Fig 1.
Subthreshold dendritic integration in granule cells.
a: Left: Z-projection of 2-photon scan of representative cell, top part shows magnified inset with uncaging spots labeled by blue stars. Right: Somatic compound uEPSPs and global Na+-spikes generated by simultaneous activation of 1, 3, 5, 7, and 9 spines (AP). Inset: Single uEPSPs recorded at the soma (see Materials and methods). b: sO/I of n = 29 individual experiments. Gray lines and circles ○: Sublinear to linear integration. Black lines and ○: Supralinear integration (solid circles: data from a). Blue lines and ○: Averaged sO/I of 1 to 9 coactivated spines across all GCs. Dashed line: linear y = x. Gray lines: Cut-off supra- and sublinear regime for classification of cells (y = 1.2x, y = 0.8x, see Materials and methods). 1 data point of 1 experiment exceeds the scale. c: sO/I cumulative plot of experiments in b with data arranged from -9 to 0 spines relative to global Na+-spike threshold. Significance levels refer to O/I EPSP amplitude ratio distributions with means beyond the linear regime (0.8–1.2) tested against linearity (see inset, Materials and methods). Blue diamonds ◆: average sO/I of all experiments (see also inset): Supralinear at −2 (p = 0.006) and 0 spines (p < 0.001, mean O/I ratio 1.53 ± 0.63). Black diamonds ◆: average of supralinear sO/Is only (n = 19), significantly exceeding linear summation beyond −3 spines: −2 spines (p < 0.001), −1 spine (p = 0.007), 0 spine (i.e. at threshold, p < 0.001, mean O/I ratio 1.86 ± 0.52). Gray diamonds ◆: average of sublinear to linear sO/Is only (n = 10), significantly below linear summation below −3 spines: −7 spines (p = 0.027), −6 spines (p = 0.008), −5 spines (p = 0.020), −4 spines (p = 0.021, mean O/I ratio 0.79 ± 0.37). Inset: average O/I ratios of all experiments versus spine number relative to global Na+-spike (AP) threshold. AP, action potential/global Na+-spike; EPL, external plexiform layer; GCL, granule cell layer; MCL, mitral cell layer; O/I, output/input; sO/I, subthreshold O/I relationship; uEPSP, uncaging-evoked excitatory postsynaptic potential. In all figures, data means are presented ± standard deviation; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2.
Dendritic Ca2+-spikes: A nonlocal mode of dendritic activation.
a: Left: Scan of representative granule cell (as in Fig 1). Sp1 and D1-D4 indicate line scan sites, and stars indicate uncaging spots. Right, bottom: Somatic Vm traces of single-spine and multisite uncaging (global Na+-spike truncated). Right, top: Averaged ΔF/F in the spine upon activation of 1, 6, 10, and 10 spines at the respective locations. Right, middle: Averaged ΔF/F in the dendrite measured at increasing distance from the activation site upon subthreshold activation of 6 and 10 spines. Truncated uncaging artefact in fluorescence traces. Gray: signals subthreshold for Ca2+-spike, green: at Ca2+-spike threshold, black: EPSPs and associated ΔF/F signals just subthreshold for global Na+-spike threshold, magenta: suprathreshold for global Na+-spike. b: Dendritic Ca2+ signals versus distance from the center of the stimulated spine set. Green circles ○: responses at Ca2+-spike threshold, black circles ○: responses for EPSPs just subthreshold for the global Na+-spike threshold or evoked by maximal available spine number. Data from 12 cells with ΔF/F data imaged at various distances from the set of stimulated spines. Solid symbols: Data from cell in a. Green and black lines: Exponential fits to respective data sets (at Ca2+-spike threshold: decay constant ± SD: λ = 61 ± 30 μm, ΔF/F(200 μm) = 0%, n = 38 data points; at or closer to global Na+-spike threshold: λ = 69 ± 47 μm, ΔF/F(200 μm) = 8%, n = 44 data points). c: Comparison of spine numbers (left) and somatic thresholds (right; both n = 28, p < 0.001, paired t-test) for Ca2+-spikes and global Na+-spikes. All error bars denote standard deviation, also in panels d, e, f. d: Mean somatic cuEPSP amplitudes with spine numbers aligned relative to Ca2+-spike threshold ([0]Ca2+-spike; n = 25). Difference between [−2/−1] and [0] not significantly different from extrapolated linear fit (p = 0.29; Wilcoxon test, see Materials and methods for details of the test, see S2A Fig for data points from individual experiments). Gray symbols: subthreshold Ca2+-spike, green symbols: suprathreshold Ca2+-spike, dashed line: linear fit of subthreshold mean amplitudes, also for e, f. e: Mean O/I ratios aligned relative to Ca2+-spike, not significantly different from subthreshold (p = 0.78, n = 25). f: Kinetics of compound uEPSPs (n = 25 granule cells, see S2A Fig for data points from individual experiments): No significant increase above extrapolated linear fits at Ca2+-spike threshold for rate of rise (left, p = 0.52, n = 25) or rise time (middle, p = 0.49, n = 25) or half duration τ_1/2 (right, p = 0.42, n = 22). cuEPSP, compound uEPSP; EPL, external plexiform layer; GCL, granule cell layer; MCL, mitral cell layer; n.s., not significant; O/I, output/input; uEPSP, uncaging-evoked excitatory postsynaptic potential.
