The Journal of Neuroscience, January 12, 2005, ():
Regulation of Gephyrin Cluster Size and Inhibitory Synaptic Currents on Renshaw Cells by Motor Axon Excitatory Inputs
J. Neurosci. Gonzalez-Forero et al.
25: 417
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1: Quantitative analysis of gephyrin-IR clusters in confocal images.
A, High magnification confocal optical section close to the top surface of the control Renshaw cell shown in Figure 2D. Gephyrin immunofluorescence is shown in A1 and calbindin immunofluorescence in C2. Calbindin immunoreactivity allowed unequivocal identification of cellular boundaries. To define the area containing Renshaw cell gephyrin-IR clusters for measurement, the Renshaw cell profile was traced at 2-mm intervals in optical section stacks (i.e. every fourth section; green outline in A2). This area of interest (AOI) was then applied to optical section images displaying gephyrin immunoreactivity (A1). In this section (2 mm from the top of the cell) many clusters are already on the edge of the cell. Only clusters on the cell bodies and viewed en face were measured. B: Optical section from the top surface of the cell. Several en face clusters appear in focus in the center of the cell (B1). Thresholding the gephyrin cluster brightness to 33% of the brightest immunofluorescence selects the clusters from the background and traces the clusters just outside the out-of-focus halo (B2,B3). Red indicates pixels above the brightness threshold. Comparison with the optical section 0.5 mm below (shown in C) and above (not shown) indicate which clusters are in better focus in adjacent optical sections. Only en face clusters in best focus in the optical section are measured (red outlines with numbers). This method ensures that each cluster is only counted once at the optical section in which it displays the maximal resolution of its structure. C: The same process is repeated in the next optical section and clusters already analyzed and brighter in B removed (C2,C3). Scale bars = 5 mm in A2, B3 and C3.
- supplemental material
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Supplementary Figure 2. Alterations in calbindin-immunoreactivity after toxin injections.
A, Calbindin-immunoreactivity in the ventral horns of a spinal cord section from a TeNT animal. The side ipsilateral to the toxin injection is labeled TeNT, the contralateral side is labeled control. Renshaw cells are brightly immunofluorescent in both sides of the spinal cord. Ipsilateral to the injection some neurons located dorsal to Renshaw cells and with larger cell bodies express some weak calbindin. These neurons were rare in the control side. Somatic Renshaw cell calbindin-immunofluorescence is similar in both sides, although the calbindin-IR fiber plexus around motoneuron pools appears slightly brighter in the side injected with the toxin. Similar results were obtained at P20 and P15. B, In contrast, calbindin immunoreactivity was decreased in Renshaw cells in the side ipsilateral to the BoNT-A injections. The non-injected (control) side has several brightly immunofluorescent Renshaw cells near the ventral white matter border and a large plexus of calbindin-IR neurites around motor pools. Renshaw cells on the BoNT-injected side were less intensely labeled. Decreased calbindin-immunoreactivity was detected in 4 of 6 P20 animals and in 3 of 4 P15 animals. No differences in calbindin-immunolabeling were noted in dorsally located interneurons in the same sections. Calbindin immunoreactivity in Renshaw cells located several segments above the affected motor pools appeared not different from immunoreactivity in Renshaw cells in the control side, suggesting that the toxin did not induce significant contralateral changes in calbindin expression.
In conclusion, BoNT-A injections reduced calbindin expression in Renshaw cells suggesting reduced levels of activity. A clear effect was however not found after TeNT injections, perhaps suggesting that Renshaw cells already express calbindin-immunoreactivity close to maximal levels during normal development. Scale bars = 200 µm
