In Vivo Measurement of Glycine Receptor Turnover and Synaptic Size Reveals Differences between Functional Classes of Motoneurons in Zebrafish  Dawnis M. Chow, Kathryn A. Zuchowski, Joseph R. Fetcho  Current Biology  Volume 27, Issue 8, Pages 1173-1183 (April 2017) DOI: 10.1016/j.cub.2017.03.032 Copyright © 2017 Terms and Conditions

Figure 1 The Expression of dendra2-GlyRα1 in Motoneurons, Physiology of dendra2-GlyRα1 Channels in Muscle Fibers, and Synapse Level Targeting for dendra2 Photoconversion (A) A single motoneuron expressing dendra2-GlyRα1 (green) along with membrane targeted tdTomato (magenta), exhibiting distributed glycinergic puncta along the labeled soma and dendrites. See also Figure S1 for costaining with gephyrin. (B) Expression of dendra2-GlyRα1 in muscle to test channel formation. Left: transgenic muscle fiber targeted for patch recording. Right: voltage-clamp recordings at the indicated holding potentials in a muscle fiber. (C) Precise targeting with a 405-nm laser allowed us to convert synaptically localized dendra2-GlyRα1 from green (top) to red fluorescence (bottom, represented as magenta) with near single-synapse accuracy. Current Biology 2017 27, 1173-1183DOI: (10.1016/j.cub.2017.03.032) Copyright © 2017 Terms and Conditions

Figure 2 Analysis of Photoconversion Recovery Time Courses (A) The normalized maximum projection time series of photoconverted synapses in five different PMNs and estimated recovery constant τ. (B) Data from (A) plotted along with estimated recovery curves. See also Figure S2. Current Biology 2017 27, 1173-1183DOI: (10.1016/j.cub.2017.03.032) Copyright © 2017 Terms and Conditions

Figure 3 GlyR Turnover Kinetics in 8-dpf PMNs (A) The natural log of the estimated recovery parameter τ of 77 synapses sampled from seventeen 8-dpf PMNs (each color represents photoconverted synapses from the same cell, with X marking the median for that cell) plotted against its normalized beginning intensity (relative intensity). (B) The same data from the top plot re-coded to illustrate synapses present on the dendritic arbors (red) or on the ventral dendrite/axon hillock region (black). Current Biology 2017 27, 1173-1183DOI: (10.1016/j.cub.2017.03.032) Copyright © 2017 Terms and Conditions

Figure 4 The Distribution of GlyR Turnover Kinetics Measured from Primary and SMNs (A) Images of a 9-dpf dendra2-GlyR α1-labeled PMN (left) and SMN (right) prior to photoconversion experiments. PMNs have larger somas than SMNs and have large specialized glycinergic synapses on the ventral dendrite/axon hillock region (see ventral cluster arrow). (B) The overlaid histograms of estimated τ (on log axis) from photoconversion experiments on 94 PMN synapses (gray) and 77 SMN synapses (green). PMN synapses were more likely to have long recovery times (larger values of ln τ) than SMN synapses (two-sample KS test, p < 0.001). See also Figure S3. Current Biology 2017 27, 1173-1183DOI: (10.1016/j.cub.2017.03.032) Copyright © 2017 Terms and Conditions

Figure 5 Synaptic Volumes and Synaptic Distances to Soma Measured in 8- to 9-dpf Motoneurons (A) Box-and-whisker plots of the median (red line), interquartile (box), and full range (whiskers) of data along with automatically determined outliers (+ symbols) for the distribution of mean synaptic volumes of all synapses per neuron (left) or the largest 10% (right) of 14 PMNs (1,239 synapses) and 11 SMNs (665 synapses) in 8- to 9-dpf animals. A two-sample t test confirmed a statistically significant difference in the overall distributions (medians 0.23 μm3 and 0.45 μm3, ∗p = 0.017) and the 10% tail (medians 0.60 μm3 and 1.45 μm3, ∗∗p = 0.004) between PMNs and SMNs. (B) The average synaptic volume (bars, left abscissa) and average number of synapses (lines, right abscissa) at increasing dendritic distances to soma plotted for the same set of PMNs and SMNs. (C) The average frequency histogram of synaptic volume for the same PMNs and SMNs after fourth root transformation to produce normality. The non-transformed average cumulative distribution for each cell type is shown in the inset. See also Figure S4. Current Biology 2017 27, 1173-1183DOI: (10.1016/j.cub.2017.03.032) Copyright © 2017 Terms and Conditions

Figure 6 Relationships between τ and Synaptic Volume or Synaptic Distance to Cell Soma (A) τ values for photoconverted PMN and SMN synapses plotted against their volumes. The particularly large synapses enclosed in the dashed box are specialized synapses on the ventral dendrite/axon hillock region of PMNs. (B) The τ of all photoconverted synapses for PMNs and SMNs plotted against their distances along the dendrite to the cell soma. Note that τ is plotted on a log axis for both panels. See also Figures S5 and S7. Current Biology 2017 27, 1173-1183DOI: (10.1016/j.cub.2017.03.032) Copyright © 2017 Terms and Conditions

Figure 7 Synaptic Age and Kinetics (A) Example PMN (left) and SMN (right) imaged at 5, 7, 8, and 9 dpf were examined for the formation of new synapses. Individual synapses are numbered to show examples of tracking over time. Note the appearance of new synapses 9 and 10 at 8 dpf on the PMN (left, blue inset) due to the growth of a new branch from a more ventral process and the loss of the branch containing synapses 5, 6, and 7 previously. White bars in bottom left of panels are 2 μm. (B) Synaptic volumes on 8-dpf (top) and 9-dpf (bottom) motoneurons organized by the timing of formation for each synapse (n = 8 PMNs and 7 SMNs total, see text for statistics; see also Figure S6). Error bars represent SEM. (Ci and Cii) Cumulative frequency histograms for τ of new, 1-day-old, and 2+-day-old synapses (Ci) and the τ (on log axis) of photoconverted synapses plotted against their volume (Cii). The blue line represents the fit of a LME model to 2+-dpf synapses. See also Figure S7. Current Biology 2017 27, 1173-1183DOI: (10.1016/j.cub.2017.03.032) Copyright © 2017 Terms and Conditions