Fig 3.
D-spikes in approximately two thirds of granule cells: Spikelets and/or increased rate of rise associated with onset of supralinearity in O/I plot.
a: Examples of somatic spikelets recorded from 2 different cells at different numbers of coactivated spines. Green traces and spine numbers: Ca2+-spike threshold. Black traces and numbers: Na+-spikelets. Magenta traces and numbers: Full-blown global Na+-spikes, truncated (Na+-spike: short for global Na+-spike, also in c). b: Example cuEPSP Vm recording in rising phase (top) and first derivative (bottom). Colors as in a, black here: D-spike, indicated by increase in rate of rise. c: Comparison of spine numbers sufficient to elicit Ca2+-spike, D-spike and global Na+-spike in the same granule cell (n = 18 cells; F(2,53) = 20.75, p < 0.001, Ca2+-spike: 5.1 ± 1.4, D-spike: 6.5 ± 2.7, global Na+-spike: 8.9 ± 1.7 Holm–Sidak post hoc: Ca2+-spike versus D-spike: p = 0.027, Ca2+-spike versus global Na+-spike: p < 0.001, D-spike versus global Na+-spike: p < 0.001). d: Mean somatic cuEPSP amplitudes with spine numbers aligned relative to D-spike threshold for granule cells with supralinear sO/Is (n = 18; x-axis as in panel below). Difference between [−2/−1] and [0] highly significantly different from extrapolated linear fit (p < 0.001; Wilcoxon test, see Materials and methods, see S2B Fig for data points from individual experiments). Gray symbols: subthreshold D-spike, black symbols: suprathreshold D-spike, dashed line: linear fit of subthreshold mean amplitudes, also for e, f. e: Mean O/I ratios aligned relative to D-spike (x-axis as in panel below). Increase from 1.03 ± 0.13 to 1.77 ± 0.54, not tested, because the onset of supralinear O/I ratios was the criterion for the selection of cells and the setting of the D-spike threshold. f: Kinetics of cuEPSPs (n = 18 cells): Highly significant increases beyond extrapolated linear fits at D-spike threshold for rate of rise (left, p = 0.002, n = 18), rise time (middle, p < 0.001, n = 18) and half duration τ_1/2 (right, p = 0.009, n = 16). See S2B Fig for data points from individual experiments. Amp, amplitude; D-spike, dendritic Na+-spike; O/I, output/input; rel, relative; cuEPSP, compound uncaging-evoked excitatory postsynaptic potential.
Fig 4.
Additional ΔCa2+ in spine and dendrite caused by nonlocal spikes.
