Fig 1.
Electrochemical coupling of bAPs and intracellular Ca2+ store release in dendritic spines.
(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%, n = 16/3 spines/cells) and ryanodine (blue, -10 ± 5%, n = 21/5 spines/cells) is significant when compared to the same time interval under control conditions (black, +11 ± 6%, n = 12/3 spines/cells, CPA versus control: p < 0.001, ryanodine versus control: p < 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, n = 21/5 spines/cells) and ryanodine (blue, n = 19/6 spines/cells) is not significant when compared to the same time interval under control conditions (black, n = 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%, n = 34/8 spines/cells) is significant when compared to the same time interval under control conditions (-4 ± 4%, n = 31/7 spines/cells, p < 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) * p < 0.05; ** p < 0.01; *** p < 0.001.
Fig 2.
Enhancement of bAP-Ca2+ transients in dendritic spines.
(a1) Illustration of recording pipette positioning in layer 2 of the MEC (black) and in the hippocampal CA1 region (green). (a2) Experimental paradigm for enhancement of bAP-Ca2+ transients. Top: Test stimulus AP doublets evoked by current injection before (pre, grey) and after (post, black) 5 AP bursts (bold black). Bottom: Experimental timeline, vertical bars correspond to test stimulus AP doublet (short bars) and 5 APs (long bars). Doublet test stimuli were delivered every 60 s. After a 6 min baseline measurement, we applied a bursting paradigm consisting of 5 APs delivered every 30 s for 5 min. After the bursting paradigm, we again measured the bAP-Ca2+ transient evoked by the doublet test stimulus. Grey box illustrates the interval 15 to 20 min after bAP stimulation onset which is compared to baseline stimulation to quantify enhancement. (b1) Z-projection of the imaged dendritic segment (scale bar corresponds to 2 μm). Asterisks mark imaged spine and dendritic segment. (b2) bAP Ca2+ transient enhancement in a cortical neuron. Averaged fluorescence traces of Ca2+ transients in spine (top) and dendrite (middle). Representative AP traces from the time intervals averaged for Ca2+ imaging (bottom). (c1) Z-projection of the imaged dendritic segment in a hippocampal CA1 pyramidal cell. (scale bar corresponds to 2 μm). Asterisks mark imaged spine and dendritic segment. (c2) Overlay of averaged fluorescence traces 0–5 (grey) and 15–20 min after bAP stimulation onset in a CA1 pyramidal cell spine (green) and the adjacent dendrite (red). (c3) Time plot of normalized single sweep amplitudes. (d) Cumulative distribution plot of normalized bAP Ca2+ transient enhancement in spines of MEC layer 2 cortical neurons (19 ± 4%, n = 92/43 spines/cells, black) and hippocampal CA1 pyramidal cells (8 ± 4%, n = 59/22 spines/cells, green), comparison did not reach significance (n.s., Mann Whitney U Test). (e) Bar graph illustrates percentage of spines displaying bAP-Ca2+ transient enhancement >20% in MEC layer 2 cells (42%, 39/27 out of 92/43 spines/cells, black) and in hippocampal CA1 pyramidal cells (24%, 14/8 out of 59/22 measured spines/cells, green).
Fig 3.
A subpopulation of spines undergoes activity-dependent enhancement.
