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Xiwu Zhao, Kwoon Wong; Whole-cell recordings of dark-adapted mouse ipRGCs. Invest. Ophthalmol. Vis. Sci. 2013;54(15):308.
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© ARVO (1962-2015); The Authors (2016-present)
Five types of mouse ipRGCs (M1 - M5) have been identified but their spontaneous and light-evoked electrical activities have not been studied properly because for all published whole-cell recordings, these cells were identified using epifluorescence and were thus severely light-adapted. Here, we overcame this issue by using invisible two-photon laser to identify ipRGCs.
Under infrared illumination, retinas were isolated from dark-adapted mice whose ipRGCs were selectively labeled with GFP (Ecker et al. 2010 Neuron). GFP+ cells were visualized using 915-nm laser and targeted for whole-cell recording. All stimuli were encoded using MATLAB and were white light generated by an OLED monitor. In the receptive field mapping experiment, the stimuli were light spots of various diameters centered within each cell’s receptive field. In all other experiments, the stimuli were full-field step increases in light intensity.
In darkness, the resting potentials of M1 - M5 cells were -59, -55, -62, -52 and -57 mV, respectively, whereas their spontaneous spike rates were about 5, 30, 20, 90 and 70 Hz. All cell types generated fairly sustained excitatory responses to 10-s light steps, with a threshold intensity of ~7 log quanta cm-2 s-1. At all suprathreshold light intensities, all cells’ responses peaked within 1 s after light onset and then gradually decayed at similar rates. At all intensities, M1 and M3 generated the largest light-evoked depolarizations whereas those of M4 and M5 were the smallest. In the mapping experiment, all non-M1 cells had ON-center/OFF-surround receptive fields, whereas none of the M1 cells showed any evidence for an inhibitory surround. Receptive field center diameters ranged from ~200 µm for M3 and M5, to ~400 µm for M1 and M4. For all M2 - M5 cells, surround diameters were between ~2 and ~3 mm. In the presence of synaptic blockers, all cells generated sluggish melanopsin-based light responses, and the threshold intensities for these responses varied from ~10.5 log quanta cm-2 s-1 for M1 to ~13 log quanta cm-2 s-1 for M3.
Although all ipRGCs receive rod input and respond to full-field white light with similar kinetics, the five ipRGC types are different for most of the other parameters examined, suggesting functional diversity. Most of our measurements differed drastically from those reported previously, illustrating the advantage of using two-photon laser to identify GFP-labeled neurons.
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