Inactivation of retinogeniculate synapses induces caspase-3 activity.

(A) Schematics of experimental setup. AAVs expressing tetanus toxin light chain (TeTxLC) and/or mTurquiose2 (Turq) were injected into the right eye of E15 mice (left). By P5, retinogeniculate synapses in dLGN were inactivated to varying extents depending on injection and side (right). (B-D) Confocal images of Turq (left panels) and activated caspase-3 (right panels) in left dLGN (B) and right dLGN (C) of a TeTxLC-injected P5 animal and in left dLGN of a control P5 animal (D). Images from the same fluorescent channel were adjusted to the same contrast. Dotted lines delineate dLGN boundaries. The compass in B marks tissue orientation. Scale-bars: 100 μm. D, dorsal; V, ventral; M, medial; L, lateral. (E) Quantification of caspase-3 activity in indicated dLGNs. Activated caspase-3 signals in each dLGN (highlighted areas in B-D) were summed and normalized to dLGN area. Each point represents the result from one dLGN. Data from two dLGNs of the same animal were paired for analysis (grey lines). n=10 for TeTxLC-injected animals and n=9 for control animals. Mean and standard deviation (S.D.) are shown. P-values were calculated from two-tailed t-tests (paired when applicable). (F) Example images showing punctate caspase-3 activities in ventral-medial regions of indicated dLGNs. Images were adjusted to the same contrast. Scale-bar: 20 μm. (G) High-resolution images of dLGN showing TeTxLC-expressing RGC axons (yellow) and activated caspase-3 (magenta). Two regions of interest (dotted squares) are magnified to illustrate that caspase-3 activity was found juxtaposing TeTxLC-expressing axon terminals but not within them. Scale-bar: 5 μm.

Synapse inactivation-induced caspase-3 activation requires synaptic competition.

(A) Schematics illustrating different experimental conditions. Presynaptic (green) and postsynaptic (cyan) compartments and synaptic strength (yellow arrowheads) of retinogeniculate synapses are shown. No synapses are inactivated in the control (left); expressing TeTxLC only in right eye inputs results in synapse inactivation and competition (middle); expressing TeTxLC in both eyes results in synapse inactivation but no competition (right). (B) Confocal images of dLGNs in the three conditions showing activated caspase-3 signal. The dLGN shown for the single inactivation condition was from the left side. Dotted lines mark dLGN boundaries. Scale-bars: 100 μm. (C) Quantification of caspase-3 activity in the dLGN in the indicated conditions. Activated caspase-3 signal in each dLGN were summed and normalized to the dLGN area. For the single inactivation condition, values were from the left dLGN only. For the other two conditions, values from both dLGNs were averaged. n=8 animals for control, n=11 animals for single inactivation, and n=10 animals for dual inactivation. Mean and S.D. are shown. P-values were calculated from Tukey’s multiple comparison tests.

Caspase-3 is required for segregation of eye-specific territories.

(A-B) Representative confocal images of retinogeniculate inputs in the dLGN of P10 wild-type (A) and Casp3−/− (B) mice. Contralateral inputs are labeled with AlexaFlour488 (AF488) conjugated cholera toxin subunit B (CTB) and ipsilateral inputs with AF594-CTB. Original images were thresholded into 0-or-1 images using the Otsu method (34), and the overlap between thresholded contralateral and ipsilateral inputs is shown. (C) Percentage overlap between eye-specific territories in wildtype and Casp3−/− mice under a series of increasing signal cutoff thresholds. Note that the percentage overlap is plotted on a log scale. Each circle represents one animal. Mean and S.D. are shown. n=9 for wildtype mice and n=6 for Casp3−/− mice. (D) Mean percentage overlap values in wildtype and Casp3−/− mice and p-values of two-tailed t-tests between the two genotypes are listed for each cutoff threshold.

Caspase 3 is required for retinogeniculate circuit refinement.

