Figure 1.
Syt I is strongly expressed in neonatal SACs and IPL.
Ai–Aiii. Merged images of Syt I (green) and ChAT (red) staining in retinal cross-sections from P0–P2 rats. The colocalization of Syt I and ChAT labeling was found in the IPL (yellow). Scale bars, 25 µm. ChAT, choline acetyltransferase. NBL, neuroblast layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bi–Biii. Immunofluorescence labeling of Syt I (green) in the IPL of retinal cross-sections from P0–P2 rats. Ci–Ciii. Immunofluorescence staining of ChAT (red) labeling SACs and the IPL in retinal cross-sections from P0–P2 rats. Di–Diii. Merged images of Syt I and ChAT staining in the same retinal cross-sections. The Syt I and ChAT labeling was colocalized to the SAC somata (yellow) as indicated by arrows. Scale bars for B-D, 50 µm. Ei–Eiii. The high magnification for merged images of Syt I and ChAT staining in the boxes of Di–Diii. Scale bars for E, 5 µm.
Figure 2.
Gene transfer into whole-mount retinas by the homemade electroporation device.
A–B. Preparation of platinum (Pt) electrodes. Ai. The arrangement of the six slides for the (+) electrode base. The colors of slides represent the slide arrangement in different layers. The numbers in the circles indicate the corresponding sequence to arrange slides. Aii. The Pt foil (15×15 mm with one extra 5×5 mm-overhanging square) was aligned to one side of the 7th slide with the overhanging square left out. Epoxy glue was applied to attach the Pt foil onto the slide and connect all the slides together. Aiii. The wire was soldered to the edge of the overhanging Pt square. Aiv. The last (8th) slide was glued onto the (+) electrode base. Bi–ii. The arrangement of the (−) electrode. A pen tube (green) with a diameter of 10 mm was used to attach the same-sized Pt foil by epoxy glue. The electric wire was inserted through the pen tube and soldered onto the Pt foil. C. The setup for electroporation in a horizontal configuration. The retinal explant was placed in a well [with dimensions 12 (length) ×12 (width) ×3 (height) mm] on the (+) electrode. The (−) electrode made contact with solution above the well and covered the retinal explant. The distance between the (+) and (−) electrodes was adjusted by a micromanipulator that held the (−) electrode. D. The P1 rat retinas were transfected with pCMV-HA (vector) or pCMV-HA-Syt I (HA-Syt I) with this electroporation device. The retinal explants were incubated for 72 hr to allow gene expression. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with antibodies indicated on the right (HA or α-tubulin). Only the retinas transfected with HA-Syt I displayed the HA signal with a size of ∼65 kD, which corresponded to the molecular weight of Syt I. Data shown were representative blots from 3 different experiments.
Table 1.
Comparison of wave characteristics following transfection.
Figure 3.
The mGluR2 promoter drives gene expression selectively in SACs.
Whole-mount retinas from P0–P2 rats were double-labeled with HA (green) and ChAT (red) after 72 hr of transfection. Ai–Ci. Whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Ai. Labeled with HA. Bi. Labeled with ChAT. Ci. Merged images of Ai and Bi. Scale bar, 50 µm. EGFP, enhanced green fluorescent protein. Aii–Cii. High magnification view of whole-mount retinas transfected with pCMV-HA-Syt I-IRES2-EGFP. Aii. Labeled with HA. Bii. Labeled with ChAT. Cii. Merged images of Aii and Bii. Scale bar, 25 µm. Di–Fi. Whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Di. Labeled with HA. Ei. Labeled with ChAT. Fi. Merged images of Di and Ei. Scale bar, 50 µm. Dii-Fii. High magnification view of whole-mount retinas transfected with pmGluR2-HA-Syt I-IRES2-EGFP. Dii. Labeled with HA. Eii. Labeled with ChAT. Fii. Merged images of Dii and Eii. Scale bar, 25 µm. G–H. Retinal cross-sections transfected with either pCMV-HA-Syt I-IRES2-EGFP (G), or pmGluR2-HA-Syt I-IRES2-EGFP (H). The SACs and IPL were stained for ChAT (red). The transfected cells were stained for HA/GFP (green). The cell nuclei were stained with DAPI (blue). Right, Merged images (yellow). The transfected SACs were indicated by arrows. Insets showed the higher magnification of merged images. Scale bars, 25 µm.
