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Rationale: Fig. 1f,g,h,i LRRTM3 has a excitatory synapse-promoting activity Literal "However, we found that the newly engineered L3S and L3L constructs displayed a strong ability comparable to that of LRRTM2 (L2) and LRRTM4 (L4S and L4L) to trigger presynaptic differentiation in cultured hippocampal neurons, suggesting that LRRTM3 also possesses potent synaptogenic activity (Figures 1F and 1G). Next, we examined whether LRRTM3 exhibits synapse-promoting activities by transfecting hippocampal neurons at 10 days in vitro (DIV10) with mVenus-fused L3L or L3S and immunostaining them at DIV14 for excitatory synaptic markers (VGLUT1 and Shank) and for inhibitory synaptic markers (GAD67 and gephyrin) (Figures 1H, 1I," Fig. 2, the knockdown of LRRTM3 with shRNA reduces excitatory, but not inhibitory, synapse numbers in DG granule neurons Literal: "To accomplish this, we transfected cultured DG neurons at DIV8 with vectors that express only EGFP (control) or coexpress EGFP with shRNAs against Lrrtm3 (L3 KD) or Lrrtm4 (L4 KD). We then immunostained the transfected neurons for VGLUT1 or GAD67 at DIV14 and quantified the density of excitatory and inhibitory synapses on dendrites of the transfected neurons (Figures 2A–2D). We found that the synapse loss induced by L3 or L4 KD was specific for excitatory synapses and not for inhibitory synapses (Figures 2A–2D). Strikingly, double KD of LRRTM3 and LRRTM4 (L3/L4 DKD) did not further decrease the excitatory synapse numbers than KD of either protein alone, suggesting that both LRRTM3 and LRRTM4 may share common pathways in maintenance of excitatory synapse structure at DG neurons (Figures 2A–2D)." Fig. 4e,f, in lrrtm3-KO mice the development of excitatory, but not inhibitory synapses is mpaired in the DG Literal "A quantitative immunofluorescence analysis revealed a significant decrease in the intensity of VGLUT1 puncta in DG molecular layers (MML), but not in the CA1 stratum oriens (oriens), a phenotype similar to that observed in Lrrtm4-KO mice (Figures 4E and 4F; Siddiqui et al., 2013). In contrast, the intensity of GAD67 puncta was unchanged in both DG MML and CA1 oriens (Figures 4E and 4F). We next analyzed dendritic spine density in Golgi-stained brain sections." Fig. 5, lrrtm3-KO mice have reduced excitatory synapse density in the DG, but not the CA1 Region of the Hippocampus Literal: "Moreover, electron microscopy analyses showed that the number of dendritic spines (i.e., asymmetric/excitatory synapses) in the DG middle molecular layer (MLL) was modestly, but significantly, decreased at P14 and P42 in Lrrtm3-KO mice (Figures 5A–5C, S5A, and S5B). This reduction in excitatory synapse density at the ultrastructural level was not observed in the CA1 pyramidal neurons of Lrrtm3-KO mice (Figures 5A–5C). In contrast, the number of GABA-positive presynaptic boutons (i.e., symmetric/inhibitory synapse) in the DG was not reduced in Lrrtm3-KO mice (Figures 5D–5F). The thickness and length of excitatory PSD- or GABA-positive boutons were unchanged by ablation of LRRTM3 protein (Figures 5D–5F). Collectively, these results demonstrate that the loss of LRRTM3 protein decreases the number of excitatory synapses in vivo without altering their gross ultrastructure." Fig. 6, the lrrtm3-KO Mice shows Impaired excitatory synaptic transmission in DG granule neurons Literal "To determine whether excitatory synaptic function is changed as a result of genetic perturbation of LRRTM3, we performed whole-cell recordings from DG granule cells in hippocampal slices from WT and Lrrtm3-KO mice (Figure 6). We found that the amplitude of miniature excitatory postsynaptic currents (mEPSCs) was markedly reduced in LRRTM3-deficient neurons (Figures 6A–6C). The amplitude of spontaneous excitatory events (sEPSCs) was reduced to the same extent in Lrrtm3-KO neurons (Figures 6D and 6E). Strikingly, the frequency of mEPSCs was significantly increased in neurons from Lrrtm3-KO mice compared with those from WT mice, whereas the frequency of sEPSCs was not altered (Figures 6A–6E). No changes were observed in the frequency or amplitude of miniature inhibitory postsynaptic currents (mIPSCs) (Figures 6D and 6F).". Experimental description: The function of LRRTM3 in excitatory synapse formation was done in LRRTM3 KO mice. Hovever synapse-promoting activities of LRRTM3 was done in rat hippocampal neurons. Literal from Supplemetary M&M "Cultured hippocampal neurons were prepared from E18 rat brains, as described previously" "Lrrtm3 Knockout Mice. Lrrtm3-KO mice have been generated previously" 8/6/2017 Pim - species of the over-expressed LRRTM3 is **human**. Literal Suppl,M&M: "pGW1-LRRTM3[Long]-mVenus encodes full-length human LRRTM3 fused to mVenus ... Full-length LRRTM3 (LRRTM3[Long] and LRRTM3[Short]) and LRRTM4 (LRRTM4[Long] and LRRTM4[Short]), excluding the corresponding signal peptide sequences, were PCR-amplified,..." - shRNA KD experiments essay LRRTM3 function in rat neurons --> rat species of the protein. - Data in Fig.1 on presynapse assebly I chose *human* species based on the over-expression construct used in this figure.