(A-B) Example recordings of dLGN relay neuron responses in P30 wildtype (A) and Casp3−/− (B) mice. Excitatory postsynaptic currents (EPSCs) were evoked by increasing stimulation currents in the optic tract. Both AMPAR-mediated inward current at −70 mV membrane potential and AMPAR and NMDAR-mediated outward current at +40 mV membrane potential are shown. Peak response amplitudes at each stimulation intensity are plotted to the right of recording traces. Scale bars represent 0.5 nA and 10 ms. (C) Distribution of RGC input numbers on individual dLGN relay neurons in wildtype and Casp3−/− mice. The number of RGC inputs was inferred by manually counting the number of steps in AMPAR-mediated EPSC response curves (lower right in A and B) while blind to the genotypes. P-value was calculated from two-tailed t-test. n=37 cells for wildtype mice and n=32 cells for Casp3−/− mice. (D) Example recordings from paired pulse measurements at −70 mV membrane potential in wildtype and Casp3−/− mice. Traces from experiments with 50, 150, 250, 500, and 1000 ms inter-stimulus intervals are overlayed. Stimulus artifacts were removed from the traces for clarity. Scale-bars represent 100 ms (horizontal) and 0.3 nA (vertical). (E) Paired-pulse ratio (calculated as peak amplitude of the second response over that of the first response) in wildtype and Casp3−/− mice at various inter-stimulus intervals. Mean and standard error of the mean (SEM) are shown. P-values were calculated from Bonferroni’s multiple comparison test. p=0.0067 for 50 ms interval, p=0.0369 for 150 ms interval, and p=0.0097 for 250 ms interval. n=16 cells for wildtype mice and n=13 for Casp3−/− mice.

Microglia-mediated synapse elimination depends on caspase-3.

(A) Representative 3D-reconstructed images of a P5 Casp3+/−; Cx3cr1-Gfp+/− mouse dLGN with microglia displayed in green, contralateral RGC axon terminals in red, and ipsilateral RGC terminals in blue. In the merged image, the region from which microglia are selected for analysis is indicated with the dashed line. The scale-bar represents 100 μm. (B) Representative surface rendering of microglia (green) from P5 dLGNs of Casp3+/−; Cx3cr1-Gfp+/− and Casp3−/−; Cx3cr1-Gfp+/− mice. Intracellular contralateral (red) and ipsilateral (blue) RGC axon terminals are shown. Microglia from caspase-3 deficient mice engulf visibly less synaptic material. Scale-bars represent 10 μm. (C) Total volume of engulfed synaptic material in individual microglia from Casp3+/−; Cx3cr1-Gfp+/− and Casp3−/−; Cx3cr1-Gfp+/− mice. (D) Total volume of engulfed synaptic material in each microglia (from C) is normalized to the volume of that microglia. In C-D, each point represents one microglia. Mean and S.D. are shown. p-values were calculated from unpaired two-tailed t-tests. n=61 microglia from 8 Casp3+/−; Cx3cr1-Gfp+/− mice and n=54 microglia from 5 Casp3−/−; Cx3cr1-Gfp+/− mice.

Caspase-3 activation determines specificity of microglia-mediated synapse elimination.

(A) Schematics illustrating the experimental rationale. In wildtype mice (upper panel), inactivating retinogeniculate synapses from the right eye activates caspase-3 (magenta pie) and recruits microglia to engulf more right eye-originated synapses (red dots) than left eye-originated synapses (green dots). If caspase-3 activation is blocked (lower panel), we expect preferential engulfment of inactive synapses to be attenuated. (B-E) Surface rendering of representative microglia from P5 left dLGN of Casp3+/+ (B-C) or Casp3−/− (D-E) mice injected with AAV expressing mTurquoise2 (B and D) or TeTxLC (C and E) in the right eye at E15. RGC axon terminals from the left eye are shown in yellow and terminals from the right eye in red. Scale bars represent 15 μm. (F-G) Ratio between volumes of right-eye and left-eye-originated synaptic material engulfed by microglia from Casp3+/+ (F) or Casp3−/− (G) mice injected with AAV expressing mTurquoise2 (blue) or TeTxLC (red). Each dot represents one microglia. Engulfment ratios are displayed on a log scale. 0, 25, 50, 75, and 100 percentiles are shown. p-values were calculated from unpaired two-tailed Mann-Whitney tests. n=52 microglia from 7 Turq-injected Casp3+/+ mice, n=50 microglia from 8 TeTxLC-injected Casp3+/+ mice, n=64 microglia from 5 Turq-injected Casp3−/− mice, and n=51 microglia from 6 TeTxLC-injected Casp3−/− mice.

Caspase-3 deficiency protects against Aβ induced synapse loss.

