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. Author manuscript; available in PMC: 2012 May 26.
Published in final edited form as: Neuron. 2011 May 26;70(4):758–772. doi: 10.1016/j.neuron.2011.04.004

Non-apoptotic function of BAD and BAX in long-term depression of synaptic transmission

Song Jiao 1, Zheng Li 1,†
PMCID: PMC3102234  NIHMSID: NIHMS290059  PMID: 21609830

Summary

It has recently been found that caspases not only function in apoptosis, but are also crucial for non-apoptotic processes such as NMDA receptor-dependent long-term depression (LTD) of synaptic transmission. It remains unknown, however, how caspases are activated and how neurons escape death in LTD. Here we show that caspase-3 is activated by the BAD-BAX cascade for LTD induction. This cascade is required specifically for NMDA receptor-dependent LTD but not for mGluR-LTD, and its activation is sufficient to induce synaptic depression. In contrast to apoptosis, however, BAD is activated only moderately and transiently and BAX is not translocated to mitochondria, resulting in only modest caspase-3 activation. We further demonstrate that the intensity and duration of caspase-3 activation determin whether it leads to cell death or LTD, thus fine-tuning of caspase-3 activation is critical in distinguishing between these two pathways.

Introduction

Although caspases are well-known for their role in apoptosis (Pop and Salvesen, 2009), they can also be activated for non-apoptotic functions, such as for differentiation of lens and muscle cells (Murray et al., 2008; Weber and Menko, 2005); proliferation and differentiation of T and B cells (Beisner et al., 2005; Salmena et al., 2003); developmental pruning of dendrites in Drosophila neurons (Kuo et al., 2006; Williams et al., 2006); derivation of induced pluripotent stem cells (Li et al., 2010a); chemotropic responses of retinal growth cones in Xenopus (Campbell and Holt, 2003); habituation to repetitive songs in zebra finches (Huesmann and Clayton, 2006); and modification of synaptic transmission such as LTD in hippocampal neurons (Li et al., 2010b; Lu et al., 2006). However, the signaling pathway underlying caspase activation and the question of why active caspases do not cause cell death in such non-apoptotic functions remain largely unexplored.

Here we address these questions in LTD. LTD is a long-lasting form of synaptic plasticity in neurons, which is the ability of synapses to change in strength and which plays a crucial role in the refinement of neuronal connections during development and in cognitive functions such as learning and memory (Kessels and Malinow, 2009; Malenka and Bear, 2004). N-methyl D-aspartate (NMDA) receptor-dependent LTD is a prototypical form of LTD that is primarily mediated by the removal of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors from the postsynaptic membrane (Bredt and Nicoll, 2003; Collingridge et al., 2004; Malenka and Bear, 2004; Shepherd and Huganir, 2007). This type of LTD involves Ca2+ influx, protein phosphatases (PP2B/calcineurin and PP1) (Malenka and Bear, 2004), GSK3β (Peineau et al., 2007), small GTPases such as Rap1 (Zhu et al., 2002) and Rab5 (Brown et al., 2005), and p38 MAP kinase (Zhu et al., 2002). Recently, we reported that in hippocampal neurons of rodents, caspase-3 is required for AMPA receptor endocytosis and LTD induction, and that cytochrome c release from mitochondria is necessary for the activation of caspase-3 (Li et al., 2010b). However, the questions of how stimulation of NMDA receptors leads to caspase-3 activation and, importantly, how neurons survive despite caspase-3 activation have not been addressed in detail.

Cytochrome c release from mitochondria in apoptosis is mediated by mitochondrial outer membrane permeabilization (MOMP), which is regulated by members of the B-cell lymphoma-2 (BCL-2) family of proteins [for recent reviews, see (Chipuk et al., 2010) and (Youle and Strasser, 2008)]. Some members of this family, such as BAX and BAK, promote apoptosis, while others, such as BCL-2 and BCL-XL, inhibit apoptosis by antagonizing the pro-apoptotic BCL-2 family members. BAX and BAK are multi-BCL-2-homology (BH) domain proteins that form pores in mitochondrial membranes during MOMP (Chipuk et al., 2006). In hippocampal and cortical neurons, BAX is the major pore-forming BCL-2 family protein, as BAK is not expressed in these cells (Sun et al., 2001; Uo et al., 2005). In the absence of death signals, BAX resides predominantly in the cytosol (Hsu et al., 1997), but upon stimulation of apoptosis, it translocates to mitochondria (Goping et al., 1998; Wolter et al., 1997). BAD and BID are two other well-known pro-apoptotic BCL-2 family proteins that regulate the pore-forming activity of BAX. BAD is activated by protein phosphatases (Danial, 2008; Klumpp and Krieglstein, 2002), while BID is activated by proteolytic cleavage (Yin, 2006). Upon activation, BAD translocates to mitochondria and binds to anti-apoptotic BCL-2 family proteins, such as BCL-XL, to counteract their inhibition of BAX (Danial, 2008). Likewise, BID migrates to mitochondria upon activation and promotes the pore-forming activity of BAX (Youle and Strasser, 2008).

In this study, we demonstrate a novel function for BAD and BAX in LTD induction. By using siRNA-mediated knockdown and knock-out approaches as well as protein infusions, we show that the BAD-BAX cascade, but not BID, is both necessary and sufficient to activate caspase-3 and to induce LTD in hippocampal neurons. Intriguingly, activation of the BAD-BAX cascade is required specifically for NMDA receptor-dependent LTD, but not for mGluR-LTD. Unlike in apoptosis, however, BAD is activated to a lower extent and BAX translocation to mitochondria is not induced in LTD, leading in turn to milder caspase-3 activation. Furthermore, by manipulating the level and duration of caspase-3 activation, we provide evidence that strong and prolonged activation of caspase-3 is required to induce cell death, and modest and transient caspase-3 activation is critical for preventing cell death in LTD. These findings reveal unexpectedly that despite the fact that activation of the BAD-BAX-caspase-3 pathway usually leads to cell death, neurons adopt this entire pathway for induction of LTD, and underscore the importance of quantitative differences in caspase-3 activation for determining the cellular function of this pathway.

Results

BAD and BAX siRNAs inhibit LTD in CA1 hippocampal neurons

To determine the mechanism for caspase-3 activation in LTD, we first examined whether knocking down the expression of BAD, BAX and BID would affect LTD, as these proteins activate caspase-3 in apoptosis. To this end, we generated constructs expressing siRNAs that target the mRNAs encoding these proteins. The efficiency and specificity of these siRNAs were tested against corresponding cDNAs expressed in heterologous cells and against their endogenous targets in cultured hippocampal or cortical neurons. As shown in Figure S1, the siRNAs were highly effective and specific. To test their effect on synaptic transmission, we biolistically transfected cultured hippocampal slices with the siRNA constructs along with a plasmid expressing venus (a YFP mutant) (Nagai et al., 2002) and measured excitatory postsynaptic currents (EPSCs) evoked by stimulating the Schaffer collateral pathway. As shown in Figure 1A–D and Table S1, the amplitudes of both AMPA and NMDA receptor-mediated currents (EPSCAMPA and EPSCNMDA, respectively) were comparable in untransfected cells and in cells transfected with control or siRNA plasmids. These results indicate that NMDA receptor functions and basal AMPA receptor-mediated currents are intact in the transfected cells.

