Article Figures & Data
Figures
- Figure 1.
Depth-dependent co-occurrence of PI+ cells and GCaMP-filled neurons in acute neonatal brain slices. A, Experimental design. B, Representative MIPs (15 µm depth) of images acquired at different depths. B’, Example of a PI+ cell, a GCaMP-filled neuron, and colocalized puncta. False-positive detections were eliminated using a ratiometric approach (Extended Data Fig. 1-1). PI labeling due to cytotoxic injury was validated by staurosporine-induced apoptosis in acute brain slices (Extended Data Fig. 1-2). C, Reduction in the number of PI+ cells and GCaMP-filled neurons with depth in acute brain slices (RM one-way ANOVAs, PI+: F(2,52.2) = 42.7, p < 0.0001, GCaMP-filled: F(2.58,67) = 18.8, p < 0.0001). D, Normalized fraction of total PI+ cells across different depths. Single exponential decay fits show a similar depth-dependent decrease in PI+ cells and GCaMP-filled neurons (τPI+: 41 ± 10 µm, R2: 0.75, τGCaMP-filled: 65 ± 25 µm, R2: 0.54; inset, unpaired t test). E, Reduction in colocalized puncta (PI+ and GCaMP-filled) with depth (R2: 0.63, τ: 30 µm). Right axis, fractional plot (R2: 0.58, τ: 21 µm, 79% in the first 30 µm). F, Significant correlation between PI+ cells and GCaMP-filled neurons, identified from individual MIPs, normalized to slice totals (Spearman's ρ = 0.66, p < 0.0001, linear regression slope: 0.75, R2 = 0.6, p < 0.0001). PI+ cells and GCaMP-filled neurons show no cortical layer-specific localization (Extended Data Fig. 1-3) or postnatal age-dependent distribution change across depth (Extended Data Fig. 1-4). However, their distribution across depth depends on the acute slice thickness (Extended Data Fig. 1-4). Mean ± 95% CI. Scale bar, 100 µm. See also Movies 1 and 2.
- Figure 2.
Spatial correlation between PI+ cells and the neuropil Ca2+ signal. A, Representative MIPs showing PI+ cells and neuropil GCaMP6s signal in an acute neonatal brain slice across different depths. B, Depth-dependent decrease in the median GCaMP6s intensities within the neuropil (RM one-way ANOVA, F(2,52.2) = 42.7, p < 0.0001, τneuropil: 131 ± 57 µm, R2: 0.45). PI+ data and fit from Figure 1D are displayed for comparison. C, Number of PI+ cells and median neuropil GCaMP intensities of individual MIPs (both normalized) showed a significant correlation (Spearman's ρ = 0.68, p < 0.0001, linear regression slope: 1.3, R2 = 0.4, p < 0.0001). D, Placement of identical regions of interest (ROIs) in a 4 × 4 grid over a MIP. E, The number of PI+ cells and median neuropil GCaMP intensities of individual grids (normalized) showed a significant correlation (Pearson's r = 0.61, p < 0.0001, linear regression slope: 2.7, R2 = 0.4, p < 0.0001). Numbers in parentheses represent data points in each quadrant. Mean ± 95% CI. Scale bar, 100 µm.
- Figure 3.
Prolonged incubation times increase the number of PI+ cells and GCaMP-filled neurons in deeper tissue. A, Top, Experimental design. Bottom, MIPs representing different depths acquired from slices after 0–1 and 3–5 h incubations. B, Normalized number of PI+ cells across depth at different incubation times (two-way RM ANOVA, interaction: F(27,198) = 1.58, p = 0.043; treatment: F(3,22) = 2.75, p = 0.07). C, Increase in the percentage of PI+ cells with increasing incubation times. R2 and τ from a single exponential fit to the means. D, Similarly, a slower exponential decay of CaMP6s-filled neurons was observed with more extended incubation (R2 and τ of exponential decay fit to the means). E, Increase in the decay constants measured from individual slices (% PI+ cells) during prolonged incubation (one-way ANOVA, F(3,22) = 3.06, p = 0.049, Dunnett's test, 0–1 vs 3–5 h: p = 0.033). Although not significant, the decay constants for the GCaMP-filled neurons also showed a trend toward an increase with prolonged incubations (one-way ANOVA, F(3,18) = 1.7, p = 0.2). Mean ± 95% CI. Scale bar, 100 µm.
- Figure 4.