(A and C) Representative 3D-reconstructed images (3 μm in z) showing presynaptic (SV2, in green) and postsynaptic (Homer1, in red) signals in dentate gyrus of female 6 month-old APP/PS1 mice on caspase-3 wildtype (A) and deficient (C) backgrounds. Ellipsoids were fitted to the original images (upper panels) to isolate pre- (green) and post-synaptic (red) puncta (lower panels). Homer1 ellipsoids found with 300 nm of a SV2 ellipsoid are highlighted in white in the fitted images. Original images are adjusted to the same contrast. For the fitted images, only ellipsoids from the upper half of the z-stack are shown. Scale-bar represents 4 μm. (B and D) Quantification of synapse density in APP/PS1 mice on caspase-3 wildtype (B) and deficient (D) backgrounds. Mean and S.D. are shown. p-values were calculated from unpaired two-tailed t-tests. n=5 for App/Ps1−/− mice, n=6 for App/Ps1+/− mice, n=6 for Casp3−/−; App/Ps1−/− mice, and n=4 for Casp3−/−; App/Ps1+/− mice.

Segregation of eye-specific territories in the mouse retinogeniculate pathway.

In the mouse retinogeniculate pathway, retinal ganglion cells (RGCs) in the retina of each eye innervate relay neurons in both the contralateral (opposite side as the originating RGC) dorsal lateral geniculate nucleus (dLGN) and the ipsilateral (same side from the originating RGC) dLGN to form retinogeniculate synapses (upper right). Within each dLGN, the majority of retinogeniculate synapses receive inputs from the contralateral eye, while the minority receive inputs from the ipsilateral eye (upper right). At the age of P3, regions in each dLGN receiving inputs from each of the two eyes overlap significantly (left). Through a process that requires synapse elimination and spontaneous RGC activity, these regions are refined into non-overlapping eye-specific territories by the age of P8 (upper right). If the refinement process is defective because neural activity or synapse elimination is disrupted, eye-specific territories fail to completely segregate, and regions innervated by the two eyes remain overlapping (lower right).

Quantifying eye-specific segregation with multi-threshold overlap analysis.

For each dLGN, RGC inputs from the two eyes were imaged using separate fluorescence channels. A small area in the thalamus outside of each dLGN was chosen, and average signal intensity in each channel within that area was calculated and used as background (upper panel). For each channel, background was subtracted, and signals were normalized to between 0 and 1. To calculate overlap between eye-specific territories, a threshold, x, was chosen between 0 and 1 and applied to both channels (middle panel). The overlap between eye-specific territories were defined as the set of pixels with above-threshold signals in both channels. Percentage overlap was then calculated as the ratio between the area of the dLGN where eye-specific territories overlapped and the total area of the dLGN (lower panel). To avoid introducing biases by artificially selecting one threshold, we repeated the analysis with a set of increasingly stringent thresholds (e.g., from 0.1 to 0.25 in Fig. 3C and 3D).

Blocking spontaneous RGC activity in one eye with TeTxLC disrupts eye-specific segregation in the retinogeniculate pathway.

(A and B) Confocal images of P8 left dLGN (left panels) and right dLGN (right panels) of an animal receiving control injections (A) and an animal receiving TeTxLC injection in the right eye (B). Eye-specific regions were labeled with tdTomato (red, left eye input) and mTurquiose2 (cyan, right eye input). Right eye territory (cyan) contracted and left eye territory (red) expanded in TeTxLC-injected animals (B) compared to controls (A). Scale-bars: 100 μm. (C) Quantification of overlap between eye-specific territories in dLGN of P8 control and TeTxLC-injected animals. Analysis was done at multiple thresholds to avoid biases introduced with threshold selection. Note that percentage overlap is displayed on a log scale. For details on the analysis, see Fig. S2 and text associated with Fig. 3. n=7 for control animals and 5 for TeTxLC-injected animals. Mean and S.D. are shown. (D) Statistics of the overlap analysis at multiple thresholds. Fold difference and the statistical significance of the difference in percentage overlap between the two groups of animals increased as threshold increased. P-values were calculated from two-tailed t-tests. We did not implement multiple comparison corrections as values at different thresholds are derived from the same dataset and are not independent.

