Lentiviral infection with APP₆₉₅ rescued LTCC expression and I_Ca ²⁺ in APP^−/− striatal cultures. A. Immunoblots of striatal cultures of 12 DIV from WT, littermate APP^−/− and APP^−/− neurons infected with APP₆₉₅. Lysates were blotted for LTCC (α1C), human APP (6E10) and mouse/human APP (APPc); α-tubulin was used as loading control. B. I_Ca ²⁺ was reduced in APP^−/− striatal cultures infected with APP₆₉₅ (N=15) compared to APP^−/− alone (N=12). Scales: 100 pA/50 msec. Data shown as mean ± SEM. C. Representative traces of whole-cell I_Ca ²⁺ recorded from APP^−/− and APP^−/−+APP₆₉₅ neurons, respectively.

Representative immunofluorescence staining of hippocampal sections of two-month-old APP^−/− and WT controls with anti-α1C (green) and anti-GABA (red) antibodies, showing increased α1C labeling in GABAergic interneurons in APP^−/− hippocampus as compared to the controls (Merge). Blue: DAPI showing CA1 pyramidal cells. Representative α1C- and GABA-double positive cells are labeled by arrows. Scale bar, 10 µm.

Interaction of APP with Ca_v1.2. A. α1C interacts with full length APP. HEK293 cells were transfected with full length APP and α1C cDNA; cell lysates were immunoprecipitated with an anti-α1C antibody (IP: α1C) and probed with APP antibody 22C11. Rabbit IgG (IP: IgG) was used as a negative control. B. Conversely, cell lysates were immunoprecipitated with an anti-APP antibody (IP: 4G8) and probed with α1C antibody. Mouse IgG (IP: IgG) was used as a negative control. C. Brain striatal tissue lysates were immunoprecipitated with anti-α1C and anti-APP (APPc) antibodies and probed with APP 22C11 antibody. Rabbit IgG was used as a negative control (IP: IgG). D. Striatal lysates were immunoprecipitated with antibodies against pore-forming subunits of three different VGCCs, α1A, α1B and α1C, and probed with the anti-APP antibody, 22C11. Only α1C antibody was able to immunoprecipitate APP.

Upregulated LTCC expression in APP^−/− striatal tissue and enhanced I_Ca ²⁺ in APP^−/− striatal cultures. A. Representative immunoblots of tissue extracts from striatum of 3-week-old APP^−/− (−/−) and WT (+/+) littermates. The blots were probed with anti-α1C, α 1A and α1B subunit antibodies representing LTCC, P/QTCC and NTCC, respectively. α-tubulin was used as loading control. B. Quantification of the immunoblots revealed a significant increase in LTCC expression, but not P/QTCC or NTCC in APP^−/− mice. Each value represents the mean ± SEM of six samples/genotype. *p<0.05. C. Plot of peak current density vs. voltage in WT (N=24) and APP^−/− (N=39) striatal neurons and example traces of whole-cell Ca²⁺ currents elicited by a series of depolarizing steps in WT and APP^−/− striatal neurons. D. Plot of peak current density vs. test pulse voltage under control conditions (WT, N=9; APP^−/−, N=21), or in the presence of LTCC blocker (WT, N=9; APP^−/−, N=21), LTCC blocker + P/QTCC blocker (WT, N=9; APP−/−, N=18), or LTCC blocker + P/QTCC blocker + NTCC blocker (WT, N=8; APP^−/−, N=14). E. Bar graphs illustrate the blockade of peak current exerted by L-, N- and P/Q-blockers, respectively, in WT and APP^−/− striatal cultures. L-blocker-sensitive components increased significantly in APP^−/− striatal cultures compared to WT controls. Error bars indicate SEM. **p<0.01. Scale: 100 pA/100 msec.

Altered STP in APP^−/− striatal GABAergic synapses and normalization of STP by LTCC blocker nifedipine. A. Representative traces of WT and APP^−/− striatal neurons in response to paired-pulse (before tetanus), tetanus (20 Hz, 1 sec) and after tetanus paired-pulse stimulation. B. Similar IPSC1 amplitude in the presence of CNQX and APV in APP^−/− striatum (N=20) compared to WT controls (N=12). C. The ratio of IPSC1 amplitude after tetanus (IPSC1_aft) vs. before the tetanus (IPSC1_bef) in APP^−/− striatal (N=18) and WT controls (N=12). D. Paired-pulse ratio (P2/P1) before and after tetanic stimulation in WT [WT Bef., 0.62±0.09 (N=15); WT Aft., 0.82±0.21 (N=6)] and APP^−/− [APP^−/− Bef., 0.95±0.11 (N=17); APP^−/− Aft., 0.6±0.05 (N=12)]. E. Representative traces showing paired-pulse response in the absence of nifedipine (Ctr.), in the presence of nifedipine before train stimulation (Bef. tetanus), during the train (20@20 Hz) and after the train stimulation (Aft. tetanus). ↓: indicating 15 min application of 10 µM nifedipine. F. Paired-pulse ratio (P2/P1) in APP^−/− striatal cultures in the absence [Ctr., 0.74±0.04 (N=6)] and presence of nifedipine (before and after tetanus) (Bef., 0.57±0.09; Aft. 0.49±0.03 (N=6)]. G. IPSC1 amplitude in the absence (Ctr) and presence of nifedipine in APP^−/− hippocampal cultures (before and after tetanus). Data shown as mean ± SEM. Scales: 100 pA/50 msec. *p<0.05.

Altered STP in APP^−/− hippocampal GABAergic synapses and normalized STP by LTCC blocker nifedipine. A. Representative traces of WT and APP^−/− hippocampal neurons in response to paired-pulse (Bef. tetanus), tetanic (20@20) and after tetanus (Aft. tetanus) paired-pulse stimulation. B. Identical IPSC1 amplitude in the presence of CNQX and APV in hippocampal cultures of APP^−/− and WT mice (WT, N=13; APP^−/− N=26). C. IPSC_aft./IPSC_bef. ratio in APP^−/− hippocampal neurons (N=22) compared to WT controls (N=12). D. P2/P1 ratio before and after tetanic stimulation in WT [WT Bef., 0.48±0.06; WT Aft.0.48±0.12 (N=7)] and APP^−/− neurons [APP^−/− Bef., 0.72±0.09; APP^−/− Aft., 0.48±0.04 (N=13)]. E. Representative traces showing paired-pulse response in the absence of nifedipine (Ctr.), in the presence of nifedipine before tetanic stimulation (Bef. tetanus), during the train (20@20 Hz) and after the tetanic stimulation (Aft. tetanus) in APP^−/− cultures. ↓: indicating 15 min application of 10 µM nifedipine. F. Paired-pulse ratio (P2/P1) in APP^−/− hippocampal cultures in the absence [Ctr., 0.69±0.02 (N=5)] and presence of nifedipine (before and after tetanus) (Bef., 0.56±0.048; Aft. 0.61±0.06 (N=5)]. G. IPSC1 amplitude in the absence (Ctr.) and presence of nifedipine (Bef. and Aft.) in APP^−/− hippocampal cultures. Data shown as mean ± SEM. Scales: 100 pA/50 msec. *p<0.05.