Cultured hippocampal neurons were transfected with U6-shScramble-hSyn-CFP and
hSyn-YFP as control, U6-shClptm1-hSyn-CFP and hSyn-YFP as knockdown (KD), or
U6-shClptm1-hSyn-CFP and hSyn-YFP-p2a-Clptm1* as rescue at 0 DIV. CFP
and YFP dual positive neurons were selected for recording or immunostaining.
mIPSCs were recorded at 13 DIV, mEPSCs were recorded at 14 DIV, and
immunostaining was performed at 14 DIV.
(A) Knockdown of Clptm1 significantly increased mIPSC amplitude compared with the
control group, an effect rescued by expressing the shRNA-resistant
Clptm1*. n=17–19 cells from 4 independent experiments,
p<0.05 one-way ANOVA and *
p<0.05 post hoc Holm-Sidak tests. The cumulative probability
curve of KD amplitude was scaled by dividing by a factor of 1.65. KS test showed
no significant difference between control and scaled KD groups.
(B) Knockdown of Clptm1 did not affect mEPSC amplitude or frequency.
n=16–18 cells from 4 independent experiments.
(C) Neurons were immunostained live using anti-GABAAR γ2
antibody, followed by fixation and immunostaining for VGAT and MAP2. Knockdown
of Clptm1 significantly increased synaptic surface γ2 intensity, an
effect normalized by expressing the shRNA-resistant Clptm1*. Scale bar
represents 10 μm. n=30 cells from 3 independent experiments,
p<0.0001 one-way ANOVA and ***
p<0.001, ****
p<0.0001 post hoc Holm-Sidak tests.
Results are expressed as mean ± SEM.
See also Figure S3 and
Table S2.