Inactivation of retinogeniculate synapses induces postsynaptic caspase-3 activity in dendritic compartments of dLGN relay neurons.

(A) High-resolution images of three representative field-of-views (i-iii) in P5 TeTxLC-expressing dLGNs showing co-localization of punctate caspase-3 activity (magenta) and a dendritic marker, MAP2 (green). Dotted crosses mark x and y positions where the x-z and y-z cross-sections were generated. Scale-bars: 2 μm. (B) Example images showing caspase-3 activity in entire neurons (i-iii) or in multiple dendritic branches (but not the soma) of a neuron (iv) in P5 TeTxLC-expressing dLGNs. The neurons positive for active caspase-3 (i-iii) have relatively large and round somas and multipolar dendritic arbors that are characteristic of dLGN relay neurons. Scale-bars: 20 μm. (C) Example images of four representative neurons (i-iv) in P5 TeTxLC-expressing dLGNs that are positive for active caspase-3 (magenta) and a neuronal nuclear marker, NeuN (green). Scale-bars: 10 μm.

Blocking spontaneous RGC activity in both eyes with TeTxLC disrupts eye-specific segregation in the retinogeniculate pathway.

(A and B) Confocal images of the left dLGN (left panels) and right dLGN (right panels) of an animal receiving control injections in both eyes (A) and an animal receiving TeTxLC injection in both eyes (B). Eye-specific territories were labeled with tdTomato (magenta, left eye input) and eGFP (green, right eye input). In animals injected with TeTxLC in both eyes, territories of the two eyes are similar in size and are more dispersive compared to those in controls, leading to substantial overlap between eye-specific territories. Scale-bars: 100 μm. (C) Quantification of overlap between eye-specific territories in the dLGN of control and dual TeTxLC-injected animals. Analysis was done at multiple thresholds to avoid biases introduced with threshold selection. Note that percentage overlap is displayed on a log scale. For details on the analysis, see Fig. S2 and text associated with Fig. 3. n=8 for control animals and n=9 for dually TeTxLC-injected animals. Mean and S.D. are shown. (D) Statistics of the overlap analysis at multiple thresholds. Fold difference between the two groups of animals increases as the threshold increases. Statistical significance does not show clear increase with threshold as percentage overlap values are more variable at higher thresholds. P-values were calculated from two-tailed t-tests. We did not implement multiple comparison corrections as values at different thresholds are derived from the same dataset and are not independent.

Caspase-3 deficiency does not alter RGC density in the retina.

(A) Images of whole-mount retinae from P10 Casp3+/+ (left) and Casp3−/− (right) animals. RGCs were labeled in red by immunostaining against an RGC-specific marker, RBPMS (RNA-binding protein with multiple splicing) (37). The dark regions on the bottom of the left panel and on the left of the right panel are optic discs. Scale-bars: 200 μm. (B) Quantification of RGC densities in retinae of P10 Casp3+/+ and Casp3−/− animals. Each point represents one retina. Left and right retinae were analyzed separately. n=4 animals for Casp3+/+ mice and n=5 animals for Casp3−/− mice. Mean and S.D. are shown. P-values were calculated from unpaired two-tailed t-tests.

Measuring electrophysiological properties of retinogeniculate synapses.