Figure 1.

Figure 1

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LTD in CA1 neurons is inhibited by BAD siRNA and BAX siRNA, but not by BID siRNA. Cultured hippocampal slices were biolistically transfected with the EGFP construct (as a visual marker) and either the empty vector, or a plasmid expressing BAD siRNA, BAX siRNA or BID siRNA. Transfected and nearby untransfected CA1 neurons were recorded simultaneously in the whole-cell patch-clamp mode 2 days after transfection. (A–D) Pair-wise analysis of the effect of BID siRNA, BAD siRNA and BAX siRNA on basal EPSCAMPA (recorded at −70 mV) and EPSCNMDA (recorded at +40 mV). Pairs of transfected and nearby untransfected cells are individually plotted (filled circles). Open circles indicate mean ± SEM. n = 9–11 pairs of transfected and untransfected cells for each construct. (E–H) LTD was induced by low frequency stimulation (300 pulses at 1 Hz paired with a holding potential of −45 mV). The EPSC amplitude of transfected cells and nearby untransfected cells normalized to the baseline prior to LTD induction was plotted as mean ± SEM. n = 9–11 pairs of transfected and untransfected cells for each construct. See also Figure S1 and Table S1.

We then proceeded to test the effect of siRNAs on NMDA receptor-dependent LTD induced by a pairing low-frequency stimulation protocol (see Experimental Procedures). LTD was blocked by the selective NMDA receptor antagonist APV [(2R)-amino-5-phosphonovaleric acid](data not shown), confirming that this stimulation protocol induces NMDA receptor-dependent LTD. Simultaneous whole-cell recordings were conducted in pairs of transfected and nearby untransfected CA1 neurons in the same slice. As shown in Figure 1E, LTD as revealed by a reduction of EPSCs measured 30 min after stimulation was comparable in untransfected and control plasmid transfected cells [56 ± 9% of baseline (pre-induction) in untransfected cells; 49 ± 6% of baseline in control plasmid transfected cells; p = 0.52, n = 11 pairs; Figure 1E]. Similarly, LTD was not altered in BID siRNA transfected cells (62 ± 6% of baseline in untransfected neurons; 61 ± 8% of baseline in BID siRNA transfected neurons; p = 0.92, n = 10 pairs; Figure 1F). However, LTD was reduced in neurons transfected with BAD siRNA (52 ± 6% of baseline in nearby untransfected neurons; 80 ± 10% of baseline in BAD siRNA transfected neurons; p = 0.03, n = 9 pairs; Figure 1G) and BAX siRNA (55 ± 6% of baseline in nearby untransfected neurons; 89 ± 7% of baseline in BAX siRNA transfected neurons; p = 0.002, n = 11 pairs; Figure 1H). siRNA-induced LTD inhibition was abolished by cotransfection of siRNA-resistant BAD and BAX constructs which had synonymous mutations in the siRNA-targeted region (BAD siRNA plus BAD: 66 ± 7% of baseline in transfected neurons, 62 ± 7% of baseline in nearby untransfected neurons, p = 0.69, n = 10 pairs; BAX siRNA plus BAX: 57 ±7% of baseline in transfected neurons, 60 ±7% of baseline in nearby untransfected neurons, p = 0.77, n = 10 pairs; Figure S1F,G), hence the effect of siRNAs on LTD was caused by specific reduction of BAD and BAX. These results suggest that BAD and BAX, but not BID, are essential for NMDA receptor-dependent LTD in CA1 neurons.

NMDA receptor-dependent LTD is abolished in both BAD knockout and BAX knockout hippocampal slices

To confirm the results obtained with siRNAs, we examined LTD in hippocampal slices from 2–3 week old BAD knockout and BAX knockout mice. BAD knockout mice show no developmental or histological abnormalities in the brain (Ranger et al., 2003). In BAX knockout mice, the number of neurons is slightly increased, but the overall structure of the brain is normal (Forger et al., 2004; White et al., 1998). To test whether basal synaptic transmission is altered in BAD knockout and BAX knockout slices, we analyzed the input-output relationship of Schaffer collateral-CA1 synapses (Figure S2A), current-voltage curves of EPSCAMPA (Figure S2B) and EPSCNMDA (Figure S2C), and the EPSCAMPA to EPSCNMDA ratio (Figure S2D). All of these measurements were indistinguishable in wild-type, BAD knockout and BAX knockout slices. In addition, the expression of NMDA receptors (subunit NR1, NR2A and NR2B) and AMPA receptors (subunit GluR1 and GluR2) in BAD knockout and BAX knockout slices was comparable to that observed in wild-type slices (Figure S2E). These results suggest that basal synaptic transmission and the expression and properties of AMPA and NMDA receptors are normal in these knockout mice.

We then examined LTD in hippocampal slices prepared from the knockout mice. By recording the field EPSP (fEPSP) in the CA1 region, we found that slices from wild-type littermates of both BAD and BAX knockout mice showed normal LTD after low-frequency stimulation (1 Hz, 900 pulses) of the Schaffer collateral pathway, so we pooled the data from all wild-type slices (84 ± 3% of baseline, n = 10 slices from 3 mice; Figure 2A, B). In both BAD and BAX knockout slices, however, LTD-induction was blocked (BAD knockout: 98 ± 2% of baseline, n = 10 slices from 3 mice, p = 0.001 for knockout vs. wild-type, Figure 2A; BAX knockout: 94 ± 2% of baseline, n = 10 slices from 3 mice, p = 0.01 for knockout vs. wild-type, Figure 2B). In addition, we tested LTD in postsynaptic-specific BAX knockout mice generated by crossing a floxed BAX mouse line (The Jackson Laboratory) with a transgenic mouse strain selectively expressing Cre recombinase in the CA3 region, but not in the CA1 region (Nakazawa et al., 2002). LTD in the CA1 region was comparable in hippocampal slices prepared from Cre positive and Cre negative littermates (BAXflox−/−Cre+: 81 ± 3% of baseline; BAXflox+/+Cre−: 79 ± 2% of baseline; BAXflox+/+Cre+: 83 ± 3% of baseline; n = 9 slices from 3 mice for each group; Figure S2F), supporting that BAX in the presynaptic neurons was not required for LTD induction. Hence, the knockout experiments combined with the siRNA experiment indicate that BAD and BAX are required in postsynaptic neurons for NMDA receptor-dependent LTD.

