Dynamic role of postsynaptic caspase-3 and BIRC4 in zebra finch song-response habituation

Abstract

Activation of the protease caspase-3 is commonly thought to cause apoptotic cell death. Here, we show that caspase-3 activity is regulated at postsynaptic sites in brain following stimuli associated with memory (neural activation and subsequent response habituation) instead of cell death. In the zebra finch auditory forebrain, the concentration of caspase-3 active sites increases briefly within minutes after exposure to tape-recorded birdsong. With confocal and immunoelectron microscopy, we localize the activated enzyme to dendritic spines. The activated caspase-3 protein is present even in unstimulated brain but bound to an endogenous inhibitor, BIRC4 (xIAP), suggesting a mechanism for rapid release and sequestering at specific synaptic sites. Caspase-3 activity is necessary to consolidate a persistent physiological trace of the song stimulus, as demonstrated using pharmacological interference and the zenk gene habituation assay. Thus, the brain appears to have adapted a core component of cell death machinery to serve a unique role in learning and memory.

Figure 1. Increase and apparent synaptic association of active caspase-3 after novel song stimulation

Shown are example confocal microscopic images of sections from a bird hearing only silence (panel A) or another bird immediately after 10 novel song presentations (B–D). The field of view is within the caudomedial nidopallium (NCM, in the auditory forebrain). The sections were double-labeled for active caspase (using biotinylated DEVD peptide detected by fluorescent-linked streptavidin, green fluor) and a synaptic marker protein, synaptotagmin (red fluor). Panel D: merger of panels B and C showing close association but distinct distributions of active caspase-3 and synaptotagmin immunoreactivities. Bar size = 4 microns.

Figure 2. Time course of activated caspase-3 immunoreactivity in NCM during novel song playback

After overnight isolation in a sound chamber, each bird was presented with repeated playback of a song stimulus (15 seconds followed by 45 seconds of silence), and then euthanized at the time shown (relative to first stimulus onset). Brain sections containing NCM were probed with DEVD which was visualized by DAB staining using a 20x objective, and the signal above background was quantified (Methods). Numbers of birds at each timepoint (0, 2, 10, 20, 30 and 90 min, respectively): 7, 4, 8, 4, 3, 3.

Figure 3. Ultrastructural localization of activated caspase-3 by immunogold electron microscopy

Representative images within NCM from birds after song or silence. (A) showing one of the few [3/31, 9.7%] caspase-3 staining fields from silent control tissue. (B–D) showing 3 of the more plentiful [42/66, 63%] staining fields from song stimulated tissue. In each field, post-synaptic densities are evident with vesicle-filled presynaptic terminals on opposite side. Arrows note gold particles indicating caspase-3 location at the post-synaptic density (panel A) or nearby on the postsynaptic side (panels B–D). Size bars = 200 nm in all panels. No gold particles were found in soma, or presynaptic locations supporting a post-synaptic localization of this caspase-3 response.

Figure 4. Caspase-3 is present in brain homogenates, but concentration does not change with song stimulation

Auditory lobules (AL) were dissected from birds, homogenized, fractionated and immunoblotted. All lanes were blotted with anti-active caspase-3 antibody. Each lane represents the AL from a separate bird. The experiment was repeated a total of four times on 16 birds, and this figure is representative; quantitative densitometric analysis detected no significant effect of treatment. Activated caspase-3 often appears as multiple cleaved bands migrating variably in the 17–20 kD range (Jarskog et al., 2006; Faleiro et al., 1997); the doublet visible in lanes 3 and 4 aligns with the doublet indicated by arrows in Fig. 5B. Lane 1, positive control showing human caspase-3 from transfected Jurkat cell line; Lane 2, negative control (non-transfected Jurkat cell homogenate); Lane 3, AL homogenate from a silent control bird; Lane 4, AL homogenate after 10 min. song stimulation; Lane 5, AL homogenate from a silent control bird after enrichment by immunoprecipitation with total caspase-3 antibody (both active and pro-form); Lane 6, as in lane 5 except AL homogenate was from a bird after 10 min. song stimulation.

