Figure 5. Genetic Deletion of STIM2 Causes Impaired nSOC and Loss of Mushroom Spines in Hippocampal Neurons.

(A) Time-course of Fura-2 Ca²⁺ signal in the soma and GCamP5.3 Ca²⁺ signal in the spines are shown for Stim2^fl/fl hippocampal neurons transduced with NLS-GFP or NLS-Cre as indicated. Individual cell traces (gray) and average trace (black) are shown for each group. (B) The average peak SOC responses in soma and spines of Stim2^fl/fl hippocampal neurons transduced with NLS-GFP or NLS-Cre (normalized to NLS-GFP). (C) Expression levels of STIM1, STIM2 and TRPC1 were analyzed by Western blotting of lysates prepared from Stim2^fl/fl hippocampal cultures infected with Lenti-NLS-GFP or Lenti-NLS-Cre as indicated. Tubulin was used as a loading control. ns is a non-specific band. (D) Spine morphology of the primary hippocampal neurons from Stim2^fl/fl mice transduced with NLS-GFP or NLS-Cre was visualized with TD-tomato. Mushroom spines are marked by arrows. Scale bar: 10 μm. (E) Total spine density and percentage of various spine types in hippocampal neuronal cultures from Stim2^fl/fl mice transduced with NLS-GFP or NLS-Cre. M – mushroom, S- stubby, T – thin. For spine quantification, n=18-20 neurons was analyzed. The data were collected from 4 batches of cultures for each group. (F) Expression levels of STIM1, STIM2 and TRPC1 were analyzed by Western blotting of hippocampal lysates prepared from Stim2^fl/fl mice injected with AAV1-NLS-GFP or AAV1-NLS-Cre as indicated. Tubulin was used as a loading control. Each sample was from individual mouse. (G) Spine morphology in hippocampal neurons from 4 months old Stim2^fl/fl mice injected with AAV1-NLS-GFP or AAV1-NLS-Cre was visualized by Lucifer yellow injections and 2-photon imaging. The infected neurons were identified by GFP fluorescence. Mushroom spines are marked by arrows. Scale bar: 5 μm. (H) Total spine density and percentage of various spine types in hippocampal slices from 4 months old Stim2^fl/fl mice injected with AAV1-NLS-GFP or AAV1-NLS-Cre (n=4 mice for each group). M – mushroom, S- stubby, T – thin. Values are shown as mean ±SEM. *p<0.05, **p<0.01, *** p<0.001, **** p<0.0001 by t-test.