Figure 2.

Figure 2

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Low frequency stimulation (LFS)-induced LTD is abolished in hippocampal slices from BAD knockout and BAX knockout mice. Acute hippocampal slices were prepared from BAD knockout mice, BAX knockout mice and their wild-type littermates (2–3 weeks old). fEPSPs were recorded in the CA1 region. (A, B) LFS (900 pulses at 1 Hz) was delivered to induce LTD. (C, D) LTP was induced by two trains of high frequency stimulations (100 pulses at 1 Hz, 20 s interval). (E, F) mGluR agonist DHPG (100 μM, 20 min) was added to the bath solution to induce mGluR-dependent LTD. Representative traces for indicated time points were shown on the top. Student’s t-test was used for statistical analysis. Data are shown as mean ± SEM. n = 10 slices from 3 mice for each group. See also Figure S2.

To determine whether this requirement is specific to NMDA receptor-dependent LTD, we also measured long-term potentiation (LTP) and metabotropic glutamate receptor-dependent LTD (mGluR-LTD) in CA1 neurons of knockout mice. Both LTP and mGluR-LTD were comparable in wild-type slices prepared from littermates of BAD or BAX knockout mice, again allowing us to pool the data from all wild-type slices. LTP induced by two tetanic stimulations (100 Hz, 1s) was similar in wild-type, BAD knockout and BAX knockout slices (wild type: 163 ± 6% of baseline, n = 10 slices from 3 mice, Figure 2C,D; BAD knockout: 167 ± 9% of baseline, n = 10 slices from 3 mice, p = 0.72 for knockout vs. wild-type, Figure 2C; BAX knockout: 169 ± 9% of baseline, n = 10 slices from 3 mice, p = 0.59 for knockout vs. wild-type, Figure 2D). mGluR-LTD induced by bath application of the mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG; 100 μM for 20 min) was also similar in all three genotypes (wild type: 67 ± 6% of baseline, n = 10 slices from 3 mice, Figure 2E, F; BAD knockout: 68 ± 4% of baseline, n = 10 slices from 3 mice, p = 0.89 for knockout vs. wild-type slices, Figure 2E; BAX knockout: 67 ± 4% of baseline, n = 10 slices from 3 mice, p = 1.00 for knockout vs. wild-type slices, Figure 2F). These results suggest that BAD and BAX are required specifically for NMDA receptor-dependent LTD.

BAD and BAX are essential for AMPA receptor internalization and caspase-3 activation in LTD

The above results clearly indicate that BAD and BAX play crucial roles in NMDA receptor-dependent LTD. Because AMPA receptor endocytosis is a critical step in this form of LTD (Collingridge et al., 2004; Malenka and Bear, 2004; Shepherd and Huganir, 2007), we next examined whether BAD and BAX are involved in AMPA receptor endocytosis using an antibody feeding assay to analyze the endocytosis of AMPA receptor subunit GluR2 (Li et al., 2010b). Dissociated hippocampal neurons (14 days in vitro, DIV14) were transfected with BAD, BAX or BID siRNA constructs, and 2–3 days later stimulated with NMDA [30 μM for 5 min, a method to induce “chemical LTD” that shares the molecular mechanism with electrically induced LTD (Beattie et al., 2000)]. As shown in Figure 3A,B, NMDA-induced GluR2 internalization was significantly reduced by both BAD and BAX siRNAs, but not by BID or scrambled siRNAs, or by cotransfection of BAD and BAX siRNAs with siRNA-resistant BAD and BAX constructs. However, basal internalization of GluR2, which was measured in the absence of NMDA treatment, was not altered by transfection of BAD, BAX or BID siRNA constructs (Figure 3C). Thus BAD and BAX are required for NMDA-induced but not basal AMPA receptor internalization.

Figure 3.

Figure 3

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NMDA induced GluR2 endocytosis is inhibited by BAD siRNA and BAX siRNA, but not by BID siRNA. Cultured hippocampal neurons (DIV14) were transfected with the β-gal construct (as a marker of transfected cells) and either the empty vector, siRNA constructs, siRNAs along with siRNA-resistant BAD and BAX constructs, or scrRNA constructs. 3 days after transfection, NMDA-induced (30 μM, 5 min) or basal (without NMDA treatment) GluR2 endocytosis was analyzed with the “antibody-feeding” assay. Representative images of neurons immunostained for internalized GluR2 and surface-remaining GluR2 are shown in (A). Arrows label transfected neurons. Normalized internalization index (integrated fluorescence intensity of internalized GluR2/integrated fluorescence intensity of internalized GluR2 plus surface GluR2) is quantified in (B,C). Student’s t-test was used for statistical analysis. n = 15 neurons for each group. ** p < 0.01, compared to β-gal transfected cells treated with NMDA. Data are presented as mean ± SEM. Scale bars, 20 μm. See also Figure S3.

To complement the siRNA experiments, we also measured GluR2 internalization in cultured hippocampal neurons prepared from BAD knockout and BAX knockout mice. As shown in Figure S3A–D, while NMDA treatment (30 μM, 5 min) caused GluR2 internalization in wild-type neurons, it failed to do so in BAD and BAX knockout neurons. The cell surface expression of GluR2 and its basal internalization were also unaffected by the genotype of the neurons (Figure S3E–H). We conclude, therefore, that BAD and BAX are critical for NMDA receptor-dependent AMPA receptor endocytosis.

The above results, together with our previous observation that AMPA receptor internalization and LTD induction depend on caspase-3 activation (Li et al., 2010b), suggest that BAD and BAX are involved in caspase-3 activation in LTD. Hence, we measured active caspase-3 in NMDA-treated (30 μM, 5 min) BAD knockout and BAX knockout slices, using an antibody against the active, cleaved form of caspase-3. As shown in Figure 4, cleaved caspase-3 was elevated in wild-type but not BAD or BAX knockout slices treated with NMDA. These data suggest that during NMDA receptor-dependent LTD, BAD and BAX are required for caspase-3 activation.

Figure 4.

Figure 4

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NMDA induced caspase-3 activation is abolished in BAD knockout and BAX knockout hippocampal slices. Acute hippocampal slices were prepared from BAD knockouts, BAX knockouts and their wild-type littermates (2–3 weeks old), and treated with NMDA (30 μM, 5 min) or left untreated. The CA1 region was removed and lysed. Active caspase-3 was analyzed by immunoblotting (A) and quantified in (B). n = 3 mice for each group. ** p < 0.01 for comparison of treated and untreated slices. Data are presented as mean ± SEM. Student’s t-test was used for statistical analysis.