(A) Inferring the number of RGC inputs of dLGN relay neurons by measuring stimulation-response curves. To measure relay neuron responses, we prepared acute brain slices from a tilted parasagittal plane in P30 mice (38). These slice preparations preserved a long segment of the optic tract and a high level of connectivity between RGC axons and dLGN relay neurons (38). We placed a stimulating electrode on the optic tract and patch-clamped individual dLGN relay neurons with a recording electrode. We stimulated the optic tract with a series of gradually increasing currents and recorded excitatory postsynaptic currents (EPSCs) in relay neurons. At low stimulation intensity, no RGC axons were activated. As the stimulus increased, more and more RGC axons were recruited. As different RGC axons have different excitation thresholds, RGC axons were excited one at a time. Whenever a stimulus activated an RGC axon that did not respond to lower stimuli, a step increase in the relay neuron response was detected, and the increment corresponded to the EPSC evoked by the newly recruited RGC axon. We can therefore infer the number of RGC inputs innervating the relay neuron being recorded by counting the number of steps in the response curve of that relay neuron. t: time. Stim.: stimulation intensity. (B-C) Inferring the number of release sites from RGC inputs by measuring paired-pulse response ratio (PPR). We stimulated the optic tract with two stimuli separated by a short interval and recorded EPSCs in dLGN relay neurons. The stimulus intensity was chosen to evoke maximum response in the relay neuron. In wildtype mice (B), the first stimulus triggers the release of a significant fraction of the readily releasable pool (RRP) of neurotransmitters (black dots), evoking a strong response (left). At the time of the second stimulus, the RRP does not have sufficient time to recover and remains depleted, resulting in a weaker second response (right). PPR can be calculated from this experiment by dividing the peak amplitude of the second response with that of the first. PPR is small in wildtype animals. In caspase-3 deficient mice (C), if the number of release sites is increased compared to wildtype animals, the RRP should be larger (left), and a smaller fraction of the RRP is released during the first stimulus, leaving more neurotransmitters available for the second stimulus (right), thereby enhancing the second response and PPR. Note that in this model, we made two assumptions. The first assumption is that the average size of the RRP in one release site is similar in Casp3+/+ and Casp3−/− mice, which is supported by the comparable fiber fractions (FFs) in the two groups of animals (see Fig. S8E). The second assumption is that the number of neurotransmitters released during the first stimulus is similar in Casp3+/+ and Casp3−/− mice (4 dots in the illustration), which is supported by the comparable maximum EPSCs in the two groups of animals (see Fig. S8B-C).

Additional analyses of electrophysiological properties of retinogeniculate synapses in Casp3+/+ and Casp3−/− mice.

(A) Distribution of RGC input numbers of individual dLGN relay neurons in wildtype and Casp3−/− mice measured by counting the number of steps in NMDAR-mediated EPSC response curves (upper right in Fig. 4A and 4B) while blind to genotypes. P-value was calculated from two-tailed t-test. n=22 cells for wildtype mice and n=24 cells for Casp3−/− mice. (B-C) Quantification of maximum amplitudes of AMPAR- (B) and NMDAR-mediated (C) EPSCs in wildtype and Casp3−/− dLGN relay neurons. (D) Quantification of the ratio between maximum amplitudes of AMPAR- and NDMAR-mediated EPSCs in wildtype and Casp3−/− dLGN relay neurons. (E) Quantification of fiber fractions in wildtype and Casp3−/− dLGN relay neurons. When stimulation on the optic tract is gradually increased, there will be a lowest stimulation intensity at which a non-zero response is first recorded in the relay neuron (see Fig. S8 for illustration and Fig. 4 for example data). This first response is presumed to be evoked by the activation of a single RGC axon fiber. Fiber fraction is defined as the ratio between this single-fiber response and the maximum response and ranges from 0 to 1. Only the AMPAR-mediated response is used to calculate fiber fractions. In B-E, mean and S.D. are shown. In A-E, p-values were calculated from two-tailed t-tests. In A, C, and D, n = 22 cells for wildtype mice and n=24 cells for Casp3−/− mice. In B and E, n=37 cells for wildtype mice and n=32 cells for Casp3−/− mice.

Caspase-3 deficiency does not cause microglia activation.

(A) Representative images of P5 dLGNs in a Casp3+/+ mouse (left) and a Casp3−/− mouse (right) showing Iba1 (green, a marker for microglia cell body) and Cd68 (magenta, a marker for microglia activation) staining. The dLGN areas are highlighted with dotted lines. Scale-bars represent 100 μm. (B) Quantification of microglia morphology in Casp3+/+ and Casp3−/− mice using the sphericity metric. A sphericity of 1 corresponds to a perfect sphere. The smaller the sphericity is, the more ramified the cell is. (C-D) Quantification of microglia activation by normalizing the total volume of Cd68 signal (C) or the total intensity of Cd68 signal (D) in each microglia to the volume of that microglia. By both morphology and Cd68 signal, microglia in Casp3−/− mice show no evidence of activation. In B-D, each data point represents one microglia, and mean and S.D. are shown. n=46 microglia from 4 Casp3+/+ mice and n=52 microglia from 4 Casp3−/− mice. P-values were calculated from two-tailed unpaired t-tests.

Additional analyses of microglia-mediated engulfment of synaptic material.

