A and B, hypertonic sucrose response after MCD treatment. A, sample traces. B, summary graph showing that the average charge transfer during the first 10 s of the 30 s sucrose stimulation is decreased 75% in cultures treated in 20 mm K⁺ with MCD. Cultures treated with 20 mm K⁺ alone were not significantly different from non-treated cultures. Treatment with MCD alone also reduced the response; however, the magnitude of the decrease was less (68%). The addition of cholesterol to MCD-treated cultures rescued the depleted hypertonic sucrose responses to values not significantly different from non-treated cultures. Horizontal bar represents the presence of hypertonic sucrose; data collected from at least 3 cultures for each condition: no treatment, n = 18; 20 mm K⁺ alone, n = 3; MCD alone, n = 16; 20 mm K⁺ with MCD, n = 18; 20 mm K⁺ with MCD + cholesterol, n = 10. C and D, field stimulation-evoked responses after MCD treatment. C, sample traces. D, summary graph depicting that the average maximum evoked EPSC amplitude is reduced 78% in cultures treated in 20 mm K⁺ with MCD. Cultures treated with 20 mm K⁺ alone were not significantly different from non-treated cultures. Treatment with MCD alone also reduced the response; however, the magnitude of the decrease was less (57%). The addition of cholesterol to MCD-treated cultures rescued the reduced EPSC amplitudes to values not significantly different from non-treated cultures. Arrow represents timing of the stimulation, at least 3 cultures: no treatment, n = 7; 20 mm K⁺ alone, n = 3; MCD alone, n = 12; 20 mm K⁺ with MCD, n = 8; 20 mm K⁺ with MCD + cholesterol, n = 7. Error bars represent the standard error of the mean (s.e.m.) *P < 0.05, **P < 0.01.

A–C, mEPSC recorded after BAPTA-AM loading (1 μm for 30 min) in MCD-treated cultures. A, sample traces. B, summary graph shows a 10-fold increase in the frequency of mEPSCs for cultures treated in 20 mm K⁺ with MCD then loaded with BAPTA-AM before recording compared with BAPTA-AM-loaded, non-treated cultures. The frequency of mEPSCs in non-treated cultures loaded with BAPTA-AM was 60% of the frequency of non-treated cultures not loaded with BAPTA-AM. The frequency of mEPSCs in cultures treated with MCD alone had a 6-fold increase in frequency; however, the difference was not significant from the frequency of BAPTA-AM-loaded, non-treated cultures. This demonstrates that the increase in the frequency of mEPSCs after MCD treatment is not due to an increase in Ca²⁺ levels/influx. C, the distributions of mEPSC amplitudes were not affected by the BAPTA-AM treatment as determined by the K-S test (P > 0.0001). 1 culture, no treatment, no BAPTA-AM, n = 3; no treatment, BAPTA-AM, n = 7; MCD alone, n = 4; and 20 mm K⁺ with MCD, n = 6). D–F, ionomycin responses from MCD-treated cultures. D, sample traces of ionomycin perfusion (1 μm for 3 min) (above). Detailed view of the traces before ionomycin and after ionomycin for each treatment are shown below. E, histogram of the average frequency over 10 s intervals of both the 20 mm K⁺ with MCD and the 20 mm K⁺ alone treatments before and after ionomycin perfusion. Cultures treated in 20 mm K⁺ with MCD had a 3-fold higher mEPSC frequency before ionomycin treatment than cultures treated with 20 mm K⁺ alone. F, summary graph of the fold increase in mEPSC frequency after ionomycin perfusion. The average fold increase in the frequency of mEPSCs after ionomycin perfusion was 2.5- and 5-fold for cultures treated with 20 mm K⁺ alone and 20 mm K⁺ with MCD, respectively; however, the difference is not significant. Horizontal black bar indicates the presence of ionomycin, 1 culture, 20 mm K⁺ alone, n = 6; 20 mm K⁺ with MCD, n = 3. Error bars represent the s.e.m.*P < 0.05.