Figure 4.
Ca2+ transient frequency is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.
A. Left, The expression pattern of pmGluR2-IRES2EGFP in transfected whole-amount retinas. Right, The RGC layer labeled with the Ca2+ indicator fura-2 to measure the wave-associated Ca2+ transients after transfection. Scale bar for both panels, 25 µm. B. Spontaneous, correlated Ca2+ transients were monitored from randomly selected cells in the RGC layer. The example traces of fluorescent changes over time showed the Ca2+ transients in the nearby cells from one imaged region. Retinas were transfected with pmGluR2-IRES2EGFP (Control), pmGluR2-IRES2EGFP-Syt I (Syt I), pmGluR2-IRES2EGFP-Syt I-D230S (Syt I-C2A*), or pmGluR2-IRES2EGFP-Syt I-D363N (Syt I-C2B*). C. Summary of Ca2+ transient frequency after transfection. Data were from 23–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (**p<0.01; ***p<0.001; One-Way ANOVA with post-hoc Student-Newman-Keuls test.) D. Distributions of cumulative probability for Ca2+ transient frequency from individual cells. Data were from 1196–1489 cells out of the same data sets in C. E. Summary of fraction of cells with Ca2+ transients in one imaged region after transfection. Data were from 32–58 transfected regions out of the same data sets in C. (**p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.).
Figure 5.
Ca2+ transient size is reduced by weakened Ca2+ binding to the C2AB domains of Syt I.
A. Top, Definitions of Ca2+ transient duration and amplitude. The peaks of Ca2+ transients were automatically picked by Igor Pro according to the criteria in Materials and Methods. The RMS noise (Noise) was measured from the trace between 30 sec prior to the peak and 50 sec following the peak. The starting point (x0, y0) (indicated by solid arrows) was defined by the latest time point where the first derivative was zero (i.e. dy/dx = 0, where y was the fluorescence changes in % ΔF/F and x was the recording time in sec) before the peak. To define the end point (x′, y′) (indicated by open arrows), a line was drawn to connect the time points where the trace of Ca2+ transients returned to within the RMS noise of the baseline, with the minimal fluctuation of fluorescence between the starting and end points (i.e. y′−y0 = minimum). Bottom, The Ca2+ transient duration was the interval between the starting and end points. The Ca2+ transient amplitude was the fluorescence change from the baseline to peak. B. Representative individual Ca2+ transients from retinas transfected with Control (black), Syt I (blue), Syt I-C2A* (green), or Syt I-C2B* (red). C. Summary of Ca2+ transient duration for each group. Data were from 22–39 transfected retinas and 7–15 pups (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). (*p<0.05; **p<0.01; ***p<0.001; Kruskal-Wallis method followed by post-hoc Dunn test.) D. Distributions of cumulative probability for Ca2+ transient duration from individual cells. Data were from 860–1106 cells out of the same data sets in C. E. Summary of Ca2+ transient amplitude after transfection. Data sets were the same as in C. (**p<0.01; Kruskal-Wallis method followed by post-hoc Dunn test.) F. Distributions of cumulative probability for Ca2+ transient amplitude from individual cells. Data sets were the same as in D.
Figure 6.
Pairwise correlation is not altered by weakened Ca2+ binding to the C2AB domain of Syt I.
A. Representative pairwise cross-correlograms of 49 cells in one imaged region from the transfected retinas. Each event was a Ca2+ transient in an individual cell. Delay times were the time differences between Ca2+ transient peaks in cell pairs. Note that all correlograms for control, Syt I, Syt I-C2A* and Syt I-C2B* were very similar with sharp peaks at t = 0 sec (see Table 2 for statistics). The oscillatory behavior of the correlograms beyond | t | > ∼7.5 sec might reflect the bursting behavior of the distant cells. Bin widths for all correlograms were 1 sec. B. Pairwise C.I. values as a function of intercellular distance for control, Syt I, Syt I-C2A* and Syt I-C2B*. Data points were averages of medians within the 50 µm bins from 17–23 transfected retinas (about 50% data in each group from the retinas transfected on P0 with DIV 3–4). The error bars were the standard errors of the C.I. values in each bin.
Table 2.
Comparison of wave correlograms for Syt I and its C2AB mutants.