Fig 2: Excitatory synapse function is differentially altered in the CA1 and DG of LRRTM1/2-DKO mice.(A and D) Representative mEPSC recordings from wild-type (WT) and LRRTM1/2-DKO hippocampus CA1 pyramidal neurons and DG granule cells. (B and C) Cumulative distributions of mEPSC inter-event intervals (B) and amplitudes (C) in wild-type and LRRTM1/2-DKO neurons. Insets display mean ± SEM for mEPSC frequency (B) and amplitude (C) in CA1 pyramidal cells, respectively. Double knockout of Lrrtm1 and Lrrtm2 decreased mEPSC frequency, Student’s t-test, **p=0.0090, without affecting mEPSC amplitude, p=0.6873, n=10–11 neurons per group, three mice each. (E and F) Cumulative distributions of mEPSC inter-event intervals (E) and amplitudes (F) in wild-type and LRRTM1/2-DKO DG granule cells. Insets display mean ± SEM for mEPSC frequency (E) and amplitude (F) in DG granule cells, respectively. Double knockout of Lrrtm1 and Lrrtm2 increased mEPSC frequency, Student’s t-test, ***p=0.0004, without affecting mEPSC amplitude, p=0.4731, n=11–15 neurons per group, three mice each. Figure 3—source data 1.Source data for Figure 3B, C. Figure 3—source data 2.Source data for Figure 3E, F.

Fig 3: Excitatory presynapse development is unaltered in CA1 dendritic layers and DG molecular layers of LRRTM1/2-cDKO.(A) Representative image of dorsal hippocampus (injection site) with infected CA1 and dentate gyrus neurons (green) from AAV8-hSYN-Cre-eGFP. Below, zoomed in images of the dorsal CA1 and dentate gyrus. Scale bar is 20 µm. (B) Representative images of whole brain (modified from Allen Brain Institute) showing bregma points −1.34, –2.06, and –2.80, with 2.06 AP as the injection site in LRRTM1/2-cDKO mice and controls. (C) Percent infected cells (Number of GFP +neurons / Number of DAPI +neurons) across the dorso-ventral axis for CA1 and dentate gyrus. (D and F) High-resolution immunofluorescence of VGlut1 punctate was unaltered in the dendritic layers of CA1 and DG molecular layers in LRRTM1/2-cDKO mice as compared with control mice. Scale bar is 5 µm. (E and G) Quantitation of VGlut1 punctate integrated intensity per tissue area, (Multiple t-test, n=3 mice each after averaging data from 6 sections per mouse). Data presented as mean ± SEM. Figure 6—source data 1.Figure 6 source data for 6C. Figure 6—source data 2.Figure 6 source data for 6DG.

Fig 4: Excitatory synapses are reduced in the CA1 of LRRTM1/2-DKOs.(A) Golgi staining revealed a reduced density of dendritic spines in CA1 radiatum in the LRRTM1/2-DKO mice as compared with wild-type (WT) mice at 6 weeks postnatal. No differences were observed between genotypes in hippocampal dentate gyrus granule cell medial molecular layer. Scale bar is 1 µm. (B) Quantitation of dendritic spine density along CA1 radiatum and dentate gyrus medial molecular layer dendrites (Two-way ANOVA p<0.0001, *p<0.001, comparing LRRTM1/2-DKO and wild-type mice for each region by Bonferroni posthoc test, n=3 mice each for radiatum and n=3 mice each for outer molecular layer). (C) Representative electron micrographs from CA1 radiatum of wild-type (WT) and LRRTM1/2-DKO mice, scale bar 100 nm. (D) Quantitation of asymmetric synapse density (Student’s t test *p=0.0394, n=4 mice each at 6 weeks postnatal). (E) Quantitation of spine width, Student’s t test (p=0.3533, n=4 mice each at 6 weeks postnatal). Figure 2—source data 1.Source data for Figure 2A, B. Figure 2—source data 2.Source data for Figure 2D,E.

Fig 5: Impaired plasticity in dorsal hippocampal CA1 and dentate gyrus regions in LRRTM1/2-cDKO mice.(A) Epifluoroscent image of dorsal CA1 with stimulating and recording electrodes. Scale bar 50 µm. (B) fEPSP recordings from control and LRRTM1/2-cDKO stratum radiatum CA3-CA1 synapses in acute hippocampal slices taken 20 min into baseline and 60 min after high-frequency stimulation (2 × 1 s at 100 Hz). Insets: Representative traces. (C) Comparisons of %fEPSP values taken from significant reductions in the fEPSPs in CA1 region of LRRTM1/2-cDKO mice, (Student’s t-test **p=0.0033, n=9 slices from three control mice and n=8 slices from three LRRTM1/2-cDKO mice). (D) Epifluoroscent image of dorsal dentate gyrus with stimulating and recording electrodes. Scale bar 50 µm. (E) fEPSP recordings from control and LRRTM1/2-cDKO inner molecular layer of dentate gyrus synapses in acute hippocampal slices taken 20 min into baseline and 60 min after high-frequency stimulation (4 × 1 s at 100 Hz). Insets: Representative traces. (F) Comparisons of average %fEPSP values revealed significant reductions in fEPSPs in dentate gyrus of LRRTM1/2-cKO mice, (Student’s t test *p=0.0176, n=9 slices for three control mice and n=8 slices for three LRRTM1/2-cDKO mice). Data presented as mean ± SEM. Figure 8—source data 1.Figure 8 source data.