a: Left: Scan of representative granule cell. Sp1 and D indicate line scan sites, stars indicate uncaging spots. Right: Somatic Vm recordings (top) and line scans in spine (Sp1) and dendritic location (D) for increasing spine numbers (bottom). Gray traces: below Ca2+-spike, green traces: at Ca2+-spike threshold; black traces: at and above D-spike threshold; magenta traces: suprathreshold for global Na+-AP. Na+-AP truncated for clarity. b: Spine Ca2+ signals ΔF/F normalized to Sp1-4 (see Materials and methods) and aligned to Na+-AP threshold (n = 33 spines in 16 GCs); Gray circles ○: individual spines, solid black ●: mean; open black ○: spine from a, open magenta ○: AP data from individual spines; solid magenta with black outline ●: mean global Na+-spike data (responses with more than 1 AP were not taken into account). Gradual increase from −6 spines onwards of spine ΔF/F with highly significant additional increase upon AP generation (n = 20 pairs, Wilcoxon, p < 0.001). c: Dendritic Ca2+ signals ΔF/F normalized to mean above Ca2+-spike threshold and below global Na+-spike threshold (see Materials and methods; n = 19 cells). Symbols as in right panel, with squares instead of circles. Gradual increase from −5 spines onwards with significant additional increase upon global Na+-spike generation (n = 12 pairs, Wilcoxon, p = 0.001). d: Spine ΔF/F normalized as in b, arranged relative to Ca2+-spike threshold (left panel, n = 26 in 14 cells, Wilcoxon test, p < 0.001, see Materials and methods) and relative to D-spike threshold (right panel, n = 19 in 10 cells, p < 0.001). See S2A and S2B Fig for individual data points. Gray symbols: subthreshold Ca2+-spike/D-spike, green symbols: suprathreshold Ca2+-spike, black symbols: suprathreshold D-spike, dashed line: linear fit of subthreshold mean amplitudes, also for e. e: Dendrite ΔF/F normalized as in c, arranged relative to Ca2+-spike threshold (left panel, n = 17, significance not tested, because increase in dendritic ΔF/F above noise level was criterion for onset of Ca2+-spike) and relative to D-spike threshold (right panel, n = 12, Wilcoxon, p = 0.015, see S2B Fig for individual data points). Symbols as in d, with squares instead of circles. AP, action potential; D-spike, dendritic Na+-spike.
Fig 5.
Molecular mechanisms of subthreshold integration: Navs.
a: Example Nav blockade experiment. Top left: Stimulated spine set with line scan sites Sp1, D, and uncaging spots. Top right: Somatic Vm recording of compound uEPSPs, below spine Sp1 and dendrite D ΔF/F for 1 and 10 spines. Gray traces: control. Blue traces: TTX (0.5–1 μM). b: Effect (n = 12 cells) of 0.5–1 μM TTX on somatic compound uEPSP amplitude upon activation of 1, 2, 4, 6, 8, and 10 spines. Repeated measures 2-way ANOVA (Materials and methods, also in d,f, h): no interaction effect (spine # x TTX): F(5,119) = 2.54, p = 0.091; no TTX effect: F(1,119) = 0.97, p = 0.352. c: Cumulative effect (n = 12 cells) of Nav blockade on averaged sO/I. No interaction effect (spine # x TTX): F(5,99) = 1.60, p = 0.195; no TTX effect: F(1,99) = 0.84, p = 0.385 (n.s. above). Lines and grey asterisks as in Fig 1C. The average O/I ratio for 8 spines was highly significantly supralinear in control (** above data points, p < 0.01) and significantly increased versus 6 spines (* in inset, p < 0.05, Wilcoxon test). In TTX, O/I ratios were still significantly supralinear (* below data points, p < 0.05 for all), but the increase from 6 to 8 spines disappeared (inset, n.s. below). d: Effect of Nav blockade on average ΔF/F in spine 1 (Sp1, left, n = 25 spines in 11 cells) and dendrites (D, right, n = 12) upon activation of 1–10 spines. No interaction effect on spine 1 ΔF/F (spine # x TTX): F(5,239) = 1.69, p = 0.145; TTX effect: F(1,239) = 15.16, average reduction to 0.89 ± 0.54 of control, p < 0.001. No interaction effect on dendrite ΔF/F from 4 spines onwards (spine # x TTX): F(3,79) = 0.46, p = 0.715; TTX effect: F(1,79) = 9.29, average