed cell (red) showed a large increase in amplitude in the exercise-TRAPed cells, as quantified in Figure 1E. In contrast, superimposed EPSCs from a control cell (black, 46.5 ± 4.2 pA) and an exercise-TRAPed cell co-expressing shMtss1L (orange, −59.6 ± 13.5 pA) showed no increase in amplitude (p=0.42, paired t-test, seven-cell pairs from five mice). Traces are normalized and scaled relative to control cells (black) for presentation. (E) Summary plot across experimental conditions. The exercise-TRAPed increase in EPSC amplitude in the OML was blocked by Mtss1L shRNA expression in exercise-TRAPed cells. (Percent increase in EPSC amplitudes: exercise-TRAPed - OML stimulation, 208.2 ± 52.8%, n = 13 cells, eight mice; exercise-TRAPed - MML stimulation, - 6.7 ± 10.1%, n = 7 cells, four mice; exercise TRAPed +shMtss1L - OML stimulation, 38.4 ± 32.1%, n = 7 cells, five mice; shMtss1L only - OML stimulation, 7.8 ± 8.0%, n = 6 cells, three mice, p=0.002, one-way ANOVA; exercise-TRAPed shMtss1L, p=0.025; shMtss1L only, p=0.011; exercise-TRAPed MML, p=0.004; Dunnett’s test).