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. 2015 Aug 14;4:e09590. doi: 10.7554/eLife.09590

Figure 4. Glutamate released during neuronal waves reaches MC membrane at P9 but not at P11.

(A) Left, Diagram of a retinal cross-section illustrates neuronal expression of iGluSnFR (green) in the IPL and simultaneous voltage-clamp recording of a RGC (grey). XY plane of the IPL shows iGluSnFR expression in neuronal membranes at P11. Right, Simultaneous imaging of AAV9-2YF-hSynapsin-iGluSnFR signals in neuronal membranes (green traces) and whole-cell voltage-clamp recording of a RGC (grey trace, Vm = −60 mV) monitored in the same field of view at P11 in control and in the presence of 25 μM DL-TBOA. Above the whole-cell voltage-clamp trace are histograms showing the percentage of ROIs within a neuronal iGluSnFR signal. (B) Left, Diagram of a retinal cross-section illustrates glial expression of iGluSnFR (green) and simultaneous voltage-clamp recording of a RGC (grey). XY plane of the IPL shows iGluSnFR expression in MCs. Right, Simultaneous imaging of ShH10-CMV-iGluSnFR signals in MCs (green traces) and whole-cell voltage-clamp recording of a RGC (grey trace, Vm = −60 mV) monitored in the same field of view at P9 and P11 in control and in the presence of 25 μM DL-TBOA. Above each whole-cell voltage-clamp trace are histograms showing the percentage of ROIs with glial iGluSnFR signals in response to retinal waves. (C) Plot summarizes DL-TBOA effects on the participation of neuronal (160 ROIs from 5 retinas at P9 and 160 ROIs from 5 retinas at P11) and MC (1023 ROIs from 5 retinas at P9 and 1201 ROIs from 5 retinas at P11) ROIs per retinal wave. Lines connect values from one experiment in control vs DL-TBOA. Black circle and error bars are mean ±SD. t-test ***p < 0.001. (D) Cumulative probability distribution of inter-transient intervals of iGluSnFR (green traces) and GCaMP3 (black traces) signals in MC ROIs at P9 and P11. Control in solid lines and DL-TBOA in dashed lines. See also Figure 4—figure supplement 1 and Videos 4, 5.

DOI: http://dx.doi.org/10.7554/eLife.09590.010

Figure 4—source data 1. Cumulative probability distributions of inter-transient intervals of iGluSnFR and GCaMP3 signals in MC ROIs for each experiment at P9 and P11, and in absence or in presence of DL-TBOA.
DOI: http://dx.doi.org/10.7554/eLife.09590.011
elife09590s001.xlsx (132.4KB, xlsx)
DOI: 10.7554/eLife.09590.011

Figure 4.

Figure 4—figure supplement 1. Methodological tools to study neuron-glia interaction mediated by glutamate spillover in whole mount retinas.

Figure 4—figure supplement 1.

(A) Orthogonal projection of two-photon Z-stacks shows expression of the intensity-based glutamate sensor iGluSnFR in ganglion and amacrine cell membranes of a P9 wild-type mouse retina. AAV9-2YF-hSynapsin1-iGluSnFR was tail vein injected at P2. Yellow solid and dashed lines indicate the XY planes chosen to illustrate iGluSnFR expression in neuronal membranes in the IPL and GCL, respectively (middle panel). Magenta overlay indicates an iGluSnFR-expressing ON-OFF ganglion cell targeted for whole-cell recording and filled with a red fluorescent dye (Alexa 594). Right panel: Z-projection and X-Z projection of this ON-OFF RGC (magenta) and the neuronal dendritic network expressing iGluSnFR (green). (B) Orthogonal projection of two-photon Z-stacks shows iGluSnFR expression in MCs of a P9 wild-type mouse retina. ShH10-CMV-iGluSnFR was intravitreally injected at P4. Yellow solid and dashed lines indicate the XY planes chosen to illustrate iGluSnFR expression in MC membranes in the IPL and GCL, respectively (right panel).
DOI: http://dx.doi.org/10.7554/eLife.09590.012