BAD and caspase-3 are sufficient to induce synaptic depression

Having established the role of BAD and BAX in caspase-3 activation and AMPA receptor internalization during LTD, we then examined whether BAD and caspase-3 are sufficient to induce synaptic depression. For this, we loaded active BAD and active caspase-3 directly into CA1 neurons in wild-type hippocampal slices by adding the proteins to whole-cell recording pipettes. Caspase-3 activity was measured using fluorophore-labeled DEVD (FITC-DEVD) that was perfused as described in the Experimental Procedures. As shown in Figure 5A,B, active caspase-3 was elevated by 241 ± 25% after 1 hr of infusion as indicated by the increased fluorescence signal of FITC-DEVD. This increase was comparable to that seen in NMDA (30 μM, 5min) treated cells (240 ± 27% at 10 min after treatment; Figure 5A,B). As a consequence of caspase-3 infusion, EPSCs were reduced (67 ± 5% of baseline at 1 hr of infusion, n = 9 slices from 3 mice, p = 0.0001 for comparison of 2 min and 1 hr of infusion; Figure 5C). In contrast, infusion of deactivated (boiled) caspase-3, or mutated caspase-3 (C163G, C163 is the catalytic nucleophile of caspase-3) did not alter EPSCs (Figure 5C). To monitor the quality of the recordings and the health of the recorded cells, we measured the series resistance and input resistance during recording. These parameters remained stable during the recording period (Figure S4A,C), indicating that the “run-down” of EPSCs was not caused by deterioration or demise of the recorded cells. In conclusion, infusion of active caspase-3 to a level similar to that induced by NMDA treatment is sufficient to suppress synaptic transmission.

Figure 5.

Figure 5

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Active BAD and active caspase-3 suppress synaptic transmission in CA1 neurons. Acute hippocampal slices were prepared from BAD knockouts, BAX knockouts, caspase-3 knockouts and their wild-type littermates as indicated (2–3 weeks old). Active BAD (30 μg/ml) and active caspase-3 (45 μg/ml) were each loaded into neurons through recording pipettes. (A, B) Time-lapse analysis of caspase-3 activity in neurons treated with NMDA (30 μM, 5 min), loaded with caspase-3 and loaded with BAD. FITC-DEVD was perfused for visualization of active caspase-3. Representative images of neurons before treatment and 15 min after NMDA treatment or 60 min after infusion with caspase-3 or BAD are shown in (A). Arrows in the left column of (A) label a neuron in focus. Note the variable fluorescence intensity in surrounding neurons located in different planes of focus. Arrows in the middle and right column of (A) indicate infused cells. Caspase-3 activity indicated by integrated intensity of FITC-DEVD signals is shown in (B). Graphs show mean ± SEM. n = 5 slices for each group. (C, D) Effect of infusing caspase-3 and BAD on EPSCs. CA1 neurons were recorded in the whole-cell patch-clamp mode. Active caspase-3 (C) or unphosphorylated BAD (D) were added to the internal solution of the patch pipette. Boiled caspase-3 (45 μg/ml), mutated caspase-3 (caspase-3 C163G, 45 μg/ml), boiled BAD (30 μg/ml) and mutated BAD (BAD BH3Δ, 30 μg/ml) were loaded into wild-type CA1 neurons as controls. In each graph, the normalized EPSC amplitude is shown as mean ± SEM. n = 9 slices from 3 mice for each group. Student’s t-test was used for statistical analysis. * p < 0.05 for comparison of EPSCs at 2 min and 1 hr. See also Figure S4.

We then performed similar experiments with recombinant BAD in its non-phosphorylated, active form. As shown in Figure 5A,B, active caspase-3 was increased by 182 ± 18% at 1 hour of infusion (n = 5, p = 0.0001 for comparison of pre-infusion and 1hr of infusion), but when deactivated (boiled) BAD was used, active caspase-3 was increased only slightly (119 ± 7% of baseline at 1hr of infusion, n = 5, p = 0.058 for comparison of pre-infusion and 1hr of infusion). The cells infused with active BAD showed a run-down of EPSCs (76 ± 7% of baseline at 1 hr of infusion, n = 9 slices from 3 mice, p = 0.013 for comparison of 2 min and 1 hr of infusion), while no such run-down was observed in cells infused with deactivated BAD or mutated BAD without the BH-3 domain through which BAD interacts with anti-apoptotic BCL-2 family proteins (Youle and Strasser, 2008) (Figure 5D). The series resistance and input resistance were stable during the experimental period (Figure S4B, D), thus excluding cell death. Taken together, these data show that BAD and caspase-3 are sufficient to suppress synaptic currents.

BAD, BAX and caspase-3 are sequentially recruited to induce synaptic depression

The above experiments established that BAD and BAX are required for caspase-3 activation and induction of LTD, but not whether they act in a sequential or a parallel manner. To address this question, we performed similar infusion experiments as above with hippocampal slices prepared from mice deficient in either caspase-3, BAX or BAD. As shown in Figure 5D, although infusion of active BAD suppressed synaptic currents in wild-type neurons, it did not alter them significantly in caspase-3 knockout cells (92 ± 8% of baseline at 1 hr of infusion, n = 9 slices from 3 mice, p = 0.42 for comparison of 2 min and 1 hr of infusion). Likewise, BAD infusion had no significant effect on the EPSCs of BAX knockout cells (91 ± 7% of baseline at 1 hr of infusion, n = 9 slices from 3 mice, p = 0.31 for comparison of 2 min and 1 hr of infusion). Again, the series resistance and input resistance remained constant during these infusion experiments (Figure S4). These results indicate that BAD requires BAX and caspase-3 in order to suppress synaptic transmission. Furthermore, the impairment of synaptic depression in BAD knockout and BAX knockout cells can be rescued by infusing active caspase-3 (EPSCs at 1 hr of infusion with active caspase-3 in BAD knockout cells: 46 ± 6% of baseline, n = 9 slices from 3 mice, p = 0.0001 for comparison of 2 min and 1 hr of infusion; in BAX knockout cells: 52 ± 5% of baseline, n = 9 slices from 3 mice, p = 0.0001 for comparison of 2 min and 1 hr of infusion; Figure 5C).

These results, combined with the finding that caspase-3 activation in LTD depends on BAD and BAX (Figure 4), suggest that BAD, BAX and caspase-3 are activated sequentially to induce synaptic depression.

Differential activation of the BAD-BAX-caspase-3 cascade in LTD and apoptosis

The above results show that in LTD, caspase-3 activation requires BAD and BAX, but activation of these proteins usually leads to cell death. This prompted us to investigate whether LTD and apoptosis differ in the mechanisms by which the BAD-BAX-caspase-3 pathway is activated, or in the level of its activation.