A and B, electron micrographs (EMs) of MCD-treated hippocampal cultures. A, representative synapses. B, summary graph showing a 44% decrease in the average number of vesicles per synapse for cultures treated in 20 mm K⁺ with MCD compared with the non-treated cultures. Cultures treated with MCD alone only showed an 8% decrease in the number of vesicles per synapse, indicating that treatment with MCD with stimulation is necessary for the decrease in vesicles. No treatment, n = 94; MCD alone, n = 68; and 20 mm K⁺ with MCD, n = 108. C–E, EMs of MCD-treated cultures loaded with HRP using 47 mm K⁺ depolarization for 2 min. C, representative synapses (white arrows indicate HRP-positive (HRP+) vesicles). D, summary graph showing a 61% decrease in the average number of HRP+ vesicles per synapse for cultures treated in 20 mm K⁺ with MCD compared with the non-treated cultures. Cultures treated with MCD alone had a 30% decrease in the number of HRP+ vesicles per synapse compared with the non-treated cultures. E, summary graph showing a 64% decrease in the average number of HRP+ vesicles per synapse for cultures treated in 20 mm K⁺ with MCD compared with the non-treated cultures. Cultures treated with MCD alone had a 42% decrease in the per cent of HRP+ vesicles per synapse compared with the non-treated cultures. No treatment, n = 11; MCD alone, n = 8; and 20 mm K⁺ with MCD, n = 6. F–H, EMs of MCD-treated cultures loaded with HRP spontaneously. F, representative synapse sections (white arrows indicate HRP+ vesicles). G, summary graph showing a 1.6-fold increase in the average number of HRP+ vesicles per synapse for cultures treated in 20 mm K⁺ with MCD. H, summary graph showing a 2.5-fold increase in the average per cent of HRP+ vesicles per synapse for cultures treated in 20 mm K⁺ with MCD. No treatment, n = 47; and 20 mm K⁺ with MCD, n = 72. I–K, EMs of MCD-treated hippocampal cultures depicting stranded endocytic structures. I, representative images. J, magnified images of synapses treated with 20 mm K⁺ alone. K, magnified images of synapses treated with 20 mm K⁺ with MCD. Error bars represent the s.e.m.*P < 0.05, **P < 0.01, ***P << 0.001; scale bar, 100 nm.

A and B, hypertonic sucrose stimulation after 6 h mevastatin treatment. A, sample traces. B, summary graph showing that the average charge transfer during the first 10 s of the 30 s sucrose response is decreased 80% after treatment with mevastatin for 6 h compared with non-treated cultures. The addition of cholesterol after mevastatin treatment rescued the depleted hypertonic sucrose responses to values not significantly different from non-treated cultures. Horizontal bar represents the presence of hypertonic sucrose. At least 2 cultures; no mevastatin, n = 7; mevastatin, n = 12; mevastatin + cholesterol, n = 3. C and D, field stimulation evoked responses after 6 h mevastatin treatment. C, sample traces. D, summary graph depicting a 70% reduction in the average evoked EPSC amplitude for cultures treated with mevastatin compared with non-treated neurones. The addition of cholesterol after mevastatin treatment rescued the reduced EPSC amplitudes to values not significantly different from non-treated cultures. Arrow represents timing of the stimulation. At least 2 cultures; no mevastatin, n = 5; mevastatin, n = 5; mevastatin + cholesterol, n = 4. E–G, mEPSCs after 6 h mevastatin treatment. E, sample traces. F, summary graph shows a 3-fold increase in the frequency of mEPSCs for cultures treated for 6 h with mevastatin compared with untreated neurones. The increased frequency was reduced to non-treated frequency levels after incubation with MCD: cholesterol complexes. G, the distributions of mEPSC amplitudes were not affected by these treatments as determined by the K-S test (P > 0.0001). At least 2 cultures; no mevastatin, n = 24; mevastatin, n = 13; mevastatin + cholesterol, n = 4. Error bars represent the s.e.m.*P < 0.05, **P < 0.01, ***P < 0.001.

Spontaneous miniature EPSCs (mEPSC) after MCD treatment. A, sample traces. B, summary graph showing a 5-fold increase in the frequency of mEPSCs for MCD-treated cultures compared with non-treated cultures. Treatment with 20 mm K⁺ alone did not alter the frequency of mEPSC events. Cultures treated with MCD alone had an average 3-fold higher frequency of mEPSCs; however, the rate was not significantly different from the non-treated cultures. C, cumulative histograms of the distribution of mEPSC amplitudes showed no differences using Kolmogorov-Smirnov test (K-S test, P > 0.0001). D, summary histogram depicting the persistent 5-fold increase of the average mEPSC frequency (integrated per 10 s intervals) for 20 min for non-treated cultures and cultures treated in 20 mm K⁺ with MCD (at least 3 cultures: no treatment, n = 28; 20 mm K⁺ only, n = 6; MCD alone, n = 24; 20 mm K⁺ with MCD, n = 31; and 20 mm K⁺ with MCD + cholesterol, n = 4). Error bars represent the s.e.m.*P < 0.001.