reduction to 0.75 ± 0.28 of control, p = 0.014. Asterisks above error bars: significance of difference TTX versus control. e: Example for effect of TTX on compound uEPSP kinetics below and at D-spike threshold (4 and 6 coactivated spines in this granule cell, respectively). Left traces: Vm, right traces: dVm/dt. Gray/black traces: control. Blue traces: TTX (0.5–1 μM). Thin lines: 4 spines, thick lines: 6 spines. Note the reduction in maximal rate of rise above threshold but not subthreshold. f: Cumulative data for effect of TTX (n = 7 cells with supralinear sO/Is) on compound uEPSP rate of rise (left) and rise time (right). Repeated measures two-way ANOVA (see Materials and methods, also below): no interaction effect on rate of rise (spine # x TTX): F(3,55) = 3.06, p = 0.055; TTX effect: F(1,55) = 8.25, p = 0.028. Interaction effect on rise time (spine # x TTX): F(3,55) = 12.49, p < 0.001. Asterisks indicate significance of differences between TTX and control (*p = 0.028, *** p < 0.001). Asterisks at bottom indicate significance of differences of parameter increases from -2 to 0 between control and TTX (Wilcoxon test; rate of rise: p < 0.05 [W = 17, nsr = 6], rise time: p < 0.01 [W = 28, nsr = 7]). See S2C Fig for individual data points. g: Example for effect of TTX on spine 1 and dendrite ΔF/F below and above D-spike threshold (4 and 6 coactivated spines, respectively; same cell as in e, same color code). Note the reduction in spine 1 ΔF/F by TTX at threshold but not subthreshold. h: Cumulative data for effect of TTX (n = 7 cells with D-spike) on ΔF/F in spines (left, n = 13) and dendrite (right, n = 7). Full symbols: control, open symbols: TTX. Repeated measures 2-way ANOVA (see Materials and methods): interaction effect on spine ΔF/F (spine # x TTX): F(3,103) = 3.20, p = 0.035. No interaction effect on dendrite ΔF/F: F(3,55) = 0.66, p = 0.588; no TTX effect: F(1,55) = 5.10, p = 0.065. Asterisks above indicate significance of differences between TTX and control. Asterisks at bottom indicate significance of differences of ΔF/F from −2 to 0 between control and TTX (Wilcoxon test; spine S: p = 0.029 [W = 55, nsr = 13], dendrite: not significant [W = −2, nsr = 5]). See S2C Fig for individual data points. arith., arithmetic; D-spike, dendritic Na+-spike; n.s., not significant; sO/I, subthreshold output/input; TTX, tetrodotoxin; uEPSP, uncaging-evoked excitatory postsynaptic potential.
Fig 6.
Molecular mechanisms of subthreshold integration: NMDA receptors.
a: Example NMDAR blockade experiment with strong NMDAR component. Top left: Stimulated spine set with line scan sites Sp1, D, and uncaging spots. Top right: Somatic Vm recording of uEPSP, below spine (Sp1), and dendrite (D) ΔF/F for 1 and 10 spines. Gray traces: control. Yellow traces: APV (25 μM). b: Cumulative effect (n = 8 cells) of 25 μM APV on somatic cuEPSP amplitude upon activation of 1, 2, 4, 6, 8, and 10 spines. Interaction effect (spine # x APV): F(5,95) = 8.08, p < 0.001. Black asterisks: significance of difference APV versus control. c: Effect of NMDAR blockade on averaged sO/I upon activation of 1–10 spines. Interaction effect (spine # x APV): F(5,95) = 3.37, p = 0.014, n = 8. Black asterisks above data points: significance of difference APV versus control. Lines and grey asterisks as in Fig 1C. Control O/I ratios from 4 spines upwards were supralinear, which all became linear in APV. d: Effect of NMDAR blockade on average ΔF/F in Sp1 (left, n = 15 spines) and D (right, n = 8) upon activation of 1–10 spines. Interaction effect on spine ΔF/F (spine # x APV): F(5,179) = 6.36; p < 0.001. Interaction effect on dendrite ΔF/F (spine # x APV): F(5,95) = 8.34, p < 0.001. Asterisks: significance of difference APV versus control; arith., arithmetic; comp., compound; cuEPSP, compound uncaging-evoked excitatory postsynaptic potential; sO/I, subthreshold output/input.
Fig 7.
Molecular mechanisms of subthreshold integration: Low- and high-voltage-activated Cavs.