(a1) Averaged baseline bAP-Ca2+ transients 0 to 5 min after bAP stimulation onset (grey traces) and averaged bAP-Ca2+ transients 15 to 20 min after bAP stimulation onset of 2 adjacent spines (black and blue) and the dendrite (red). (a2) Z-projection of the imaged dendritic segment (scale bar corresponds to 2 μm). Asterisks mark imaged spines and dendritic segment. (a3) Time plots of normalized single sweep amplitudes. (b) The averaged baseline amplitude is plotted against bAP-Ca2+ transient enhancement 15 to 20 min after bAP stimulation onset (r = -0.45, n = 92/43 spines/cells, p < 0.0001, Spearman’s rank order test). Spines with pre induction amplitudes <0.041 ∆G/R are depicted in black (n = 53/31 spines/cells), spines with pre-induction amplitudes >0.041 in blue (n = 39/22 spines/cells). (c) Time plot of normalized bAP-Ca2+ transients demonstrates selective enhancement of spines with baseline amplitudes <0.041 ∆G/R (black) when compared to dendrites (red) and spines with baseline amplitudes >0.041 ∆G/R (blue). Interval 15 to 20 min after bAP stimulation onset used for quantification of normalized enhancement is shaded in grey. (d1) Bar graph of normalized enhancement 15 to 20 min after bAP stimulation onset. Enhancement for spines with baseline ∆G/R<0.041 (black, +33 ± 6%, n = 53/31 spines/cells) is significantly larger than in spines with baseline ∆G/R>0.041 (blue, -1 ± 3%, n = 39/22 spines/cells, p < 0.001) and dendrites adjacent to spines with baseline ∆G/R<0.041 (red, -5 ± 5%, n = 23/23, dendrites/cells, p < 0.001, Kruskal Wallis Test with Dunn’s posthoc comparison). (d2) Cumulative distribution plot of normalized enhancement 15 to 20 min after bAP stimulation onset (shaded grey in c). Data are expressed as mean SEM *** p < 0.001.
Fig 4.
bAP-Ca2+ transient enhancement depends on neuronal output.
(a) Raster plot of a 10 min in vivo extracellular recording from a freely moving p19 rat in layer 2 of the MEC. Grey bars correspond to single spikes. AP doublets with frequencies >100 Hz are indicated in red. (b) Diagram of experimental timelines. Grey and black vertical bars correspond to test stimulus doublets (short bars) and the ten 5 AP bursts administered after baseline (long bars). Dark green vertical bars depict doublet test stimuli delivered every 60 s. A 6 min baseline measurement was followed by a stimulus-free interval of 5 min. We then again measured the bAP-Ca2+ transients evoked by the doublet test stimulus. Light green bars correspond to three doublet test stimuli delivered at an interval of 120s and followed by a 10 min stimulus-free interval. After that, six doublets were measured at a 60 s interval. Shorter pink horizontal bars correspond to singlets. For baseline measurements, three singlet test stimuli were delivered at an interval of 120 s and followed by a 10 min stimulus-free interval. After that, six singlets were measured at a 60 s interval. Interval used for normalized post/pre ratios of bAP-Ca2+ transient enhancement is shaded in grey. (c1) Z-projection of the imaged dendritic segment (scale bar corresponds to 1 μm). (c2) Singlet evoked averaged bAP-Ca2+ transients from two neighbouring spines (top and middle). Bottom trace refers to representative underlying APs. (d1) Plot of normalized bAP-Ca2+ transient enhancement 15 to 20 min after bAP stimulation onset in spines with baseline ∆G/R <0.041. In the singlet experiments, the doublet response was measured at 20 min to permit grouping for comparison with the other doublet responses. bAP-Ca2+ transient enhancement in the control group (black, n = 53/31 spines/cells) is significantly larger than in the spine group where singlets were applied (-16.5%, n = 16/11 spines/cells, magenta, p < 0.001, Kruskal Wallis Test with Dunn’s posthoc comparison). Reduced enhancement upon application of doublets when 5 AP bursts were omitted (27 ± 8%, n = 10/6 spines/cells, dark green) and doublet number further reduced (14 ± 5%, n = 16/12 spines cells, light green). Both conditions were not significantly different from controls (n.s., Kruskal Wallis Test with Dunn’s posthoc comparison). (d2) Cumulative distribution plot of normalized bAP-Ca2+ transient enhancement. Dataset corresponds to d1. Data are expressed as mean SEM *** P < 0.001.
Fig 5.
bAP-Ca2+ transient enhancement is independent of synaptic transmission and depends on bAP-mediated Ca2+ influx.