Activity-dependent Ca2+ dynamics in healthy and GCaMP-filled neurons. A, Experimental design showing placement of a puff pipette on an acute slice. B, Representative images of propagating Ca2+ transient evoked by a brief NMDA puff (125 µM, 100 ms). GCaMP-filled neurons with tonically high baseline Ca2+ signals are indicated by arrows (yellow). C, Heatmap shows Ca2+ transients evoked in multiple healthy neurons, while GCaMP-filled neurons (yellow arrows) showed no evoked Ca2+ activity (n = 12 GCaMP-filled neurons in 3 slices). See Movie 1. D, Experimental design of multiphoton acquisition of 4-AP-induced Ca2+ transients in acute slices. E, Top, Representative trace showing 4-AP-induced whole-field Ca2+ activity in an acute brain slice. Bottom, Heatmap shows 4-AP-induced Ca2+ transients acquired from multiple healthy and GCaMP-filled neurons (yellow arrows). Note that GCaMP-filled neurons showed no Ca2+ transients (n = 27 GCaMP-filled neurons in 4 slices). F, Representative images showing spatiotemporal propagation of a single 4-AP-induced Ca2+ transient. See Movie 2. Scale bar, 100 µm.
- Figure 5.
Depth-dependent alterations in seizure-induced neuronal Ca2+ transients. A, Schematic depicting experimental design. B, Representative traces showing synchronous, whole-field Ca2+ transients obtained at different depths. See Movie 3. Synchronous neuronal Ca2+ activity in deeper tissue showed a significant increase in (C) Ca2+ transient frequency, (D) mean amplitude, and (E) mean area under the curve (AUC), without a significant change in the Ca2+ transient duration measured as full-width at half-maximum (F, FWHM). Wilcoxon matched pairs test, n = 6 slices.
- Figure 6.
Inhibiting calpains during incubation mitigates cell death in acute brain slices. A, MIPs at different depths from brain slices incubated in aCSF alone (top) or with a calpain inhibitor (MDL, bottom). B, MDL significantly reduced the number of PI+ cells across different depths (two-way ANOVA, interaction: F(9,369) = 3.32, p = 0.0006; treatment: F(1,41) = 8.51, p = 0.0057) and the total PI+ cells per slice (inset, unpaired t test). C, Faster decays in the percent of PI+ cells with MDL incubation. R2 and τ of single exponential fits to the means. D, E, MDL-28170 significantly decreased the total number of PI+ cells (two-way ANOVA, interaction: F(3,34) = 0.97, p = 0.41; treatment: F(1,33) = 15.4, p = 0.0004) and also decreased the decay constants measured from individual slices, especially after 3–5 h of incubation (two-way ANOVA, interaction: F(3,33) = 1.4, p = 0.26; treatment: F(1,33) = 14.8, p = 0.0005, Sidak's test, 0.1 vs 3–5 h, p = 0.003). F, Significant reduction in the number of colocalized puncta (PI+ and GCaMP-filled) across tissue depth with MDL (two-way ANOVA, interaction: F(9,369) = 2.17, p = 0.023; treatment: F(1,41) = 4.19, p = 0.047). Inset, significant reduction in total colocalized puncta in individual slices with MDL (Mann–Whitney test, U = 86). G, MDL treatment reduced the number of GCaMP-filled neurons across different depths (two-way ANOVA, interaction, F(9,369) = 1.46, p = 0.038; treatment, F(1,41) = 2.8, p = 0.01) and total GCaMP-filled neurons per slice (inset, unpaired t test). Mean ± 95% CI (median ± IQR for panel F inset). Using an isoform-specific inhibitor, we found that calpain-2 significantly contributes to the inherent trauma-induced injury in acute brain slices (Extended Data Fig. 6-1). Scale bar, 100 µm.
