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. 2019 Jun 24;8:e45920. doi: 10.7554/eLife.45920

Figure 6. Mtss1L knockdown in exercise-TRAPed cells blocked the exercise-induced increase in dendritic spines and synaptic activity at 3 days post-exercise.

(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).

Figure 6.

Figure 6—figure supplement 1. Mtss1L shRNA knockdown efficiency.

Figure 6—figure supplement 1.

(A) shRNA knockdown efficiency was first screened using HEK293T cells overexpressing Mtss1L after transduction with lentiviral construct FUGW-mcherry-T2A-Mtss1L. Knockdown efficiency of the overexpression construct was confirmed by fluorescence microscopy Scale bar: 200 μm. (B) Primary hippocampal cultures were transduced with 10^5 particles of one or both short hairpin shRNAs against Mtss1L (designated L1 and L2) or shMtss1Lscramble lentivirus, and then treated for 7 d.i.v. with media supplemented by BDNF (25 ng/ml). The shScramble virus was ineffective, whereas the combination of the two short hairpins was the most effective. Mtss1L shRNA knockdown efficiency was analyzed by RT-qPCR for Mtss1L expression, using 18S as the housekeeping/control gene (n = 3 biological replicates, one way ANOVA with Dunnett’s multiple comparison, p<0.001).

Figure 6—figure supplement 2. shRNA-Mtss1L did not alter evoked EPSCs in granule cells that were not exercise- TRAPed.

Figure 6—figure supplement 2.

(A) Schematic of paired dentate granule cell recordings. EPSCs were evoked with a bipolar stimulation electrode in the OML. (B) Representative EPSCs from a paired control granule cell (GFP-, black trace) and an adjacent Mtss1L shRNA only (GFP+/tdT-) granule cell show similar amplitudes. Stimulation artifacts were blanked. (C) EPSC amplitudes in paired recordings were unaffected by shRNA-Mtss1L expression in granule cells that were not exercise-TRAPed, indicating that knockdown of Mtss1L had no effect unless the granule cell was exercise-activated (paired t-test, p=0.4805).