a: Example low-voltage-activated Cav blockade experiment. Top left: Stimulated spine set with line scan sites Sp1, D, and uncaging spots. Top right: Somatic Vm recording of uEPSP, below spine (Sp1) and dendrite (D) ΔF/F for 1 and 10 spines. Grey traces: control. Green traces: mib, 10 μM. b: Cumulative effect (n = 11 cells) of mib on somatic compound uEPSP amplitude upon activation of 1, 2, 4, 6, 8, and 10 spines. Repeated measures 2-way ANOVA (see Materials and methods): interaction effect (spine # x mib) F(5,131) = 3.88, p = 0.010. Asterisks: significance of difference mib versus control. c: Cumulative effect (n = 8 cells) of LVA Cav blockade on averaged sO/I upon activation of 1–10 spines. Interaction effect (spine # × mib) F(4,69) = 4.69, p = 0.006. Lines as in Fig 1C. Black asterisks above error bars indicate significance of differences between mib and control. Gray and green asterisks/significance levels above error bars refer to O/I ratio distributions with means beyond the linear regime (0.8–1.2) tested against linearity (*p < 0.05, as in Fig 1C). Integration was significantly supralinear at 10 spines for both control and mib. The increase in O/I ratios between 8 and 10 spines was also significant but significantly smaller in mib versus control (inset *, Wilcoxon test). d: Effect of LVA Cav blockade on average ΔF/F in Sp1 (left, n = 26 spines in 11 cells) and D (right, n = 11) upon activation of 1–10 spines. No interaction effect on spine ΔF/F (spine # x mib): F(5,311) = 0.26, p = 0.933; mib effect: F(1,311) = 60.16, p < 0.001. No interaction effect on dendrite ΔF/F (spine # x mib): F(3,87) = 1.11, p = 0.359; mib effect: F(1,87) = 15.84, p = 0.003. Asterisks: significance of difference mib versus control. e: Example for subsequent blockade of low- and high-voltage-activated Cavs on ΔF/F in spine (Sp1) and dendrite (D) for 1 and 10 spines. Top left inset: Scan of stimulated spine set with indicated line scan sites Sp1, D and uncaging spots. Gray traces: Control. Green traces: mib (10 μM). Dark green: added Cd2+ (100 μM). f: Effect of subsequent low- and high-voltage-activated Cav blockade on somatic compound uEPSP amplitude upon activation of 1, 6, and 10 spines (n = 4 cells). g: Effect of subsequent low- and high-voltage-activated Cav blockade upon activation of 1, 6, and 10 spines on ΔF/F in Sp1 (left, n = 8 spines in 4 cells) and dendrite (right, n = 4). No interaction effect of Cd2+ wash-in after mib on spine ΔF/F (spine # × Cd2+): F(2,47) = 1.51, p = 0.254; Cd2+ effect: F(1,47) = 14.02, p = 0.007. Asterisks: significance of difference mib + Cd2+ versus mib only. arith., arithmetic; Cav, voltage-gated Ca2+ channel; LVA, low-voltage-activated; mib, mibefradil; O/I, output/input; uEPSP, uncaging-evoked excitatory postsynaptic potential.
Fig 8.
Impact of morphological variables on threshold spine number for Ca2+-spike and global Na+-spike generation.
Ca2+-spike data (n = 47 spine sets) are denoted by green circles ○ and global Na+-spike data (n = 31 spine sets) by magenta triangles Δ. D-spike data not included for clarity (but see Table 1 and data repository). Linear correlation indicated by correlation coefficient r. See Table 1 for power of regressions. a: Influence of mean spine distance from the MCL on spine # to elicit Ca2+-spikes (r2 = 0.02, p = 0.17) and global Na+-spikes (r2 = 0.00, p = 0.89). b: Influence of the mean spine neck length of activated spine sets on spine # to elicit Ca2+-spikes (r2 = 0.03, p = 0.12) and global Na+-spikes (r2 = 0.00, p = 0.35). c: Influence of the spatial distribution of activated spines on spine # to elicit Ca2+-spikes (r2 = 0.26, p < 0.001) and global Na+-spikes (r2 = 0.08, p = 0.06). d: Influence of the mean spine head size (estimated diameter) that the spine set is distributed across on spine # to elicit Ca2+-spikes (r2 = 0.00, p = 0.75) and global Na+-spikes (r2 = 0.00, p = 0.86). e: Influence of number of different dendritic branches that the spine set is distributed across on spine # to elicit Ca2+-spikes (r2 = 0.31, p < 0.001) and global Na+-spikes (r2 = 0.00, p = 0.91). f: Influence of age of animal PND on spine # to elicit Ca2+-spikes (r2 = 0.00, p = 0.83) and global Na+-spikes (r2 = 0.00, p = 0.58). MCL, mitral cell layer; n.s., not significant; PND, postnatal day.
Table 1.
Regression between coactivated threshold spine numbers for Ca2+-spike, D-spike, and global Na+-spike and various morphological variables and input patterns (see Materials and methods).
Fig 9.
Summary of findings: Active signal types in granule cell dendrites upon simultaneous stimulation of apical dendritic spines.
Graphical summary of main results. Left: Spine #: number of coactivated spines. Somatic Vm: somatic membrane potential response; no shape changes observed here for regional dendritic Ca2+ spikes. Channels/mechanisms: components involved in generating the respective Ca2+ and Vm signals, located in the excited spines and, for all nonlocal signals, excited dendritic segments. Right: Color scale for ΔCa2+ entry. AMPAR, AMPA receptor; AP, action potential; D-spike, dendritic Na+-spike; GC, granule cell; HVA, high voltage activated; LVA, low voltage activated; NMDAR, NMDA receptor.