Clptm1 regulation of synaptic plasticity and hippocampus-dependent contextual fear memory is modulated by GABA_AR α5. A, CA1 fEPSP was recorded while stimulating the Schaffer collateral fibers at P43–49. The GABA_AR α5-negative modulator L-655,708 (10 nM) or vehicle (0.1% DMSO) was added in the ACSF after obtaining 10 min stable baseline recording, and 10 min later LTP was induced by HFS (1 s, 100 Hz). Bar graphs of the slope of the fEPSP recorded 0–2 and 55–60 min after HFS showed that heterozygous Clptm1 KOfirst mice had a deficit in the expression of LTP. Adding L-655,708 in the perfusion solution had no effects in WT slices but rescued LTP in KOfirst/+ slices. WT, n = 8 slices from seven mice; KOfirst/+, n = 7 slices from four mice; WT L-655,708, n = 8 slices from seven mice; KOfirst/+ L-655,708, n = 9 slices from six mice; 0–2 min, drug p = 0.4543; genotype p = 0.1672; drug and genotype interaction p = 0.1650, two-way ANOVA; 55–60 min, drug p = 0.0377; genotype p = 0.0140; drug and genotype interaction p = 0.0474, two-way ANOVA and *p < 0.05 post hoc Tukey's test. B, Immunostaining of surface GABA_AR α5 in cultured hippocampal neurons showed surface levels of GABA_AR α5 were significantly increased in KOfirst/+ neurons compared with WT neurons. Scale bar represents 10 µm. n = 36 cells each from three independent experiments; ****p < 0.0001, Mann–Whitney test. C, A schematic representation showing the timing for the fear-conditioning protocol. Thirty minutes before fear conditioning, mice were intraperitoneally injected with L-655,708 (0.7 mg/kg) or vehicle (3% DMSO). The baseline activity during the 3 min preceding the conditioning procedure was recorded. Three 2 s, 0.5 mA foot shocks, separated by 60 s intervals, were used for contextual fear conditioning. The after-shock activity for 1 min immediately after the footshocks was recorded. The contextual fear memory was tested by placing mice in the same chamber 24 h later. D, There were no differences in baseline freezing between WT and Clptm1 heterozygous KOfirst mice. Furthermore, L-655,708 had no effect on the freezing response. WT male vehicle, n = 11; WT female vehicle, n = 11; WT male L-655,708, n = 10; WT female L-655,708, n = 10; KOfirst/+ male vehicle, n = 10; KOfirst/+ female vehicle, n = 10; KOfirst/+ male L-655,708, n = 10; KOfirst/+ female L-655,708, n = 9; drug p = 0.6393; genotype p = 0.6876; sex p = 0.2783; drug and genotype interaction p = 0.8549; dug and sex interaction p = 0.6480; genotype and sex interaction p = 0.0929; drug, genotype, and sex interaction p = 0.7152, three-way ANOVA. E, There were no differences between WT and Clptm1 heterozygous KOfirst mice in the freezing behavior immediately after footshocks. Furthermore, L-655,708 had no effect on the freezing response. WT male vehicle, n = 11; WT female vehicle, n = 11; WT male L-655,708, n = 10; WT female L-655,708, n = 10; KOfirst/+ male vehicle, n = 10; KOfirst/+ female vehicle, n = 10; KOfirst/+ male L-655,708, n = 10; KOfirst/+ female L-655,708, n = 9; drug p = 0.9093; genotype p = 0.6954; sex p = 0.0864; drug and genotype interaction p = 0.1042; dug and sex interaction p = 0.9799; genotype and sex interaction p = 0.8030; drug, genotype, and sex interaction p = 0.6484, three-way ANOVA. F, The contextual fear memory was impaired in vehicle-treated Clptm1 heterozygous KOfirst mice compared with WT mice. In addition, L-655,708, while having no effects on the performance of WT mice, improved the performance of Clptm1 heterozygous KO mice to the level observed in WT mice. WT male vehicle, n = 11; WT female vehicle, n = 11; WT male L-655,708, n = 10; WT female L-655,708, n = 10; KOfirst/+ male vehicle, n = 10; KOfirst/+ female vehicle, n = 10; KOfirst/+ male L-655,708, n = 10; KOfirst/+ female L-655,708, n = 9; drug p < 0.0001; genotype p = 0.0746; sex p = 0.2552; drug and genotype interaction p < 0.0001; dug and sex interaction p = 0.7823; genotype and sex interaction p = 0.7146; drug, genotype, and sex interaction p = 0.7085; three-way ANOVA and **p < 0.01; ****p < 0.0001 post hoc Sidak's test.