(a1) Z-projection of the imaged spine (scale bar corresponds to 2 μm). Asterisk marks imaged spine. (a2) Averaged baseline bAP-Ca2+ transients 0 to 5 min after bAP stimulation onset (grey traces) and averaged bAP-Ca2+ transients 15 to 20 min after bAP stimulation onset (red) of a spine with synaptic transmission blocked using 1 μM Gabazine, 100 μM APV, 20 μM NBQX, and 500 μM (S)-MCPG/25μM MPEP and 100 μM LY 367385. (b1) Time plot of normalized doublet evoked bAP-Ca2+ transient enhancement in spines with small baseline amplitudes comparing control (black) spines to spines with synaptic transmission blocked (red). One doublet was applied every 60 s, the data is plotted in 3 min bins. In the synaptic transmission blocked group, doublets were applied 11 to 14 min after stimulation onset, but not imaged. (b2) Cumulative distribution plot of normalized bAP-Ca2+ transients enhancement 15 to 20 min after bAP stimulation onset in spines with small baseline amplitudes. Control spine enhancement (black, +33 ± 6%, n = 53/31 spines/cells) is not significantly different from spines with synaptic transmission blocked (red, +32 ± 10%, n = 17/9 spines/cells, p = 0.9, Mann Whitney U test). (c1) Z-projection of the imaged spine (scale bar corresponds to 2 μm). Asterisk marks imaged spine.(c2) Averaged baseline bAP-Ca2+ transients 0 to 5 min after bAP stimulation onset (grey traces) and averaged bAP-Ca2+ transients 15 to 20 min after bAP stimulation onset (blue) of a spine using 500 μM fluo-5F as a Ca2+ indicator. (d1) Time plot of normalized doublet evoked bAP-Ca2+ transient enhancement in spines comparing control spines measured with fluo-4FF (black) to spines measured with fluo-5F (blue). Fluo-5F data is selected based on the median baseline value of all spines with fluo-5F to match the fluo-4FF control group with small baseline bAP-Ca2+ transients. One doublet was applied every 60 s, the data is plotted in 3 min bins. In the fluo-5F group, doublets were applied 11 to 14 min after stimulation onset, but not imaged. (d2) Cumulative distribution plot of normalized bAP-Ca2+ transients enhancement 15 to 20 min after bAP stimulation onset in spines with small baseline amplitudes. Fluo-5F data is selected based on the median baseline value of all spines with fluo-5F to match the fluo-4FF control group with small baseline bAP-Ca2+ transients. Spines in the small pre-burst bAP-Ca2+ transient group also showed no enhancement with fluo-5F (blue, 1 ± 3%, n = 35/14 spines/cells) compared to the fluo-4FF control group (black, +33 ± 6%, n = 53/31 spines/cells; p < 0.001, Mann Whitney U Test). *** p < 0.001.
Fig 6.
bAP-Ca2+ transient enhancement depends on Ca2+ release from intracellular stores.
(a1) Z-projection of the imaged spine segment (scale bar corresponds to 2 μm). Asterisk marks imaged spine. (a2) Averaged baseline bAP-Ca2+ transients 0 to 5 min after bAP stimulation onset (grey) and averaged bAP-Ca2+ transients 15 to 20 min after bAP stimulation onset (red) of a spine in the presence of 30 μM CPA. (a3) Time plot of normalized doublet evoked bAP-Ca2+ transient enhancement in spines with small baseline amplitudes comparing control (black) spines to spines in the presence of 30 μM CPA (red). One doublet was applied every 60 s, the data is plotted in 3 min bins. (b1) Z-projection of the imaged spine segment (scale bar corresponds to 2 μm). Asterisk marks imaged spine. (b2) Averaged baseline bAP-Ca2+ transients 0 to 5 min after bAP stimulation onset (grey) and averaged bAP-Ca2+ transients 15 to 20 min after bAP stimulation onset (blue) of a spine in the presence of 100 μM ryanodine. (b3) Time plot of normalized doublet evoked bAP-Ca2+ transient enhancement in spines with small baseline amplitudes comparing control (black) spines to spines in the presence of 100 μM ryanodine (blue). One doublet was applied every 60 s, the data is plotted in 3 min bins. (c1) Z-projection of the imaged spine segment (scale bar corresponds to 2 μm). Asterisk marks imaged spine. (c2) Averaged baseline bAP-Ca2+ transients 0 to 5 min after bAP stimulation onset (grey) and averaged bAP-Ca2+ transients 15 to 20 min after bAP stimulation onset (green) of a spine in the presence of 10 μM Xestospongin-C. (c3) Time plot of normalized doublet evoked bAP-Ca2+ transient enhancement in spines with small baseline amplitudes comparing control (black) spines to spines in the presence of 10 μM Xestospongin C (green). One doublet was applied every 60 s, the data is plotted in 3 min bins. (d1) Bar graph of normalized enhancement 15 to 20 min after bAP stimulation onset for spines with small baseline amplitudes in control spines (black, +33 ± 6%, n = 53/31 spines/cells), CPA (red, 0 ± 6%, n = 24/10 spines/cells), ryanodine (blue, -2 ± 7%, n = 27/16 spines/cells) and Xestospongin C (green, +14 ± 4%, n = 56/18 spines/cells). In comparison to the control group, CPA and ryanodine blocked the enhancement (p < 0.01 and p < 0.001, respectively), whereas enhancement was not significantly reduced by Xestospongin C (n.s., Kruskal-Wallis test with Dunn’s posthoc correction). (d2) Cumulative distribution plot of normalized bAP-Ca2+ transient enhancement. Dataset corresponds to d1. Data are expressed as mean SEM ** p < 0.01, *** p < 0.001.
