Volume 21, Issue 13, Pages 3637-3645 (December 2017) Loss of Synapse Repressor MDGA1 Enhances Perisomatic Inhibition, Confers Resistance to Network Excitation, and Impairs Cognitive Function  Steven A. Connor, Ina Ammendrup-Johnsen, Yasushi Kishimoto, Parisa Karimi Tari, Vedrana Cvetkovska, Takashi Harada, Daiki Ojima, Tohru Yamamoto, Yu Tian Wang, Ann Marie Craig  Cell Reports  Volume 21, Issue 13, Pages 3637-3645 (December 2017) DOI: 10.1016/j.celrep.2017.11.109 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 3637-3645DOI: (10.1016/j.celrep.2017.11.109) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Genetic Deletion of MDGA1 Selectively Increases Inhibitory Synapse Density in CA1 Pyr (A) Pattern of β-gal activity expressed from the Mdga1 locus in an Mdga1+/− adult mouse coronal slice. (B and C) Immunohistochemical staining of the Mdga1+/− CA1 region for β-gal; DAPI, which labels nuclei; and GAD67 (B) or parvalbumin (C), which label interneurons and inhibitory terminals. The enlarged regions show no overlap between β-gal-expressing pyramidal neurons and GAD67- or parvalbumin-expressing interneurons (white arrowheads). (D) In situ hybridization showing a restricted expression pattern for native MDGA1 RNA in the Pyr cell body layer and absence from regions containing scattered interneurons expressing GAD65. (E) Representative confocal images from CA1 immunolabeled for GAD65. (F) Quantification of punctate integrated intensity per tissue area showed a significant increase in GAD65 in KO mice relative to WT controls in Pyr (unpaired t test, t8 = 2.388, ∗p < 0.001, n = 5 mice per group) but no difference in Rad (t8 = 0.6364, p = 0.5423). (G) Representative electron microscope images from hippocampal CA1 showing symmetric (inhibitory; yellow arrows) and asymmetric (excitatory; red arrows) synapses. (H) Symmetric (symm) synapse density was significantly elevated in KO mice compared with WT in Pyr (unpaired t test, t2 = 8.897, ∗p < 0.001, n = 3 mice per group) with no difference in Rad (t2 = 1.204, p = 0.3518). Asymmetric (asymm) synapse density was unaltered. Scale bars: (A) 1 mm, (B and C) 80 μm, (D) 0.2 mm, (E) 10 μm, (G) 1 μm. Data are presented as mean ± SEM. See also Figure S1. Large area images in (B) and (C) are mosaics stitched in an automated way using the Zeiss LSM700 tiling software. Cell Reports 2017 21, 3637-3645DOI: (10.1016/j.celrep.2017.11.109) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Perisomatic Inhibitory Transmission Is Selectively Increased in Mdga1−/− CA1 (A) Representative fEPSPs generated by increasing stimulation intensity. Scale bar: 1 mV, 5 ms. (B) The slope of fEPSPs was reduced in KO slices (n = 10) relative to WT (n = 12; two-way repeated-measures (RM) ANOVA, F3,15 = 29.31, p < 0.0001; Tukey’s post hoc test, ∗p < 0.01 and ∗∗p < 0.001 for KO relative to WT). Treatment with bicuculline (BICU) enhanced fEPSPs (Tukey’s post hoc test, #p < 0.01 and ##p < 0.001 for KO BICU relative to KO alone), resulting in no difference between genotypes with BICU treatment (p > 0.05, n = 15 per group). (C and D) Comparison of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) from CA1 pyramidal cells while stimulating in either Pyr (C, somatic) or distal Rad (D, dendritic). Insets show example traces while increasing stimulation as a multiple of threshold (∼15 pA) stimulation. (C) Somatic IPSCs were significantly elevated at stimulation intensities greater than a 3× threshold in KO mice (n = 13) relative to WT (n = 13; two-way RM-ANOVA, F1,5 = 96.31, p < 0.0001; Tukey’s post hoc test, ∗p < 0.01 and ∗∗p < 0.001 for KO relative to WT). (D) No differences were detected in dendritic IPSCs (n = 13 per group). Scale bars: 50 ms, 50 pA. (E) Representative traces of mIPSCs. Scale bar: 40 pA, 1 s. (F) KO neurons exhibited a significant increase in mIPSC frequency relative to WT (unpaired t test, t33 = 2.782, ∗∗p < 0.01, n = 20 cells per group). This increase resulted in a leftward shift in the cumulative probability curve corresponding to reduced interevent intervals (Kolmogorov-Smirnov test, p < 0.001). (G) mIPSC amplitude did not differ significantly between groups. (H–J) Representative mEPSC traces (H). Scale bar: 20 pA, 1 s. Neither the frequency (I; unpaired t test, t30 = 1.026, p = 0.3131, n = 18 cells for WT and 15 cells for KO) nor the amplitude (J; t23 = 0.956, p = 0.3445) of mEPSCs was altered in KO neurons relative to WT. (K) Histogram plotting rise slopes across mIPSC events. (L) Comparison of rise slope means revealed a significant increase in onset slope of mIPSCs in KO neurons relative