Supplementary Movies for “Purines Induce Directed Migration and Rapid Homing of Microglia to Injured Pyramidal Neurons in Developing Hippocampus” Kurpius D, Nolley E, and Dailey M. Dept. of Biological Sciences Univ. of Iowa Accepted for publication in GLIA © 2007

Figure 2 Supplementary Movie 1 To view in 3D, use red-green glasses (red over left eye). SP SR 30  m Time-lapse sequence showing activation and directional migration of MG into the pyramidal cell body layer (SP) in an acutely isolated neonatal mouse hippocampal tissue slice. MG in the stratum radiatum (SR) near the SP (lower right) polarize and migrate directionally into the SP. Note the slow wave of activation that progresses away from the SP through the SR (lower right to upper left). Total time is ~4hr.  Click to play

Figure 3 Supplementary Movie 2 & 3 Two-channel time-lapse imaging shows IB4-labeled MG (green) engaging nuclei of injured/dead neurons (red). (A) is a still image showing the relative position of fields ‘B’ and ‘C’. (B) Time-lapse sequence starting ~3.5 hr after tissue excision. (C) Time-lapse sequence starting ~6.5 hr after tissue excision. Note that MG appear to engulf the dead cell nuclei.  Click to play C. B. Supplementary Movie 2 Supplementary Movie 3

Figure 5C Supplementary Movie 4 Apyrase inhibits MG motility and migration in neonatal tissue slices. High magnification, time- lapse sequence shows highly mobile MG in and near the CA3 pyramidal cell body layer (SP). During the baseline, MG are highly locomotory. Application of apyrase (100 U/ml) inhibits MG movements. Following washout, MG movements rapidly recover. Total time = ~6 hr. Experiment Baseline (2 hr) Apyrase (2.5 hr) Washout (1.5 hr) SP SR 30  m  Click to play

Suppl. Figure 3 Supplementary Movie 5 Time-lapse sequence showing diffusion of BodipyTR-ADP (100  M) from exterior of tissue slice (intense red region at left) to interior of slice (stratum radiatum, SR). Imaging starts shortly after application of ADP. Note the subtle increase in red fluorescence in the central and right portion of the field of view, especially during the first 60 min. In response to diffusion of ADP into the slice, microglia (green) in the SP and SR move toward the edge of the tissue slice.  Click to play BodipyTR-ADP (100  M) + ADP (1mM) 3 hr Experiment IB4-Microglia

Figure 6 Supplementary Movie 6 ADP (10hr) (from the start) Time-lapse sequence showing directed branch extension followed by migration in response to exogenous ADP (1mM). ADP was applied to the slice immediately after excision, and imaging commenced 1hr later. When ADP is first applied at early stages of activation (as here), MG first extend long branches directed toward the source of ADP at the edge of the tissue slice (below and to the right). Subsequently, MG in the SP and SR migrate past the injured neurons in the SP (not labeled) and accumulate at the edge of the tissue slice. Total sequence time is ~10hr.  Click to play Experiment

Figure 7A Supplementary Movie 7 Time-lapse sequence showing sequential responses of MG to application of apyrase and ATP in a neonatal rat hippocampal tissue slice. During the baseline, note the migration of MG and their accumulation near injured CA3 pyramidal neurons in the SP. Application of apyrase (indicated in upper right corner) inhibits MG movements. Following application of ATP (1mM), MG are drawn away from injured neurons in the SP and move toward the edge of the tissue slice (just below field of view). Following washout of ATP, MG show a low level of undirected movements.  Click to play Baseline (3.5 hr) Apyrase (2 hr) Washout (2 hr) ATP (4.5 hr) Experiment Washout (5.5 hr)