Dephosphorylation and translocation to mitochondria are critical steps in the activation of BAD during apoptosis. To test whether BAD is activated by similar mechanisms in LTD, we analyzed the level of phosphorylated BAD and the amount of BAD in the mitochondrial fraction. In fact, NMDA treatment (30 μM for 5 min as used for LTD induction) decreased phosphorylated BAD as detected by immunoblotting with an antibody against BAD phosphorylated at Ser112 (Figure 6A–B and Table S2), but the total amount of BAD was not affected (Figure S5A). It is notable that the level of phosphorylated BAD was higher at 30 min than at 10 min after NMDA stimulation (Figure 6A,B), suggesting that dephosphorylated BAD was rapidly rephosphorylated following NMDA treatment. Concomitant with the decrease in phosphorylated BAD, there was a transient increase of BAD in the mitochondrial fraction (Figure 6G–H). Taken together, these data suggest that BAD undergoes transient dephosphorylation and mitochondrial translocation during LTD.

Figure 6.

Figure 6

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Differential activation of BAD and BAX in LTD and apoptosis. Cultured cortical neurons were incubated with 30 μM NMDA for 5 min or 10 μM actinomycin D for 6 hr and lysed at the indicated time points. (A,B,I,J) BAD dephosphorylation induced by NMDA and actinomycin D. Cell lysates collected at the indicated time were immunoblotted for phosphorylated BAD (pBAD-S112) and total BAD (A, I). The ratio of pBAD-S112 to total BAD was quantified in (B) and (J). (C-F) Inhibition of NMDA-induced BAD dephosphorylation by okadaic acid and FK506. Neurons were incubated with 50 nM okadaic acid or 50 nM FK506 for 15 min before NMDA treatment. Immunoblots of phosphorylated BAD and total BAD (C,E) were quantified in (D,F). (G,H,K,L) Analysis of BAD and BAX in the mitochondrial fraction after treatment with NMDA or actinomycin D. The mitochondrial fraction was prepared from neurons treated with NMDA or actinomycin D and analyzed by immunoblotting for BAD, BAX and the mitochondrial protein COX IV. Quantification is shown in (H,L). (M,N) Active BAX analyzed by immunoprecipitation with the 6A7 antibody. Quantification in (N) shows active BAX normalized to total BAX in the input lysate. n = 3 for all conditions. Graphs show mean ± SEM, * p < 0.05. See also Figure S5 and Table S2.

It is known that in apoptosis, BAD can be dephosphorylated by PP1, PP2A and PP2B/calcineurin. We therefore tested whether these phosphatases were also involved in BAD dephosphorylation during LTD. In fact, NMDA-induced dephosphorylation of BAD was blocked by okadaic acid (50 nM, an inhibitor of PP1 and PP2A) and FK506 (50 nM, an inhibitor of PP2B/calcineurin) (Figure 6C–F), suggesting that these phosphatases may be responsible for BAD dephosphorylation in LTD. Interestingly, PP1 and PP2B/calcineurin are well known for their roles in the induction of NMDA receptor-dependent LTD, thus the mechanism that activates BAD is in line with the canonical pathway for LTD induction.

With respect to the activation of BAX in apoptosis, two processes are known to lead to an increase in active BAX in mitochondrial membranes: translocation of BAX activated in the cytosol to mitochondria, and activation of BAX associated with the mitochondrial membranes by pro-apoptotic BCL-2 family proteins such as BAD and BID. We measured the amount of active BAX in the whole cell lysates of NMDA-treated neurons (30 μM, 5 min) using immunoprecipitation with the antibody 6A7 that specifically recognizes BAX in the active conformation. It is known that once activated, BAX translocates to mitochondria very efficiently (George et al., 2009). Hence, immunoprecipitation of whole cell lysates with 6A7 measures active BAX predominantly in mitochondria. The amount of active BAX immunoprecipitated by 6A7 from treated cells was higher than that detected in control cells (Figure 6M–N and Table S2). However, we found, unexpectedly, that the total amount of BAX in the mitochondrial fraction was not altered by NMDA treatment (30 μM, 5 min; Figure 6G–H and Table S2). This is in contrast to the mitochondrial recruitment of activated BAX from the cytosol that is observed during apoptosis.

To compare the levels of active BAD and BAX in LTD and apoptosis, we treated neurons with actinomycin D (a transcription inhibitor) to induce apoptosis. Prolonged incubation with actinomycin D (10 μM) decreased phosphorylated BAD (Figure 6I–J and Table S2), increased BAD in the mitochondrial fraction (Figure 6K–L and Table S2) and enhanced cell death as detected by propidium iodide (PI) staining (Table S3). The total amount of BAD was not changed by actinomycin D (Figure S5B). Notably, both BAD dephosphorylation and translocation to mitochondria were induced to a higher level by actinomycin D than by NMDA (30 μM for 5 min; Figure 6I–L and Table S2). Actinomycin D treatment also increased BAX in the mitochondria fraction (Figure 6K–L and Table S2) and elevated active BAX more robustly than LTD-inducing NMDA treatment (Figure 6M-N and Table S2).

The differences in the levels of BAD and BAX activation in LTD and apoptosis were expected to result in different levels of caspase-3 activation. To determine the activity of caspase-3, we used the Caspase-Glo™ 3/7 Assay kit, which in rat hippocampi only measures caspase-3 activity, as caspase-7 is not detectable in this tissue (Li et al., 2010b). In fact, we found that after NMDA stimulation (30 μM for 5 min), caspase-3 activity reached a peak at 10 min (Figure 7A and Table S3) and declined by 30 min to a level close to that observed before treatment (Figure 7A). Conversely, active caspase-3 was increased to a high level and stayed high for at least 8 hr in cells treated with 10 μM actinomycin D (Figure 7C, Table S3 and data not shown). Thus, unlike in apoptosis, caspase-3 is activated to a moderate level and only transiently in LTD as previously reported (Li et al., 2010b).

Figure 7.

Figure 7

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The apoptotic activity of caspase-3 is dependent upon the intensity and duration of its activation. Cultured hippocampal neurons (DIV 17) were treated with NMDA or actinomycin D. Caspase-3 activity was assessed using the Caspase-Glo™ 3/7 Assay kit and cell death was analyzed using propidium iodide. (A) Caspase-3 activity at indicated times after NMDA treatment. (B) Cell death induced by treatment with 100 μM NMDA. Neurons were incubated with 10 μM LLY-FMK (a calpain inhibitor that prevents calpain-induced cell death) for 1 hr prior to NMDA treatment. Q-VD (20 μM) was added to the medium at indicated times, and propidium iodide staining was conducted 22 hr after NMDA treatment. (C) Caspase-3 activation induced by 0.1 μM or 10 μM actinomycin D. (D) Cell death induced by actinomycin D. Hippocampal neurons were treated with actinomycin D (at indicated concentrations) for 2 hr. Cells were incubated with medium containing Q-VD (20 μM) for 22 hrs following actinomycin D treatment for propidium iodide staining. (E,F) Caspase-3 activation and cell death induced by repetitive NMDA stimulations. Hippocampal neurons were treated with NMDA (30 μM, 5 min) or sham-treated every 35 min for 1–4 times. Caspase-3 activity was measured using the Caspase-Glo™ 3/7 Assay (E). To analyze cell death, Q-VD (20 μM) was added to the medium 30 min after 1, 2, 3 or 4 NMDA stimulations. Cell death was measured 22 hr after treatment and was shown in (F). * p < 0.05 for Student’s t-test. Data are presented as mean ± SEM (n = 3). See also Table S3.