A and B, hypertonic sucrose stimulation after MCD treatment in the absence of glia. A, sample traces. B, summary graph showing that the average charge transfer during the first 10 s of the 30 s sucrose response is decreased 94% after treatment with MCD compared with cultures treated with 20 mm K⁺ alone. Horizontal bar represents the presence of hypertonic sucrose; 1 culture, 20 mm K⁺ alone, n = 6; 20 mm K⁺ with MCD, n = 6. C and D, field stimulation evoked responses after MCD treatment in the absence of glia. C, sample traces. D, summary graph showing an 85% reduction in the average evoked EPSC amplitude for cultures treated with MCD compared with cultures treated with 20 mm K⁺ alone. Arrow represents timing of the stimulation; 1 culture, 20 mm K⁺ alone, n = 6; 20 mm K⁺ with MCD, n = 3. E–G, mEPSCs after MCD treatment in the absence of glia. E, sample traces. F, summary graph shows a 3-fold increase in the frequency of mEPSCs for cultures treated with MCD compared with cultures treated with 20 mm K⁺ alone. G, the distributions of mEPSC amplitudes were not affected by these treatments as determined by the K-S test (P > 0.001). 1 culture; 20 mm K⁺ alone, n = 6; 20 mm K⁺ with MCD, n = 9. Error bars represent the s.e.m.*P < 0.05, **P < 0.005.

A and B, hypertonic sucrose stimulation of synaptobrevin-2-deficient (Syb2^−/−) and wild-type littermates (WT) neurones after MCD treatment. A, sample traces. B, summary graphs of the charge transfer during the first 10 s of the 30 s sucrose stimulation depicts an 80% decrease in WT neurones treated in 20 mm K⁺ with MCD, and an 80% lower response of Syb2^−/− neurones compared with WT. However the sucrose responses of Syb2^−/− cultures treated in 20 mm K⁺ with MCD were not significantly different from the responses from non-treated Syb2^−/− cultures. Horizontal bar represents the presence of hypertonic sucrose; 1 culture, WT, n = 8; WT MCD, n = 4; Syb2^−/−, n = 7; Syb2^−/− MCD, n = 5. C–E, mEPSCs in Syb2^−/− and WT after MCD treatment. C, sample traces. D, summary graphs show a 2-fold and 14-fold increase after treatment in 20 mm K⁺ with MCD in the frequency of mEPSCs in WT and Syb2^−/− neurones, respectively, compared with untreated neurones. Note: the mEPSC frequency in Syb2^−/− neurones is 95% less than WT, and the frequency of Syb2^−/− neurones after MCD treatment is still 71% less than non-treated WT neurones. E, the distributions of mEPSCs amplitudes did not reveal significant differences under all conditions using K-S test (P > 0.0001). 1 culture; WT, n = 7; WT MCD, n = 5; Syb2^−/−, n = 7; Syb2^−/− MCD, n = 4. Error bars represent the s.e.m.*P < 0.05, **P < 0.005, ***P << 0.001.

A–G, electrophysiological recordings from NPC1-deficient (NPC1) and wild-type littermate (WT) hippocampal cultures. A and B, hypertonic sucrose stimulation. A, sample traces. B, summary graph showing an 80% decrease in the average charge transfer during the first 10 s of the 30 s sucrose response in NPC1 neurones compared with WT neurones. The incubation of NPC1 neurones with MCD: cholesterol complexes resulted in an increased response to hypertonic sucrose that was not significantly different from WT neurones. Horizontal bar represents the presence of hypertonic sucrose; 4 cultures; WT, n = 10; NPC1, n = 9; NPC1 + cholesterol, n = 5. C and D, field stimulation evoked responses. C, sample traces. D, summary graph depicting a 65% decrease in the average evoked EPSC amplitude for NPC1 neurones compared with WT neurones. The addition of cholesterol to NPC1 neurones rescued the reduced EPSCs to values not significantly different from WT cultures. Arrow represents timing of the stimulation; 4 cultures; WT, n = 4; NPC1, n = 4; NPC1 + cholesterol, n = 4. E–G, mEPSCs from NPC1 and WT cells. E, sample traces. F, summary graph shows a 2.5-fold increase in the frequency of mEPSCs for NPC1 neurones compared with WT neurones. The increased frequency in NPC1 neurones was reduced to the WT frequency levels after incubation with MCD: cholesterol complexes. G, the distributions of mEPSC amplitudes were not different under all conditions as determined by the K-S test (P > 0.0001). 4 cultures; WT, n = 16; NPC1, n = 17; NPC1 + cholesterol, n = 9. H–J, electrophysiological recordings from NPC1 and WT hippocampal slices. H, sample mEPSC traces. I, summary graph of the frequency of mEPSCs showing a 3.4-fold increase in the frequency of mEPSCs in the NPC1 neurones. J, the distributions of mEPSC amplitudes were not different as determined by the K-S test (P > 0.0001). WT, n = 6; NPC1, n = 5. Error bars represent the s.e.m.*P < 0.05, **P < 0.01, ***P < 0.005.