Tables
Figure Structure Type of test Results Fig. 1C Normal RM one-way ANOVA PI+: F(2,52.2) = 42.7, p < 0.0001 GCaMP-filled: F(2.58,67) = 18.8, p < 0.0001 Fig. 1D (inset) Normal Unpaired t test (two-tailed) t = 0.037, df = 32, p = 0.97 Fig. 1F Nonparametric Spearman's correlation Spearman's ρ = 0.66, p < 0.0001 Extended Data Fig. 1-2C Normal Two-way RM ANOVA Interaction: F(9,99) = 4.025, p = 0.0002 Depth: F(9,99) = 16.8, p < 0.0001 Treatment: F(1,11) = 12.3, p = 0.0049 Extended Data Fig. 1-2D Normal Unpaired t test (two-tailed) t = 3.51, df = 11, p = 0.005 Extended Data Fig. 1-2E Normal Unpaired t test (two-tailed) t = 2.76, df = 11, p = 0.02 Extended Data Fig. 1-2F Normal Two-way RM ANOVA Interaction: F(9,99) = 4.15, p = 0.0001 Depth: F(9,99) = 6.18, p < 0.0001 Treatment: F(1,11) = 12.3, p = 0.062 Extended Data Fig. 1-2G Normal Two-way RM ANOVA Interaction: F(9,90) = 2.18, p = 0.034 Depth: F(9,90) = 14.1, p < 0.0001 Treatment: F(1,10) = 0.041, p = 0.93 Extended Data Fig. 1-3C Normal Two-way ANOVA Interaction: F(6,87) = 0.3, p = 0.936 Time point: F(3,87) = 9.2, p < 0.0001 Layer: F(2,87) = 2.42, p = 0.095 Extended Data Fig. 1-3D Normal Two-way ANOVA Interaction: F(6,82) = 0.06, p = 0.99 Time point: F(3,82) = 0.95, p = 0.42 Layer: F(2,82) = 0.78, p = 0.45 Extended Data Fig. 1-4A Normal Two-way RM ANOVA Interaction: F(9,351) = 2.04, p = 0.034 Depth: F(9,351) = 129.4, p < 0.0001 Slice thickness: F(1,39) = 1.74, p = 0.125 Extended Data Fig. 1-4B Normal Two-way RM ANOVA Interaction: F(9,342) = 2.04, p = 0.0007 Depth: F(9,342) = 52.51, p < 0.0001 Slice thickness: F(1,38) = 1.81, p = 0.186 Extended Data Fig. 1-4C Normal Two-way RM ANOVA Interaction: F(9,216) = 0.08, p = 0.99 Depth: F(9,216) = 91.3, p < 0.0001 Age: F(1,24) = 0.36, p = 0.56 Extended Data Fig. 1-4D Normal Two-way RM ANOVA Interaction: F(9,306) = 0.87, p = 0.56 Depth: F(9,306) = 54.16, p < 0.0001 Age: F(1,34) = 10.49, p = 0.0027 Fig. 2B Normal RM one-way ANOVA PI+: F(2,52.2) = 42.7, p < 0.0001 Neuropil: F(2,52.2) = 42.7, p < 0.0001 Fig. 2C Nonparametric Spearman's correlation Spearman's ρ =0.68, p < 0.0001 Fig. 2E Normal Pearson's correlation Pearson's r = 0.61, p < 0.0001 Fig. 3B Normal Two-way RM ANOVA Interaction: F(27,198) = 1.578, p = 0.043 Depth: F(9,198) = 107.1, p < 0.0001 Treatment: F(3,22) = 2.75, p = 0.07 Fig. 3E Normal One-way ANOVA PI+: F(3,22) = 3.06, p = 0.049 Dunnett's multiple-comparisons test 0–1 vs 1–2: p = 0.91, 0–1 vs 2–3: p = 0.99 vs 3–5: p = 0.033 GCaMP-filled: F(3,18) = 1.7, p = 0.2 Dunnett's multiple-comparisons test 0–1 vs 1–2: p = 0.97, 0–1 vs 2–3: p = 0.96 0–1 vs 3–5: p = 0.13 Fig. 5C Nonparametric Wilcoxon matched pairs test W = 21, p = 0.031 Fig. 5D Nonparametric Wilcoxon matched pairs test W = 21, p = 0.031 Fig. 5F Nonparametric Wilcoxon matched pairs test W = 21, p = 0.031 Fig. 5F Nonparametric Wilcoxon matched pairs test W = 8, p = 0.375 Fig. 6B Normal Two-way RM ANOVA Interaction: F(9,369) = 3.32, p = 0.0006 Depth: F(9,369) = 48.1, p < 0.0001 Treatment: F(1,41) = 8.51, p = 0.0057 Fig. 6B (inset) Normal Unpaired t test (two-tailed) t = 3.19, df = 41, p = 0.002 Fig. 6D Normal Two-way ANOVA Interaction: F(3,34) = 0.97, p = 0.41 Depth: F(3,34) = 4.57, p = 0.0091 Treatment: F(1,34) = 15.4, p = 0.0004 Sidak's multiple-comparisons test <1 h: p = 0.67, 1–2 h: p = 0.21, 2–3 h: p = 0.023, 3–5 h: p = 0.011 Fig. 6E Normal Two-way ANOVA Interaction: F(3,33) = 1.4, p = 0.26 Depth: F(3,33) = 0.71, p = 0.55 Treatment: F(1,33) = 14.8, p = 0.0005 Sidak's