Figure 5.
LTP requires centaurin-α1. (A) Left: Representative Western blot of protein extracts from dissociated hippocampal neurons (15 DIV) infected for 7 d with shCentaurin-α1 lentivirus (purple) or with a control lentivirus (lacking the shRNA sequence, black). Right: Quantification of centaurin-α1 protein levels relative to uninfected condition. Bars show mean ± SEM, together with individual values from each experiment; n = 6 independent experiments; shCentaurin-α1 values were significantly different from uninfected and control (P = 0.03, Wilcoxon test). (B) Western blot similar to the one shown in A, including neuronal cultures infected with the rescue GFP-centaurin-α1 lentivirus. (C) GFP-channel (top) and immunohistochemistry (IHC) against centaurin-α1 (bottom) from organotypic hippocampal slices infected with the rescue GFP-centaurin-α1 lentivirus. (D) Left: Time course of the LTP induction in organotypic hippocampal slices (7‒11 DIV) infected for 7‒10 d with lentivirus for the expression of shCentaurin-α1 (purple symbols) or shCentaurin-α1 plus shRNA-resistant GFP-centaurin-α1 (blue symbols), compared with uninfected neurons (gray symbols). The amplitude of the synaptic response is normalized to a 3-min baseline before the LTP induction (300 pulses at 3 Hz, coupled to postsynaptic depolarization at 0 mV). Each point represents the mean of the normalized EPSCs ± SEM per condition. Right: Average response from the last 5 min of the recording and normalized to the baseline. Bars represent the mean ± SEM, together with individual values for each experiment. Left bars (LTP, paired) correspond to the stimulation pathway in which LTP protocol was induced; right (shaded) bars (control, unpaired) correspond to the pathway that was not stimulated during induction. n = 20, 10, or 12 cells for control, shCentaurin-α1, or centaurin-α1 rescue neurons, respectively, for the LTP (paired) pathway. n = 20, 6, or 6 cells for control, shCentaurin-α1, or centaurin-α1 rescue neurons, respectively, for the control (unpaired) pathway. Uninfected and rescue neurons are significantly potentiated with respect to baseline (P = 0.0003 and P = 0.005, respectively, Wilcoxon test). P values displayed in the figure indicate significant difference between shCentaurin-α1 with respect to uninfected (P = 0.001) and rescue (P = 0.009) conditions, respectively (Mann–Whitney test). (E and F) Basal synaptic responses mediated by AMPARs (E) and NMDARs (F) recorded simultaneously from adjacent neurons, uninfected and infected with either shCentaurin-α1 (purple) or GFP-centaurin-α1 rescue (blue). Gray symbols represent absolute values of the responses from each individual pair. Colored symbols show the mean ± SEM of the measured currents. n = 16 cell pairs for shCentaurin-α1 and n = 15 for centaurin-α1 rescue, in the case of AMPAR currents (E). n = 12 cell pairs for shCentaurin-α1 and n = 15 for centaurin-α1 rescue in the case of NMDAR currents (F). n.s., not significantly different (Wilcoxon test). Insets: Representative traces for uninfected (black lines), shCentaurin-α1 (purple lines), and centaurin-α1 rescue (blue lines) neurons; scale bars: vertical, 10 pA; horizontal, 10 ms.

LTP requires centaurin-α1. (A) Left: Representative Western blot of protein extracts from dissociated hippocampal neurons (15 DIV) infected for 7 d with shCentaurin-α1 lentivirus (purple) or with a control lentivirus (lacking the shRNA sequence, black). Right: Quantification of centaurin-α1 protein levels relative to uninfected condition. Bars show mean ± SEM, together with individual values from each experiment; n = 6 independent experiments; shCentaurin-α1 values were significantly different from uninfected and control (P = 0.03, Wilcoxon test). (B) Western blot similar to the one shown in A, including neuronal cultures infected with the rescue GFP-centaurin-α1 lentivirus. (C) GFP-channel (top) and immunohistochemistry (IHC) against centaurin-α1 (bottom) from organotypic hippocampal slices infected with the rescue GFP-centaurin-α1 lentivirus. (D) Left: Time course of the LTP induction in organotypic hippocampal slices (7‒11 DIV) infected for 7‒10 d with lentivirus for the expression of shCentaurin-α1 (purple symbols) or shCentaurin-α1 plus shRNA-resistant GFP-centaurin-α1 (blue symbols), compared with uninfected neurons (gray symbols). The amplitude of the synaptic response is normalized to a 3-min baseline before the LTP induction (300 pulses at 3 Hz, coupled to postsynaptic depolarization at 0 mV). Each point represents the mean of the normalized EPSCs ± SEM per condition. Right: Average response from the last 5 min of the recording and normalized to the baseline. Bars represent the mean ± SEM, together with individual values for each experiment. Left bars (LTP, paired) correspond to the stimulation pathway in which LTP protocol was induced; right (shaded) bars (control, unpaired) correspond to the pathway that was not stimulated during induction. n = 20, 10, or 12 cells for control, shCentaurin-α1, or centaurin-α1 rescue neurons, respectively, for the LTP (paired) pathway. n = 20, 6, or 6 cells for control, shCentaurin-α1, or centaurin-α1 rescue neurons, respectively, for the control (unpaired) pathway. Uninfected and rescue neurons are significantly potentiated with respect to baseline (P = 0.0003 and P = 0.005, respectively, Wilcoxon test). P values displayed in the figure indicate significant difference between shCentaurin-α1 with respect to uninfected (P = 0.001) and rescue (P = 0.009) conditions, respectively (Mann–Whitney test). (E and F) Basal synaptic responses mediated by AMPARs (E) and NMDARs (F) recorded simultaneously from adjacent neurons, uninfected and infected with either shCentaurin-α1 (purple) or GFP-centaurin-α1 rescue (blue). Gray symbols represent absolute values of the responses from each individual pair. Colored symbols show the mean ± SEM of the measured currents. n = 16 cell pairs for shCentaurin-α1 and n = 15 for centaurin-α1 rescue, in the case of AMPAR currents (E). n = 12 cell pairs for shCentaurin-α1 and n = 15 for centaurin-α1 rescue in the case of NMDAR currents (F). n.s., not significantly different (Wilcoxon test). Insets: Representative traces for uninfected (black lines), shCentaurin-α1 (purple lines), and centaurin-α1 rescue (blue lines) neurons; scale bars: vertical, 10 pA; horizontal, 10 ms.

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