Electrochemical coupling of bAPs and intracellular Ca²⁺ store release in dendritic spines.

<p>(a) Top: Illustration of recording pipette positioning in layer 2 of the MEC. Bottom: Representative voltage trace of an AP doublet (100 Hz) evoked by current injection to induce the bAP-Ca²⁺ transients displayed in b₂. (b₁) Z-projection of the imaged dendritic segments in MEC layer 2 under control conditions (top), with CPA wash-in (middle), and with ryanodine wash-in (bottom; scale bars correspond to 2 μm). Asterisks mark imaged spines. (b₂) Averaged traces (corresponding to consecutive 5 min recording intervals) of a control spine (black, top), a spine in the presence of 30 μM CPA as indicated by the red bar (red, middle), and a spine in the presence of 10 μM ryanodine (blue, bottom). (c₁) Time plot of normalized doublet evoked bAP-Ca²⁺ amplitudes comparing control (black), CPA wash-in (red), and ryanodine wash-in (blue) after 5 min of baseline (indicated by the red bar). One doublet was applied every 60 s; the data is plotted in 3 min bins. Interval used for normalized post/pre ratios of bAP-Ca²⁺ is shaded in grey. (c₂) Cumulative distribution plot of the averaged bAP-Ca²⁺ amplitudes 20 to 25 min after stimulation onset (corresponding to 16 to 21 min after drug wash-in; grey area in c₁) normalized to baseline. Reduction by CPA (red, -21 ± 3%, <i>n</i> = 16/3 spines/cells) and ryanodine (blue, -10 ± 5%, <i>n</i> = 21/5 spines/cells) is significant when compared to the same time interval under control conditions (black, +11 ± 6%, <i>n</i> = 12/3 spines/cells, CPA versus control: <i>p</i> < 0.001, ryanodine versus control: <i>p</i> < 0.01, ANOVA with Bonferroni's Multiple Comparison Test). (d₁) Time plot of normalized single AP-evoked bAP-Ca²⁺ amplitudes comparing control (black), CPA wash-in (red), and ryanodine wash-in (blue) after 5 min of baseline (indicated by red bar). One AP was applied every 60 s; the data is plotted in 3 min bins. Interval used for normalized post/pre ratios of bAP-Ca²⁺ in d₂ is shaded in grey. (d₂) Cumulative distribution plot of the averaged bAP-Ca²⁺ amplitudes 15 to 20 min after drug wash-in (grey area in d₁) normalized to baseline. Reduction by CPA (red, <i>n</i> = 21/5 spines/cells) and ryanodine (blue, <i>n</i> = 19/6 spines/cells) is not significant when compared to the same time interval under control conditions (black, <i>n</i> = 21/5 spines/cells, not significant (n.s.), ANOVA with Bonferroni's Multiple Comparison Test). (e₁) Representative voltage trace of a single AP evoked by current injection to induce the bAP-Ca²⁺ transient displayed in e₃. (e₂) Z-projection of the imaged dendritic segment (scale bar corresponds to 2 μm). Asterisk marks imaged spine. (e₃) Averaged single AP-traces before and after wash-in of 10 μM ryanodine as indicated by the blue bar. (f) Cumulative distribution plot of the averaged single bAP-Ca²⁺ amplitudes in the first 5 min of drug wash-in (blue area in d₁) normalized to baseline. The increase in the initial phase of ryanodine wash-in (+10 ± 6%, <i>n</i> = 34/8 spines/cells) is significant when compared to the same time interval under control conditions (-4 ± 4%, <i>n</i> = 31/7 spines/cells, <i>p</i> < 0.05, one-tailed Mann Whitney U test). (g) Bar graph of the fraction of spines responding with an effect size >1 standard deviation than the time-matched controls for 1 bAP during the first 5 min of ryanodine wash-in (35%, light blue), 2 bAPs 20 to 25 min after stimulation onset with ryanodine (38%, dark blue) and CPA (44%, red) wash-in. Data are expressed as mean standard error of the mean (SEM) * <i>p</i> < 0.05; ** <i>p</i> < 0.01; *** <i>p</i> < 0.001.