multiple-comparisons test <1 h: p = 0.61, 1–2 h: p = 0.75, 2–3 h: p = 0.64, 3–5 h: p = 0.001 Fig. 6F Normal Two-way RM ANOVA Interaction: F(9,369) = 2.17, p = 0.023 Depth: F(9,369) = 9.09, p < 0.0001 Treatment: F(1,41) = 4.19, p = 0.047 Fig. 6F (inset) Nonparametric Mann–Whitney test U = 86, p = 0.002 Fig. 6G Normal Two-way RM ANOVA Interaction: F(9,369) = 1.46, p = 0.038 Depth: F(9,369) = 23.7, p < 0.0001 Treatment: F(1, 41) = 2.8, p = 0.01 Fig. 6G (inset) Normal Unpaired t test (two-tailed) t = 2.15, df = 41, p = 0.038 Extended Data Fig. 6-1B (right) Lognormal One-way ANOVA (Tukey's post hoc test) ANOVA, F(2,30) = 4.65. p = 0.017 Tukey's multiple-comparisons test aCSF vs MDL, p = 0.033 aCSF vs Cal 2-1, p = 0.046 MDL vs Cal 2-1, p = 0.910 Extended Data Fig. 6-1B (right) Lognormal One-way ANOVA (Tukey's post hoc test) ANOVA, F(2,31) = 5.79. p = 0.007 Tukey's multiple-comparisons test aCSF vs MDL, p = 0.005 aCSF vs Cal 2-1, p = 0.188 MDL vs Cal 2-1, p = 0.179
Movies
- Movie 1.
Evoked transient Ca2+ activity in “healthy” and GCaMP-filled neurons. An example of transient Ca2+ activity evoked by a single NMDA puff (125 µM, 100 ms, 3 psi). The movie is split into raw signals (left, pseudocolor) and ΔF / F0 projection (right). Arrows indicate GCaMP-filled neurons. Notice that dark areas on the right during NMDA puffs correlate with filled neurons on the left. Data were acquired at 2.67 Hz. A median filter was applied (radius, 2) for clarity. Playback speed: 24×. Scale bar (white), 100 μm. [View online]
- Movie 2.
Seizure-associated Ca2+ activity in “healthy” and GCaMP-filled neurons. Example of neuronal Ca2+ transients associated with seizure-like activity induced in acute slices by 4-AP application (100 µM, ∼20 min). The movie is split into raw signals (left, in pseudocolor) and ΔF / F0 projection (right). Arrows indicate GCaMP-filled neurons. Data were acquired at 2.67 Hz. A median filter was applied (radius, 2) for clarity. Playback speed: 24×. Scale bar (white), 100 μm. [View online]
- Movie 3.
Synchronous Ca2+ activity associated with experimentally induced seizures in superficial and deeper acute slice tissue. Example of neuronal Ca2+ transients associated with seizure-like activity induced in acute slices by 4-AP application (100 µM, 30 min). Ca2+ transients in the superficial tissue of the acute slice are displayed on the left, while Ca2+ transients in deeper tissue are displayed on the right. Data were acquired at 2.67 Hz. A median filter was applied (radius, 2) for clarity. Playback speed: 24×. Scale bar (white), 100 μm. [View online]
Fig 1-1
Eliminating false PI+ cell detections by emission ratio analysis. A) Left: MIPs showing excitation of PI and GCaMP6 s using multiple 2P wavelengths. Right: Excitation wavelength vs. % emission intensity for PI and GCaMP6 s. There was ∼54% PI emission intensity at 920 nm, a wavelength widely used for Ca2+-dependent GCaMP6 s excitation (n = 8 slices). B) Left: To eliminate false PI + -cell detections due to a green bleed-through signal, a threshold was identified (-0.4, magenta dashed line) from plotting the distribution of Red/Green ratios from a dataset of manually selected “True-positive PI+ cells.” Right: Lognormal distribution of Red/Green ratios of automated PI+ detections across all slices. Detections below the threshold (dashed line) were eliminated as false positives (1,543 cells eliminated). Data represented as mean ± 95% CI. Scale bar = 100 µm. Download Fig 1-1, TIF file.