(A-B) Volume of contralateral (A) and ipsilateral (B) RGC axon terminals engulfed by individual microglia in the P5 dLGN of Casp3+/−; Cx3cr1-Gfp+/− and Casp3−/−; Cx3cr1-Gfp+/− mice. (C) Volume of individual microglia in the P5 dLGN of Casp3+/−; Cx3cr1-Gfp+/− and Casp3−/−; Cx3cr1-Gfp+/− mice. In A-C, each point represents one microglia. Mean and S.D. are shown. P-values were calculated from unpaired two-tailed t-tests. n=61 microglia from 8 Casp3+/−; Cx3cr1-Gfp+/− mice and n=54 microglia from 5 Casp3−/−; Cx3cr1-Gfp+/− mice.

Astrocyte-mediated synapse elimination does not appear to depend on caspase-3.

(A) Representative 3D-reconstructed images of a P5 Casp3+/−; Aldh1l1-Gfp+/− mouse dLGN with astrocytes displayed in green, contralateral RGC axon terminals in red, and ipsilateral RGC terminals in blue. The region from which astrocytes are selected for analysis is indicated with the dashed line in the merged image. Scale-bars represent 50 μm. (B) Representative surface rendering of astrocytes (green) from P5 dLGNs of Casp3+/−; Aldh1l1-Gfp+/− and Casp3−/−; Aldh1l1-Gfp+/− mice. Intracellular contralateral (red) and ipsilateral (blue) RGC axon terminals are shown. Only cell bodies and the base of processes were segmented. We made this segmentation choice because we observed that fine astrocytic processes are largely devoid of engulfed material. Scale-bars represent 5 μm. (C) Total volume of engulfed synaptic material in individual astrocytes from Casp3+/−; Aldh1l1-Gfp+/− and Casp3−/−; Aldh1l1-Gfp+/− mice. (D) Total volume of engulfed synaptic material in each astrocyte (from C) is normalized to the volume of that astrocytes. (E) Volume of individual astrocytes from the two groups of mice. In C-E, each point represents one astrocyte. Mean and S.D. are shown. P-values were calculated from unpaired two-tailed t-tests. n=595 astrocytes from 8 Casp3+/−; Aldh1l1-Gfp+/− mice and n=517 astrocytes from 6 Casp3−/−; Aldh1l1-Gfp+/− mice.

Additional analysis on activity-dependent microglia-mediated engulfment of synapses.

(A-D) Volumes of synaptic material originating from right (A and C) or left (B and D) eyes engulfed by individual microglia from the left dLGN of Casp3+/+ (A-B) or Casp3−/− (C-D) mice injected with AAV expressing mTurquoise2 (blue) or TeTxLC (red) in right eye were normalized to microglial volume and plotted. In Casp3+/+ mice, inactivating right eye-originated retinogeniculate synapses specifically enhanced microglia-mediated engulfment of RGC axon terminals from right eyes (A) but not left eyes (B), resulting in significantly higher engulfment ratios (Fig. 6F). In Casp3−/− mice, inactivating right eye-originated synapses upregulated engulfment of RGC terminals from both eyes (C and D), resulting in no significant change in engulfment ratios (Fig. 6G). In A-D, 0, 25, 50, 75, and 100 percentiles are shown. p-values were calculated from unpaired two-tailed t-tests. n=52 microglia from 7 Turq-injected Casp3+/+ mice, n=50 microglia from 8 TeTxLC-injected Casp3+/+ mice, n=64 microglia from 5 Turq-injected Casp3−/− mice, and n=51 microglia from 6 TeTxLC-injected Casp3−/− mice. Notes on interpretation of C and D: We observed that in Casp3−/− brains, particularly those injected with TeTxLC, microglia in dLGNs tended to increase in volume and have thicker processes. We therefore suspect the non-specific upregulation of synapse engulfment in TeTxLC-injected Casp3−/− mice is a consequence of microglial activation rather than synapse inactivation. This microglial activation is likely a ramification of intraocular surgeries, as changes in microglia morphology was more prominent in the left dLGN that received most of the inputs from AAV-injected right eyes, and microglia outside of dLGNs did not show characteristics of activation. We noticed that Casp3−/− brains tended to have weak structural integrity. It is likely that repetitive intraocular injections caused damage in the retinogeniculate pathway of Casp3−/− mice and activated microglia. Nevertheless, by calculating right-to-left engulfment ratios, substrate preference of microglia in TeTxLC-injected Casp3−/− mice can be deduced.