Taken together, these results suggest that the mechanism for activation of BAX and the level of activation of the BAD-BAX-caspase-3 cascade are different in LTD and apoptosis.

Level and duration of caspase-3 activation are essential for induction of cell death

The above results led to the hypothesis that the level of activation of the BAD-BAX-caspase-3 cascade differentiates the functions of caspase-3 in LTD and cell death. We finally directly tested this hypothesis by examining whether commitment to cell death depends on the intensity and duration of caspase-3 activation.

In our first approach, we treated cultured hippocampal neurons with a high concentration (100 μM) of NMDA to enhance caspase-3 activation. To inhibit calpain-mediated cell death following this NMDA dose, we included a cell-permeable calpain inhibitor, LLY-FMK (10 μM), in the medium. As shown in Figure 7A and Table S3, caspase-3 was activated to a greater extent by 100 μM NMDA than by 30 μM NMDA. Its activation reached a peak at 10 min after treatment and then gradually declined, but remained higher than the pre-treatment level even 60 min after treatment (Figure 7A). To limit the duration of caspase-3 activation, the broad spectrum cell-permeable caspase inhibitor Q-VD was added to the neural medium at 10, 30, and 60 min after NMDA treatment. The proportion of PI positive neurons analyzed was comparable to that of sham-treated controls in neurons exposed to elevated active caspase-3 for 10 min or 30 min (Figure 7B), but in those exposed to elevated active caspase-3 for 60 min, it was significantly higher (Figure 7B and Table S3). These results suggest that robust and prolonged activation of caspase-3 induces cell death.

If high levels of caspase-3 activity are required to induce cell death, we would expect that actinomycin D, if made to induce only a small increase in caspase-3 activity, would not cause apoptosis. Hence, in a second approach, we treated neurons with a low dose of actinomycin D (0.1 μM), and found that this treatment increased caspase-3 activity to a level similar to the peak level induced by 30 μM NMDA (Figure 7C and Table S3). In fact, the proportion of PI-positive cells assessed 22 hr after actinomycin D treatment was similar to that of untreated controls (Figure 7D and Table S3). These results show that low-level caspase-3 activation is not adequate to provoke apoptosis.

In a third approach we tested whether prolonged caspase-3 activation at low levels induces apoptosis. To this end, we employed a repetitive stimulation method with 30 μM NMDA (see Experimental Procedures). As shown in Figure 7E, the pattern of caspase-3 activation and its peak levels of activity were comparable during the first three NMDA stimulations, and there was no significant increase in PI-positive cells (Figure 7F). However, after the fourth stimulation, caspase-3 activity reached a higher level (147 ± 7% of sham-treated controls at 30 min after the fourth stimulation; Figure 7E), but it did not reach the level observed after application of 100 μM NMDA (178 ± 8% of control at 30 min after 100 μM NMDA treatment; Figure 7A). Nevertheless, after adding Q-VD in order to stop caspase-3 activation 30 min after the fourth NMDA stimulation, the proportion of PI positive cells stained 22 hr later was significantly increased (Figure 7F and Table S3). Because the higher increase of caspase-3 activity (178 ± 8%) induced by 100 μM NMDA was not sufficient to cause cell death when lasting for just 30 min (Figure 7A,B), it is unlikely that the 147 ± 7% increase of caspase-3 activity for 30 min following the fourth NMDA stimulation was responsible for increased cell death; rather, this increase appears to be the result of prolonged activation of caspase-3. We conclude, therefore, that it is the lower and transient activation of caspase-3 that prevents cell death in LTD.

Discussion

In this study, we identify a signaling pathway for caspase-3 activation in LTD and address the intriguing question of how hippocampal neurons undergoing LTD avoid cell death despite the activation of caspase-3. Our findings reveal an unexpected conservation between the apoptosis and LTD pathways, and show that fine-tuning of the BAD-BAX-caspase-3 cascade by modifying its activation mechanism and activation levels and duration plays an important role in determining its cellular function. Our results are based on a series of gene knockdown and knockout experiments, showing that BAD and BAX are required to activate caspase-3 in NMDA receptor-dependent LTD, and on the infusion of active BAD and caspase-3, showing that the BAD-BAX-caspase-3 cascade is sufficient for induction of synaptic depression in hippocampal neurons.

We further demonstrate that activation of the BAD-BAX-caspase-3 cascade is initiated by PP2B/calcineurin, PP1 and PP2A. Although in both LTD and apoptosis, the same group of phosphatases is responsible for BAD activation, it is likely that phosphatases respond differently to different stimulations. LTD-inducing stimulations are brief and mild, while stimulations used to induce apoptosis (e.g. high concentrations of actinomycin D or NMDA) are prolonged and strong. In fact, one would expect that mild, LTD-inducing NMDA stimulations would cause a lower level of calcium influx than strong, death-inducing NMDA stimulations. In turn, lower levels of calcium could lead to lower levels of PP2B/calcineurin activation and therefore only weak and brief activation of BAD.

The level and duration of BAD activation determine the characteristics of BAX activation during LTD, because our results suggest that the primary mechanism for BAX activation in LTD is activation by BAD. In apoptosis, however, translocation of BAX from the cytosol to mitochondria plays a major role in enhancing mitochondrial permeabilization and cytochrome c release, because under physiological conditions, BAX predominantly resides in the cytosol, with only a minor fraction being present on mitochondrial membranes (Hsu et al., 1997).

Why the level of BAX in mitochondria is not elevated in LTD remains unclear. In fact, even during apoptosis, the mechanism leading to BAX translocation to mitochondria is elusive. It has been suggested that some apoptotic stimulations induce sequential phosphorylations of BAX, for instance, by AKT and GSK3β (Arokium et al., 2007). Phosphorylation could then trigger a conformational change in BAX, thereby allowing it to interact with BAX-binding proteins, such as the p53 up-regulated modulator of apoptosis (PUMA), which promotes BAX translocation (Zhang et al., 2009). Among the known proteins that regulate BAX translocation, only GSK-3β is known to be activated in NMDA receptor-dependent LTD (Peineau et al., 2007). It is conceivable that additional BAX-interacting proteins necessary to enable BAX translocation are not sufficiently activated by stimulations that induce LTD, leading to a lack of BAX accumulation in mitochondria.

