Fig. 1: Characterization of synapse dynamics and ECM in the developing brain.
From: Extracellular matrix proteolysis maintains synapse plasticity during brain development
a, Schematic of the perisynaptic ECM which consists of a hyaluronan sugar matrix connected by proteoglycans including brevican, linking proteins such as tenascins and others. b, Representative images of zebrafish hindbrain at 14 and 60 dpf showing brevican (antibody staining) and hyaluronan labeled with a genetically encoded sensor, ubi:ssncan-GFP. c, Quantification of fluorescence intensity of brevican and hyaluronan (ubi:ssncan-GFP) over development from 7 to 90 dpf. Mean fluorescence intensities for brevican and hyaluronan were normalized to the intensity at 90 dpf. Brevican quantification, number of fish: 7 dpf, n = 9; 14 dpf, n = 8; 21 dpf, n = 8; 28 dpf, n = 7; 60 dpf, n = 7; 90 dpf, n = 6. For hyaluronan, number of fish: 7 dpf, n = 11; 14 dpf, n = 10; 21 dpf, n = 6; 28 dpf, n = 10; 60 dpf, n = 7; 90 dpf, n = 3. d, Schematic of zebrafish larval hindbrain. Gray dots indicate cell bodies and the synaptic region is shown in pink. A cholinergic neuron with a cell body and dendrites is shown in black. e, Dorsal view of zebrafish hindbrain at 10 dpf shows sparsely labeled cholinergic neurons expressing a TdT-tagged FingR construct that binds to the excitatory post-synaptic marker PSD95. Tg(chata:gal4);Tg(zcUAS:PSD95.FingR-TdT-CCR5TC-KRAB(A)), hereafter abbreviated Chat-PSD95FingR. f, Strategy for quantification of synapses using Chat-PSD95FingR. Insets of region in e show (i) several sparsely labeled neurons and (ii) a dendritic segment from one cholinergic neuron with synapses indicated by asterisks. g, Immunostaining for brevican protein and Chat-PSD95FingR at 14 dpf. Synaptic region is indicated in the image. h, Schematic of 24-h time-lapse imaging assay to quantify changes of synapse density. i, Representative images show a single Chat-PSD95FingR dendrite imaged at 7 dpf (t = 0) and 8 dpf (t = 24). Pink arrowheads: synapses present at t = 0 and absent at t = 24, ‘lost synapses’. Green arrowheads: synapses that appear at t = 24, ‘new synapses’. White dashed circles: present at both time points. j, Quantification of total excitatory synapse density and dynamics over the live imaging window of hindbrain development (5–14 dpf), based on Chat-PSD95FingR−TdT puncta normalized to µm of dendrite length. Black line indicates static synapse density per day (P = 0.3437, one-way ANOVA). Lilac bars indicate lost synapses (**F(4,78) P = 0.0040, one-way ANOVA). Green bars indicate new synapses (*F(4,78) P = 0.018, one-way ANOVA). Asterisks in the figure represent results of Tukey’s multiple comparisons with respect to 5–6 dpf. Number of fish: 5–6 dpf, n = 15; 7–8 dpf, n = 18; 9–10 dpf, n = 18; 11–12 dpf, n = 15; 13–14 dpf, n = 17. Results from individual fish are shown in Extended Data Fig. 2b. k, Schematic of time-lapse imaging to determine the fate of individual synapses at the indicated time points. Synapses at t = 0 were defined as ‘stable’ synapses. Synapses born between t = 0 and t = 6 were defined as ‘new’ synapses and subsequently followed with the 6-h time point set as t = 0 (lower time course). Experiments were performed at 10–12 dpf. l, Representative image of a single excitatory synapse imaged at t = 0, 6, 12 and 24 shows a ‘new synapse’ born between t = 0 and t = 6, which disappeared between t = 12 and t = 24. Left: a low-power image of a Chat-PSD95FingR cholinergic neuron. Dashed square indicates inset. Inset shows raw fluorescence (top) and fluorescence overlaid with 3D reconstruction of synapses (bottom). Arrowheads: newborn synapse. Dashed circle: site of newborn synapse. m, Kaplan–Meier plot of survival of individual synapses over time (data from n = 19 fish, n = 427 stable synapses and n = 25 new synapses, ****P < 0.0001, log-rank test). n, Quantification of the distance from the nearest synapse for stable and new synapses at t = 6 (stable, n = 116 inter-synapse intervals from n = 14 fish; new, n = 25 inter-synapse intervals from n = 14 fish; ****P < 0.0001, Welch’s t-test, performed for synapses). The inter-synapse intervals were normalized by the mean of stable synapses for each cell. Values were plotted as mean ± s.e.m. ****P < 0.0001; **P < 0.01; *P < 0.05. For representative images in e, f and g, similar results were observed from more than n = 3 independent experiments. Scale bars, 100 µm (b), 20 µm (e,f), 5 µm (g,i,l (low-power image)), 2 µm (l, inset). NS, not significant. Illustrations in a and d created using BioRender.com.