Figure 5. Association of caspase-3 and BIRC4 (xIAP) in brain homogenates

(A) Shows extracts of different regions of zebra finch brain immunoblotted with an antibody to BIRC4; lane 1: positive control peptide (human BIRC4); 2: cerebellum; 3: lateral forebrain; 4: medial forebrain; 5: auditory lobule of the forebrain (AL) which contains the song-responsive NCM. (B) Co-immunoprecipitation assay, where an extract of AL was first immunoprecipitated with the BIRC4 (xIAP) antibody used in panel A, and then immunoblotted with the antibody to active caspase-3 as in Fig. 4. Arrows indicate the active caspase-3 doublet. This experiment was repeated four times using a different bird each time, with similar results.

Figure 6. Colocalization of caspase-3 and BIRC4 (xIAP) immunoreactivity in NCM by double-label immunofluorescence microscopy

Representative section of NCM from a bird hearing 10 min. of novel song, stained for: A) active caspase-3 using DEVD (green channel); B) BIRC4 (red channel); C) DAPI counterstain (blue channel); D) merged image of all three channels. Bar = 8 microns

Figure 7. Comparison of distributions for active caspase-3 (DEVD binding) and total caspase-3 (antibody immunoreactivity)

Representative section of NCM from a bird hearing 10 min. of novel song, stained for: A) active caspase-3 using DEVD (green channel); B) total caspase-3, both active and inactive forms (red channel); C) DAPI counterstain (blue channel); D) merged image of all three channels. Bar = 200 microns

Figure 8. Effects of song stimulation and habituation on caspase-3 and BIRC4 immunoreactivities in NCM

Adult male zebra finches were placed in 3 groups treatment groups (n=4 per group) based on the sound stimulus to which they were exposed immediately prior to euthanasia: S, silence; N, novel song (10 minute playback); H, habituation (75 minutes of playback). Initial stimulations were performed simultaneously on 4 birds per day (with counterbalancing of treatment groups across 3 days), and subsequent manipulations were performed in parallel on the tissue from all 12 birds. The brains were sectioned and double-labeled as in Figs. 6 and 7, in an alternating sequence using DEVD and antisera to either BIRC4 or total caspase (active and inactive forms combined), with DAPI as a counterstain. Images were collected from 4–7 fields within NCM of each section, and the red and green channels of each image were analyzed separately as in Methods to generate a mean fraction of pixels above background threshold for each probe in each bird. The averages of these means for each treatment group are presented in the graph. Bars indicate standard error, and the asterisk indicates a significant effect of treatment on that measurement (ANOVA comparing the S, N and H treatment groups for each probe).

Figure 9. Requirement of caspase-3 activity for long-term zenk habituation

A) Reference sections showing in situ hybridization for zenk mRNA in NCM, in birds hearing 30 min. of either (left) novel or (right) familiar song. The lack of zenk response to familiar song indicates the presence of a song-specific memory (Kruse et al., 2004; Mello et al., 1995; Stripling et al., submitted). (B) Example in situ hybridizations showing effect of caspase-3 inhibition during training on the appearance of a song memory (persistent zenk habituation) a day later. Birds were trained on day 1 and tested with the same song on day 2. Left: caspase-3 inhibitor had been infused during training; note zenk response similar to bird hearing novel song (as in panel A, left). Right: vehicle (only) had been infused during training; note zenk response similar to bird hearing familiar song (as panel A, right). Images in A and B are tiled 6x5 grids of 10x microscope field views to visualize the whole of NCM. (C) Quantification of zenk in situ hybridization data from four trials of paired injections (n=8 birds total) performed as the example in panel B. Mean zenk hybridization intensity for each treatment group is expressed as a fractional area of labeling as in Figs. 2 and 8 (Methods). Each trial involved two birds treated in parallel, one receiving the inhibitor and the other only vehicle during training, and then tested a day later for their zenk responses to the training stimulus. The low level of zenk in the controls indicates persisting habituation to the stimulus. Birds that had been infused with inhibitor during training showed a significantly higher zenk response at test (p=0.01, two tailed paired t-test), indicating disruption of long-term zenk habituation by drug treatment during training. Error bars: S.E.M.