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. 2019 Apr 11;15:28–38. doi: 10.1016/j.isci.2019.04.013

Figure 5.

Figure 5

GRAPHIC Visualizes Synaptic Connection in Neuronal Networks

(A and B) GRAPHIC labels synaptic sites in zebrafish retina. (A) In the zebrafish retina, typical structures, ribbon synapses, are formed between cone photoreceptor axon terminals, namely, pedicles and dendrites of bipolar cells. n-GRAPHIC and membrane-targeted tdTomato were co-expressed in a subpopulation of bipolar cells using the vsx1 promoter (vsx1: memtdTomato-2A-n-GRAPHIC), and c-GRAPHIC and TagBFP2 were co-expressed in cone cells using the guanine nucleotide-binding protein G(t) subunit alpha-2 (gnat2) gene promoter (gnat2: TagBFP2-2A-c-GRAPHIC). Both expression vectors were co-injected into one-cell-stage zebrafish embryos and then fixed at 5 days postfertilization. (B) Reconstituted GFP signals were only detected at the dendritic tips of tdTomato-positive bipolar cell closely associated with TagBFP2-expressing cone pedicle. Scale bar, 5 μm.

(C–E) GRAPHIC signals in S1 layer IV neurons are merged with thalamocortical axons of VB neurons in mouse brain. (C) In utero electroporation (IUE) was performed at E13.5–14.5 to express n-GRAPHIC-2A-H2B-mCherry in cortical layer IV. After the electroporated mice had grown up to P14-21, c-GRAPHIC-2A-mCherry encoding AAV was stereotaxically injected into VB. After 30–40 days, the injected brains were sectioned and observed. (D) Certain number of cortical layer IV neurons (bracket) expressed n-GRAPHIC (indicated by co-expressed red nuclei label, dot line) and many mCherry labeled fibers of c-GRAPHIC expressing VB neurons reached to cortical layer IV (white arrows). (E) Higher magnification of confocal images of layer IV showed many GFP puncta largely overlapped with mCherry-positive bouton-like structures. GFP puncta that co-localize with mCherry-positive axons are indicated by arrowheads. Scale bars, 200 μm in (D) and 5 μm in (E).

(F) IUE was performed at E13.5 to express n-GRAPHIC-2A-PSD95-mCherry in cortical layer IV. After the electroporated mice had grown to adult (about 2-month-old), c-GRAPHIC-2A-H2B-mCherry encoding AAV was stereotaxically injected into thalamus VB. After about 4 weeks, the injected brains were sectioned and observed.

(G) Distribution of GRAPHIC signals in cortical layer IV neurons. GFP (GRAPHIC) signals indicate contacted sites between n-GRAPHIC-expressing layer IV neurons and c-GRAPHIC-expressing VB neurons. PSD95-mCherry signals indicate postsynaptic sites of electroporated layer IV neurons. GFP puncta that co-localize with PSD95-mCherry puncta are indicated by arrowheads. Scale bar, 5 μm.