Fig 1-2
Validation of PI + -cell detection across various depths in acute neonatal brain slices. A) Experimental design. B) MIPs representing different depths, acquired from brain slices (post-natal days 8-12), incubated for 4 hours in aCSF alone (top) and staurosporine (20 µM, bottom). C) Staurosporine significantly increased the number of PI + -cells in acute neonatal brain slices across various depths (Two-way ANOVA, interaction: F(9, 99) = 4, p = 0.0002, treatment: F(1, 11) = 12.3, p = 0.0049). D) Increase in the total number of PI + -cells (unpaired t-test, p = 0.005) and (E) in decay constants with staurosporine pre-treatment (unpaired t-test, p = 0.02) calculated from individual slices. F) Increase in colocalized puncta across tissue depth with staurosporine pre-treatment (Two-way ANOVA, interaction: F(9, 99) = 4.15, p < 0.0001, treatment: F(1, 11) = 12.3, p = 0.062). G) Although we found a modestly significant interaction between control and staurosporine-treated slices, post-hoc multiple comparisons confirmed no significant increase in GCaMP6-filled neurons due to staurosporine treatment (Two-way ANOVA, interactions: F(9, 90) = 2.18, p < 0.034, Treatment: F(1, 10) = 0.041, p = 0.93, Sidak’s test for multiple comparisons p > 0.05). aCSF: 5 slices; staurosporine: 8 slices. Mean ± 95% CI. Scale bar = 100 µm. Download Fig 1-2, TIF file.
Fig 1-3
Layer-specific distribution of PI+ and GCaMP-filled neurons A) Experimental design describing a strategy for imaging superficial and deeper layers of the primary somatosensory cortex. B) Representative image showing PI+ and GCaMP-filled distribution neurons across superficial and deeper layers. C) Density of PI+ cells and D) GCaMP-filled neurons in superficial and deeper layers, measured from acute brain slices at different durations of incubation, showed no significant difference between layers (PI+: Two-way ANOVA, interaction: F(6, 87) = 0.3, p = 0.936; GCaMP-filled: Two-way ANOVA, interaction: F(6, 82) = 0.06, p = 0.99). Data represented as mean ± 95% CI. Scale bar = 100 µm. Download Fig 1-3, TIF file.
Fig 1-4
Effects of slice thickness and postnatal age on inherent injury in acute brain slices A) Differences in normalized PI+ cells across depths between 350 and 450 µm thick acute brain slices (Two-way ANOVA, Interaction: F(9, 351) = 2.04, p = 0.034), with an increase at 300 µm depth in the 350 µm thick slices (Sidak’s multiple comparisons, p = 0.038). B) Significant difference between the distribution of GCaMP-filled neurons across depths (Two-way ANOVA, Interaction: F(9, 342) = 2.04, p = 0.0007), with an increase at 300 µm in the 350 µm thick slices (Sidak’s multiple comparisons, p = 0.0001). No postnatal age difference in C) PI+ cells (Two-way ANOVA, Interaction: F(9, 216) = 0.08, p = 0.99) or D) GCaMP-filled neurons (Two-way ANOVA, Interaction: F(9, 306) = 0.87, p = 0.56). Data represented as mean ± 95% CI. Download Fig 1-4, TIF file.
Fig 6-1
Calpain-2 isoform significantly contributes to the inherent injury in acute brain slices. A) Experimental design showing acute slice incubation with nonspecific or a calpain-2 specific inhibitor (MDL and Cal 2-1 respectively) for 2-5 hrs. B) Left: Normalized PI+ cells across multiple depths show faster exponential decays with MDL and Cal 2-1 incubation than aCSF (τaCSF: 48, τMDL: 22, τCal 2-1: 39). Right: Both MDL and Cal 2-1 reduced the number of PI+ cells after 2-5 hr incubation (One way ANOVA on log-transformed data, F(2, 30) = 4.65, p = 0.017. Tukey’s test for multiple comparisons indicated in figure). C) Left: Similarly, normalized GCaMP-filled neurons across multiple depths show faster exponential decays with MDL and Cal 2-1 incubation (τaCSFP: 53, τMDL: 35, τCal 2-1: 27). Right: Cal 2-1 treatment did not change total GCaMP-filled neurons cells after 2-5 hr incubation (One way ANOVA on log-transformed data, F = (2, 31) = 5.79, p = 0.007 with Tukey’s test for multiple comparison indicated in the figure). Data represented as mean ± 95% CI. Download Fig 6-1, TIF file.
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