By deliberately manipulating caspase-3 activation, our study also addresses the physiological significance of differential activation of caspase-3. We found that only strong or extended activation of caspase-3 (as induced by high concentrations of actinomycin D or NMDA, or repeated exposure to low concentrations of NMDA) can induce cell death, while weak or transient caspase-3 activation (as induced by low concentrations of NMDA or actinomycin D, or single application of low concentrations of NMDA) is not sufficient to do so. These findings establish a causal link between the intensity and duration of caspase-3 activation and whether caspase-3 acts as an inducer of LTD or an executor of cell death. Actinomycin D is widely used as a transcription inhibitor. Our data suggest that in addition to transcription, actinomycin D may affect synaptic transmission by activating caspase-3.

The transient elevation of caspase-3 activity following induction of LTD suggests the presence of a mechanism capable of rapidly removing active caspase-3. We have previously shown that cleaved caspase-3, which represents the active form, exhibits a rate of decay during LTD (Li et al., 2010b) similar to that of caspase-3 activity reported here. Therefore, it is likely that the reduction of caspase-3 activity is caused by rapid degradation of active caspase-3. Potential regulators of caspase degradation include the X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitors of apoptosis (IAP) family known to act as an ubiquitin- and NEDD8-E3 ligase for caspases (Broemer et al., 2010; Ditzel et al., 2008).

Because caspase-3 activation is inherently dangerous to a cell, one might ask how cells may benefit from utilizing the BAD-BAX-caspase-3 cascade for non-apoptotic functions. In fact, adapting the mitochondrial apoptotic pathway to activate caspases might be especially advantageous for non-apoptotic functions that need to be restricted to particular subcellular locations, such as LTD, which is confined to stimulated synapses. Mitochondria would seem to be ideal devices for delivering and restricting caspase activators to the vicinity of stimulated synapses to ensure synapse-specific changes, because the motility of mitochondria is controlled by synaptic activity and intracellular Ca2+, and mitochondria tend to accumulate near active synapses (Li et al., 2004). Our findings that the BAD-BAX-caspase-3 cascade is sufficient for LTD induction as demonstrated by infusion of active BAD or caspase-3 into hippocampal neurons and that overexpression of BCL-XL, which antagonizes BAD and BAX, inhibits LTD (Li et al., 2010b) provide additional support for the importance of mitochondria to synaptic plasticity.

Finally, our findings may have implications for other non-apoptotic cellular processes involving caspases; in none of these processes are the mechanisms for restricting the apoptotic function of caspases well understood. Nevertheless, it has been reported that in lens cell differentiation, for instance, caspase activation is milder than in apoptosis (Weber and Menko, 2005). Hence, it is conceivable that weak activation of caspases, either by alternative mechanisms or by modified canonical apoptosis pathways, serves as an apoptosis-inhibitory mechanism in other non-apoptotic cellular processes as well.

Experimental Procedures

Knockout mice, DNA constructs and reagents

BAD knockout mice were generously provided by Dr. Nika N. Danial (Harvard University). BAX knockout and caspase-3 knockout mice were purchased from the Jackson Laboratory. Annealed oligos containing siRNA or scrambled (scrRNA) sequences targeted to BAD (siRNA: GAATGAGCGATGAATTTGA; scrRNA: GGATATTAGAAGGGATCAT), BAX (siRNA: CTCACCATCTGGAAGAAGA; scrRNA: GAACCGACGAACGCTTATA) or BID (siRNA: CTCCTTCTATCATGGAAGA; scrRNA: GCACACCCGTAATTTAGTT) were inserted into the pSuper or pLentiLox 3.7 vector. Mutated BAD (A462G, C468T, T471C, A474G, T477C) and mutated BAX cDNAs (C555G, C558G, C561T, G567A, G570A) were inserted into the GW1 vector. The following reagents were obtained commercially: anti-BAD antibody (Cell Signalling Technology), anti-phospho-BAD antibody (Cell Signalling Technology), anti-caspase-3 antibody (Cell Signalling Technology), anti-COX IV antibody (Cell Signalling Technology), anti-BAX antibody 6A7 (Sigma), anti-BAX polyclonal antibody (Upstate Cell Signaling Solutions), anti-BID antibody (Santa Cruz Biotechnology), actinomycin D (Sigma), FK506 (Alexis Biochemicals), okadaic acid (Sigma), FITC-DEVD (ABD Bioquest), propidium iodide (Roche), LLY-FMK (SM Biochemicals), Q-VD (SM Biochemicals), active caspase-3 (R&D systems) and BAD protein (Santa Cruz Biotechnoloty).

Acute hippocampal slice

2- to 3-week-old mice were anesthetized by isoflurane overdose followed by decapitation. The brain was placed in ice-cold artificial cerebrospinal fluid (ACSF, pH 7.4, gassed with 95% O2/5% CO2), which is composed of (in mM) 124 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2.5 CaCl2, 1.3 MgSO4 and 10 D-glucose. Transverse hippocampal slices (350 μm thick) were prepared in ice-chilled, oxygenated ACSF with a vibrotome (Leica). The CA3 region of the hippocampus was removed surgically. Hippocampal slices were recovered in ACSF at 30°C for 30 min, then at room temperature for 30 min before being transferred to the recording chamber.

Hippocampal slice culture

Hippocampal slice cultures were prepared from 6- to 8-day-old Sprague-Dawley rats. After decapitation, the brain was placed immediately in the cold cutting solution composed of (in mM) 238 sucrose, 2.5 KCl, 26 NaHCO3, 1 NaH2PO4, 5 MgCl2, 11 D-glucose and 1 CaCl2. Hippocampal slices (400 μm) were cut with a McIlwain tissue chopper and placed on top of semi-permeable membrane inserts (Millipore Corporation) in a six-well plate containing culture medium (78.8% minimum essential medium, 20% heat-inactivated horse serum, 25 mM HEPES, 10 mM D-glucose, 26 mM NaHCO3, 2 mM CaCl2, 2 mM MgSO4, 0.0012% ascorbic acid, 1 μg/ml insulin; pH 7.3; 320–330 mOsm). Medium was changed every 2 days. No antibiotics were used. Neurons were biolistically transfected using the gene gun (Helios Gene-gun system, Bio-Rad) at DIV3–4. Electrophysiological recordings were performed at 2 days after transfection.