Amyloid deposition APP/PS1 mouse lines.

(A) Images of coronal brain sections of a female App/Ps1−/− mouse at 6 months (i), female App/Ps1+/− mice at 5 months (ii) or 6 months (iii), male App/Ps1+/− mice at 5 months (iv) or 6 months (v), and a female Casp3−/−; App/Ps1+/− mouse at 6 months, stained with Thioflavin S to reveal Aβ plaques (bright spots in hippocampus and cortex). Scale-bars represent 1 mm. (B) Quantification of the number of plaques per section in female 6 month-old App/Ps1+/− and Casp3−/−; App/Ps1+/− mice. Mean and S.D. are shown. p-values were calculated with unpaired two-tailed t-tests. n=6 for App/Ps1+/− mice and n=4 for Casp3−/−; App/Ps1+/− mice.

Selection of field of interest for synapse loss analysis.

To quantify synapse density, three regions of interest from each dentate gyrus on either side of the brain were chosen for each animal (total of 6 regions per animal). Regions of interest in one dentate gyrus of one animal from each genotype group are highlighted with dotted squares in the overviews above. High resolution images of SV2 (in green) and Homer1 (in red) stains were acquired for each region of interest and used for analysis. In sections from animals overexpressing mutant APP and PS1, we avoided choosing regions of interest from areas surrounding amyloid plaques (highlighted with arrows).

Additional analysis of synapse density in APP/PS1 mouse lines.

(A-D) Quantification of presynaptic (A and C) and postsynaptic (B and D) puncta densities in the dentate gyrus of APP/PS1 mice in the caspase-3 wildtype (A and B) or deficient (C and D) background. Mean and S.D. are shown. p-values were calculated from unpaired two-tailed t-tests. n=5 for App/Ps1−/− mice, n=6 for App/Ps1+/− mice, n=6 for Casp3−/−; App/Ps1−/− mice, and n=4 for Casp3−/−; App/Ps1+/− mice.

Microgliosis in APP/PS1 mouse lines.

Coronal sections from 6 month-old female App/Ps1−/− (left), App/Ps1+/− (middle), and Casp3−/−; App/Ps1+/− (right) mice were stained for Iba1 (red, to detect microglia) and Cd68 (green, as a microglia activation marker). Dentate gyrus regions are shown in the overviews above. Clusters of reactive microglia surrounding amyloid plaques are highlighted with arrows.

Aβ-induced caspase-3 activity in APP/PS1 mice.

(A-C) Density of activated caspase-3 puncti in the dentate gyrus of 4 month-old (A), 5 month-old (B), and 6 month-old (C) female App/Ps1−/− and App/Ps1+/− mice. Each column represents one animal, and each dot represent quantification from one dentate gyrus (either from left side or right side). Two out of four 4-month-old App/Ps1+/− mice showed robustly upregulation of caspase-3 activity in the dentate gyrus (A). No upregulation of caspase-3 activity was observed in 5 month-old or 6 month-old mice. (D) Images showing elevated caspase-3 activity in the molecular layer of the dentate gyrus of a 4 month-old female App/Ps1+/− mouse (A#5 in A). Images were set to the same intensity contrast. Upregulated caspase-3 activity remained in a punctate pattern and no apoptotic cell was observed. Scale-bar represents 10 μm. (E) Representative images of the dentate gyrus in 6 month-old female App/Ps1−/−, App/Ps1+/−, Casp3−/−; App/Ps1−/−, and Casp3−/−; App/Ps1+/− mice that were stained with an anti-NeuN antibody to visualize neurons. The fields of interest used to quantify neuron density are highlighted with dotted rectangles. Scale-bars represent 100 μm. (F) Neuron density in the granule cell layer of the dentate gyrus in the four groups of mice shown in E. Neither caspase-3 deficiency nor APP/PS1 overexpression caused significant changes in neuron density. Mean and S.D. are shown. p-values were calculated from Tukey’s multiple comparisons test. n=5 for App/Ps1−/− mice, n=6 for App/Ps1+/− mice, n=5 for Casp3−/−; App/Ps1−/− mice, and n=4 for Casp3−/−; App/Ps1+/− mice.