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. Author manuscript; available in PMC: 2019 Feb 7.
Published in final edited form as: Neuron. 2018 Jan 25;97(3):596–610.e8. doi: 10.1016/j.neuron.2017.12.038

Figure 4. Overexpression of Clptm1 Scales Down Inhibitory Synaptic Strength.

Figure 4

(A and B) Cultured hippocampal neurons were transfected at 7 DIV with Clptm1 or HA-CD4 together with GFP to detect transfected neurons for recording. A low efficiency transfection method was used. mIPSC recordings were performed at 9 DIV and mEPSC recordings at 10 DIV.

(A) Overexpression of Clptm1 significantly reduced mIPSC amplitude compared with control group CD4. mIPSC frequency was also reduced. n=26–28 cells from 3 independent experiments, * p<0.05, ** p<0.01, t-test. The cumulative probability curve of Clptm1 amplitude was scaled by dividing by a factor of 0.70. Kolmogorov-Smirnov (K–S) test showed no significant difference between control and scaled Clptm1 groups.

(B) Overexpression of Clptm1 did not alter mEPSC amplitude or frequency. n=20–21 cells from 3 independent experiments.

(C–F) Cultured hippocampal neurons were transfected with YFP (Control) or YFP-p2a-Clptm1 (Clptm1) at 0 DIV. mIPSC recordings and immunostaining were performed at 14 DIV.

(C) Overexpression of Clptm1 significantly reduced mIPSC amplitude without changing mIPSC frequency. n=40 cells from 6 independent experiments, **** p<0.0001, t-test. The cumulative probability curve of Clptm1 amplitude was scaled by dividing by a factor of 0.78. Kolmogorov-Smirnov (K–S) test showed no significant difference between control and scaled Clptm1 groups.

(D) Neurons were immunostained live using anti-GABAAR γ2 antibody, followed by fixation and immunostaining for VGAT and the dendritic marker MAP2. Overexpression of Clptm1 significantly reduced synaptic surface γ2 intensity without changing postsynaptic γ2 puncta number. Scale bar represents 10 μm. n=30 cells from 2 independent experiments, **** p<0.0001, t-test.

(E–F) Neurons were fixed and immunostained for VGAT, MAP2, and gephyrin (Geph; E) or neuroligin-2 (NL2; F). Overexpression of Clptm1 did not change synaptic intensity or puncta number for either gephyrin or neuroligin-2. Scale bar represents 10 μm. n=30 cells from 2 independent experiments.

Results are expressed as mean ± SEM.

See also Table S2