Fig 7.
Stores play a role in induction but not in expression of bAP-Ca2+ transient enhancement.
(a1) Z-projection of the imaged dendritic segment (scale bar corresponds to 2 μm). Asterisk marks imaged spine. (a2) Averaged traces of spine bAP-Ca2+ transients: baseline (grey, 0 to 5 min after onset of the experiment), pre-CPA wash-in (black, 10 to 15 min after onset), and post-CPA wash-in (red, 16 to 20, 21 to 25 and 26 to 30 min after the onset of the experiment). Red line indicates wash-in of 30 μM CPA. (b1) Time plot of preselected enhanced spines (enhancement >20% between 15 and 20 min after starting the experiment) comparing controls (black) and spines with CPA wash-in (red) 15 min after the onset of the experiment. Interval used for comparison between control spines and spines where CPA was washed in is shaded in grey. (b2) Cumulative distribution plot of normalized bAP-Ca2+ transient enhancement in time-matched controls and 10 min after CPA and combined CPA and ryanodine wash-in in spines (corresponds to grey area in b1). Control spine enhancement (black, +63 ± 11%, n = 14/10 spines/cells) is not significantly different from spines treated with 30 μM CPA alone (red, +42 ± 11%, n = 17/13 spines/cells, p = 0.07, one-tailed Mann Whitney U test). Combined treatment with CPA and ryanodine was tested in a small population of spines (+57 ± 23%, n = 6/3 spines/cells, magenta). Data are expressed as mean SEM.
Fig 8.
Modelling of RyR mediated intraspine Ca2+ nanodomains.
(a) Simulated linescan of the time course of [Ca2+] along a 200 nm line between the RyR located within the spine and VGCCs distributed across the opposite membrane surface. Black triangles correspond to bAPs in a 5 AP burst. Simulations were run with 200 μM fluo-4FF (top), 200 μM fluo-5F (middle) and 500 μM fluo-5F (bottom) as buffers. (b) Same as in (a) with RyRs omitted (VGCCs only). (c) [Ca2+] over time at a distance of 50 nm from the RyR channel pore (lines marked with x and o in a and b). Straight lines correspond to a scenario with RyRs and VGCCs, dotted lines to a scenario with VGCCs only. Different colours correspond to 200 μM fluo-4FF (black, top), 200 μM fluo-5F (blue, middle), and 500 μM fluo-5F (green, bottom) as buffers. (d) Spatial distribution of [Ca2+] maxima after 5th bAP with VGCC activation only (dotted line) or after RyR opening following 2nd bAP (solid lines) under different buffering conditions: 200 μM fluo-4FF (black), 200 μM fluo-5F (blue) and 500 μM fluo-5F (green). (e) Effect sizes. Plot of normalized bAP-Ca2+ transient enhancement 15 to 20 min after stimulation onset in all spines measured under different conditions. Black: 200 μM fluo-4FF, +19 ± 4%, n = 92/43 spines/cells; blue: 200 μM fluo-5F, +4 ± 5%, n = 31/7 spines/cells; grey: CPA (VGCCs only), -1 ± 5%, n = 31/11 spines/cells; green: 500 μM fluo-5F, -10 ± 3%, n = 68/24 spines/cells.