Electrophysiology

The standard method was used. Hippocampal slices were perfused with ACSF (bubbled with 95% O2/5% CO2; 30°C) at the rate of 2 ml/min. Stimulating electrode was placed on the stratum radiatum in the CA2 area. Stimuli were delivered to the electrode at 20 sec intervals. For field-recordings, recording pipettes (1–2 MΩ) were filled with the bath solution and placed in the CA1 region. Tetanic stimuli (100 pulses at 100 Hz, 2 trains at 20 sec intervals) were delivered to induce LTP, while low frequency stimulations (LFS; 900 pulses at 1 Hz) were delivered to induce LTD. For whole-cell recordings, 100 μM bicuculline was added to the ACSF to block GABAA receptors. The patch pipette (4–7 MΩ) solution is composed of (in mM) 130 cesium methanesulfonate, 8 NaCl, 4 Mg-ATP, 0.3 Na-GTP, 0.5 EGTA, 10 HEPES and 5 QX-314 at pH 7.3. EPSCs of CA1 pyramidal cells were recorded at −70 mV. A 10 min-baseline recording was conducted prior to LTD induction. For whole-cell recordings in cultured hippocampal slices, 2 μM 2-chloroadenosine was added to the ACSF to prevent bursting. Simultaneous whole-cell recordings were obtained from pairs of nearby transfected and untransfected CA1 neuron. LTD was induced by delivering low frequency stimulations (LFS; 1 Hz) for 300 sec at a holding potential of −45 mV. AMPA receptor-mediated EPSCs (EPSCAMPA) were measured at a holding potential of −70 mV. NMDA receptor-mediated EPSCs (EPSCNMDA) were measured 50–70 ms after the peak of EPSCAMPA at a holding potential of +40 mV (Terashima et al., 2004). The series resistance and input resistance were monitored on-line and analyzed with the Clampex program off-line. Only cells with a series resistance of < 25 MΩ and a < 10% drift in both series resistance and input resistance during the recording period were included. Student’s two-tailed t-test was used for statistical analysis (p < 0.05 considered significant).

Neuronal culture and transfection

Hippocampal and cortical neuron cultures were prepared from embryonic day (E) 18–19 rat or mouse embryos as previously described (Sala et al., 2001). Neurons were seeded on poly-D-lysine (30 μg/ml) and laminin (2 μg/ml) coated coverslips or plates at a density of ~750 cells/mm2. Cultures were grown in Neurobasal medium (Invitrogen) supplemented with 2% B27 (Invitrogen), 0.5 mM glutamine and 12.5 mM glutamate. Hippocampal neurons were transfected with Lipofectamine™ 2000 (Invitrogen).

GluR2 internalization assay and surface GluR2 staining

The internalization assay was performed as described previously (Lee et al., 2002). Briefly, neurons were incubated with antibodies against the N-terminus of GluR2 (Chemicon) for 15 min at 37°C, then stimulated with NMDA (30 μM) or left unstimulated for 5 min. Neurons were fixed with the fixation buffer (4% formaldehyde and 4% sucrose in PBS) immediately after the stimulation. Surface-remaining antibody-labelled GluR2 was saturated by incubation with Alexa Fluor® 555-conjugated secondary antibody (Invitrogen). Internalized antibody-labelled GluR2 was stained with Alexa Fluor® 488-conjugated secondary antibody (Invitrogen) after permeabilization with methanol (−20°C). For total GluR2 surface staining, neurons were incubated with antibodies against the N-terminus of GluR2 for 15 min at 37°C, then fixed and incubated with Alexa Fluor® 555-conjugated secondary antibody.

Image acquisition and image analysis

Images were acquired with a Zeiss LSM510 confocal microscope with a 63X (NA 1.4) objective. Confocal images were collapsed to make 2D projections. MetaMorph software (Molecular Devices) was used to measure integrated fluorescence intensity of internalized receptors and surface-remaining receptors on the dendrite. Statistical analysis was performed using Student’s t-test. All image acquisition and image analysis were done blindly to the treatment.

Preparation of cytosolic and mitochondrial fractions

Cultured cortical neurons (DIV18) were homogenized with a plastic pestle in microcentrifuge tubes using a motorized homogenizer (20 strokes). The cell lysate was centrifuged at 5000 g for 5 min to remove nuclei and unbroken cells. The supernatant was further centrifuged at 55,000 g for 1 hr to obtain the cytosolic fraction (supernatant) and mitochondrial fraction (pellet).

Luminescent assay of active caspase-3 and propidium iodide staining

Caspase-3 activity of cultured hippocampal neurons (DIV18) was analyzed with the Caspase-Glo® 3/7 Assay kit (Promega). 50 μl Caspase-Glo® 3/7 reagents was added to each well of 96-well plates seeded with primary hippocampal neurons (20,000 cells/well). After mixing and incubation at room temperature for 30 min, luminescence was measured with a 1420 Multilabel Counter luminometer (Perkin Elmer). For propidium iodide staining, cells were incubated with culture medium containing 2 μg/ml propidium iodide for 15 min, followed by wash with PBS and fixation in 4% formaldehyde. For repetitive NMDA stimulations, hippocampal neurons were stimulated with 30 μM NMDA for 5 min, returned to medium without NMDA for 30 min, then subjected to NMDA stimulation again. The stimulation was repeated 1–4 times.

Time-lapse detection of active caspase-3 in whole-cell patched neurons

Acute hippocampal slices (from mice at 2–3 weeks of age) were perfused with FITC-DEVD for 5 min followed by washout for 10 min to remove unbound FITC-DEVD and image acquisition. After the first image was taken, the slice was perfused with NMDA (30 μM, 5 min) or subject to whole-cell patch clamp for loading of BAD and caspase-3. Perfusion of FITC-DEVD and image acquisition were repeated every 15 min. Images were acquired with an Olympus BX61WI confocal microscope with an UplanSApo 60x/1.35 objective during the last 5 min of the washout period.

Preparation of mutated BAD and mutated caspase-3 proteins

The cDNAs of BAD BH3Δ (gift of Dr. Richard Youle) and caspase-3 C163G were inserted into the GB1 vector (gift of Dr. Tsan Xiao) behind the histidine tag. Proteins were expressed in bacteria and purified with HisTrap™ HP columns (GE Healthcare) followed by dialysis to remove imidazole.

Supplementary Material

01

Acknowledgments

We thank Dr. Nika N Danial (Harvard University) for providing the BAD knockout mice, Dr. Richard Youle (NINDS/NIH) for providing the BAD BH3Δ construct and for critical reading of the manuscript, Dr. Kazutoshi Nakazawa (NIMH/NIH) for providing the CA3-specific Cre mice, Dr. Tsan Xiao for providing the GB1 vector (NIAID/NIH), Dr. Heinz Arnheiter (NINDS/NIH) for provocative discussions and critical reading of the manuscript. This work is supported by NIMH Division of Intramural Research Programs.

Footnotes

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