to WT (t36 = 2.23, ∗p = 0.03, n = 20 per group). (M) No differences were observed in decay time constants between groups. Data are presented as mean ± SEM. See also Figure S2. Cell Reports 2017 21, 3637-3645DOI: (10.1016/j.celrep.2017.11.109) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Mdga1−/− Hippocampal Networks Are Resistant to Overexcitation (A) Population spikes recorded in CA1 Pyr in standard and low magnesium conditions. Scale bar: 1 mV, 5 ms. The stimulus intensities used for generating basal population spikes of comparable amplitude of at least 2 mV were 90.9 ± 5.7 μA for WT and 111.4 ± 27.9 μA for KO (mean ± SEM; t test, p = 0.085). (B) Inset: KO slices showed reduced population spike frequency in response to stimulation in Mg2+-free conditions (unpaired t test, t14 = 1.897, ∗p = 0.03, n = 9 WT and 8 KO slices). A two-way RM-ANOVA comparing spike amplitude from spikes 1–3 revealed a significant effect (F1,60 = 9.43, p < 0.01; Tukey’s post hoc analysis, ∗∗p < 0.01 between-genotype comparison, ###p < 0.0001 within-genotype comparison). The amplitude of spike 1 was significantly elevated in WT slices following exposure to 0 Mg2+ conditions relative to both WT slices in normal Mg2+ and KO slices in 0 Mg2+. KO slices failed to demonstrate a significant increase in amplitude in response to Mg2+ removal. (C) Traces from whole-cell, voltage-clamp CA1 pyramidal cells exposed to ACSF containing 2 mM and then 10 mM K+. Scale bar: 20 pA, 1 s. (D) The amplitude of postsynaptic currents (PSCs) was significantly elevated in response to increased K+ in WT cells (two-way RM-ANOVA, F1,36 = 22.93, p < 0.001, n = 10 cells per group; Tukey’s post hoc analysis, ##p < 0.001 within-genotype comparison), whereas the increase in amplitude failed to reach significance in KO neurons. Frequency of PSCs showed a similar trend with only WT demonstrating a significant increase in response to elevated K+ (two-way RM-ANOVA, F1,36 = 8.10, p < 0.01; Tukey’s post hoc analysis, #p < 0.05 within-genotype comparison). (E) KO mice and WT littermate controls were injected with KA, and behavioral seizure stages were scored. KO mice scored lower on the modified Racine scale (n = 8 per group; two-way RM-ANOVA, F1,280 = 25.83, p < 0.001; Tukey’s post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001), consistent with resistance to KA-induced seizures. Data represent mean ± SEM. Cell Reports 2017 21, 3637-3645DOI: (10.1016/j.celrep.2017.11.109) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Mdga1−/− Mice Demonstrate Impaired LTP and Cognitive Function (A) The amplitude of LTP following 1 × 100 Hz stimulation alone (WT, black, n = 10; KO, red, n = 8) and with bicuculline (WT BICU, gray, n = 13; KO BICU, blue, n = 12). Inset: traces taken 10 min pre-tetanization and 60 min post-tetanization. Scale bar: 1 mV, 5 ms. (B) LTP was impaired at early (0–20 min post-HFS) time points (one-way ANOVA, genotype F3,80 = 45.19, p < 0.0001; Tukey’s post hoc test, ∗p < 0.001) in KO slices (141.1% ± 4.2%) relative to WT (178.2% ± 6.0%). Treatment with BICU resulted in LTP of comparable magnitude between genotypes (p > 0.05) that was significantly elevated relative to drug-free slices (#p < 0.001). When LTP was compared at later time points (40–60 min post-HFS; one-way ANOVA, genotype F3,80 = 20.51, p < 0.0001), KO LTP was still impaired relative to WT controls (Tukey’s post hoc analysis, ∗p < 0.001). BICU-treated KO and WT slices exhibited similar levels of LTP that were both elevated relative to non-treated slices (#p < 0.001). Data are presented as mean ± SEM. (C) KO mice displayed normal performance in the visible platform MWM (two-way RM-ANOVA, genotype F1,54 = 3.07, p = 0.0853, n = 10 per group). (D) In the hidden platform version of the MWM, KO mice displayed increased latency to find the platform from days 2–4 relative to WT mice (two-way RM-ANOVA, genotype F1,72 = 61.43, p < 0.0001; Tukey’s post hoc test, ∗∗p < 0.001). (E) In a probe trial to assay memory in the MWM, WT mice demonstrated a preference for the target quadrant, but KO mice did not (unpaired t test, t16 = 2.84, ∗p = 0.02). (F) In the CFC task, KO mice behaved indistinguishably from WT mice 1 hr post-training but froze significantly less when placed back in the conditioning chamber 24 and 48 hr post-training (two-way ANOVA, genotype F1,64 = 14.62, p < 0.001, n = 10 per group; Tukey’s post hoc test, ∗p < 0.05). Data are presented as mean ± SEM. See also Figure S3. Cell Reports 2017 21, 3637-3645DOI: (10.1016/j.celrep.2017.11.109) Copyright © 2017 The Author(s) Terms and Conditions