Figure 5. Sequestration of endogenous miR-137 leads to phenotype reversal. Sponge_Control (black), Sponge_miR-137 (blue).

(a) Relative mRNA levels of miR-137 target genes in transfected N2a cells, t_Cplx1(8.80) = 3.02, *P = 0.0149, r = 0.71; t_Nsf(2.46) = 9.00, *P = 0.036, r = 0.64; t_Syn3(7.70) = −3.24, *P = 0.0125, r = 0.76; t_Syt1(7.96) = 3.00 *P = 0.0173, r = 0.73; n, number of experiments. (b) Relative mRNA levels in transduced induced minor allele SNP neurons, W_CPLX1 = 27, **P = 0.0064, r = −0.53; W_NSF = 13, ***P < 0.001, r = −0.70; W_SYN3 = 117, ***P < 0.001, r = −0.71; W_SYT1 = 66.5 *P = 0.0209, r = −0.39; t_PCLO(4.82) = 0.52 ^nsP = 0.6282, r = 0.23; t_STX8(5.63) = 1.54 ^nsP = 0.1782, r = 0.54; t_SYNJ1(5.56) = 1.06 ^nsP = 0.3338, r = 0.41; Sponge_Control, n = 11; Sponge_miR-137, n = 18; number of transduced minor allele SNP fibroblast lines with either construct from three reprogramming. (c) FM_4-64 imaging analysis, H(1) = 152.06, ***P < 0.001; n, number of analyzed transduced induced neurons of the two minor allele fibroblast lines from three reprogramming. (d) miR-137 target genes protein level in mossy fiber-CA3 pathway, t_Cplx1(21.97) = −2.69, *P = 0.0140, r = 0.5; t _Nsf(29.0) = −2.36, *P = 0.0251, r = 0.40; t_Syn3(16.11) = 3.43, **P = 0.0034, r = 0.65; t_Syt1(21.41) = −2.17, *P = 0.0415, r = 0.42; n, number of dissected dorsal DG-CA3 regions from at least three animals. Right, cropped western blot image. Full-length blots are presented in Supplementary Figure 9. (e,f) Ultrastructural analysis of mossy fiber presynaptic terminal. (e) Representative images of virus-transduced synapse labeled with gold particles (arrowhead); *, active zone; scale bars, 100 nm. (f) Vesicle distribution from the synaptic release site for Sponge_Control (n = 50) and Sponge_miR-137 (n = 43), V = 0.25, F(11,91) = 2.40, *P = 0.0125, post hoc analysis: F_Docked(1,91) = 4.01, *P = 0.0483; F_50nm(1,91) = 4.13, *P = 0.045; F_100nm(1,91) = 1.68, ^nsP = 0.1977; F_150nm(1,91) = 3.04, ^nsP = 0.0845; F_200nm(1,91) = 2.18, ^nsP = 0.1429; F_250nm(1,91) = 0.09, ^nsP = 0.7687; F_300nm(1,91) = 0.29, ^nsP = 0.5886; F_350nm(1,91) = 3.22, ^nsP = 0.0759; F_400nm(1,91) = 0.55, ^nsP = 0.4619; F_450nm(1,91) = 1.53, ^nsP = 0.2188; n, number of analyzed synapses from at least three animals. (g) fEPSP amplitude for 1 Hz sustained stimulation, F(1,305) = 77.73, ***P < 0.001. Representative traces embedded; gray trace, response to first stimulation; n, number of analyzed hippocampal slices from at least three animals. (h) LTP recording in DG-CA3 mossy fiber pathway, H(1) = 125.92, ***P < 0.001. Arrows, application of either high frequency stimulation or DCG-IV. Inset, representative fEPSP traces; gray traces, baseline before stimulation. Right bar chart, LTP magnitude for the last 10 min (50–60 min) recording, W = 341, ***P < 0.001, r = −0.67; n, number of analyzed hippocampal slices from at least three animals. (i,j) Fear conditioning. Freezing percentage for context (i; t(17.0) = −1.43, ^nsP = 0.1707, r = 0.33) and cue (j; t(16.21) = 0.16, ^nsP = 0.873, r = 0.04). n, number of animals. ^ns, not significant. Error bars, s.e.m.