Retinal waves coordinate patterned activity throughout the developing visual system

Abstract

The morphological and functional development of the vertebrate nervous system is initially governed by genetic factors and subsequently refined by neuronal activity. However, fundamental features of the nervous system emerge before sensory experience is possible. Thus, activity-dependent development occurring before the onset of experience must be driven by spontaneous activity, but the origin and nature of activity in vivo remains largely untested. Here we use optical methods to show in live neonatal mice that waves of spontaneous retinal activity are present and propagate throughout the entire visual system before eye opening. This patterned activity encompassed the visual field, relied on cholinergic neurotransmission, preferentially initiated in the binocular retina and exhibited spatiotemporal correlations between the two hemispheres. Retinal waves were the primary source of activity in the midbrain and primary visual cortex, but only modulated ongoing activity in secondary visual areas. Thus, spontaneous retinal activity is transmitted through the entire visual system and carries patterned information capable of guiding the activity-dependent development of complex intra- and inter-hemispheric circuits before the onset of vision.

Figure 1. Spontaneous waves of activity in retinal ganglion cell arbors in vivo

a, Calcium dye labeling and 2P imaging. b, Superior colliculus (SC) craniotomy from a P5 mouse overlaid with the corresponding 2P excitation image of CaGr-Dx labeled RGC axon arbors. c, Higher magnification image of CaGr-Dx labeled RGC axons from inset in b, 82 μm below pial surface. d, Montage (dF/F) of a spontaneous wave recorded in RGC axon arbors. From same labeled field shown in b. e, Calcium transients from RGC axon arbors during the wave shown in d. f, Raster plot and activity histogram from a 677 s recording. Points indicate calcium transient onsets from individual ROIs. Same wave from d shown on right at expanded time scale. g, Dye labeling and wide field CCD imaging for bilateral recordings. h, Raster plots from animal shown in g. Intraocular TTX application blocks waves in the contralateral hemisphere.

Figure 2. Spontaneous waves of correlated activity among SC neurons in vivo

a, Experimental overview. b, OGB1-AM bulk loading in the SC at P4. c, 2P excitation image of OGB1-AM labeled cells from inset in b, 56 μm below pial surface. d, Montage (dF/F) of a spontaneous wave recorded in the SC. From same labeled field shown in c. e, Calcium transients in local ROIs during the wave shown in d. f, Raster plot and activity histogram during a 610 s recording. From same experiment as d, e. g, Calcium imaging in single SC cells loaded with OGB1-AM. h, Single cell ROIs and calcium transients during a wave. i, Plot of neurons active during 2 sequential waves. j, Pairwise cell correlations as a function of pair distance. Dashed line indicates the chance distribution. k, Bilateral dye labeling and example widefield CCD image. l, Raster plots from a bilateral SC recording. Intraocular TTX application blocks waves in the contralateral hemisphere.

Figure 3. Retinal waves originate in the ventro-temporal retina and propagate bilaterally

a, Contour map of normalized ROI response frequencies in the SC within 2 s of wave onset. b, Wind rose histogram of all wave directions in SC. c, Montage (dF/F) of synchronized retinal waves recorded in OGB1-AM labeled cells in both hemispheres of the SC. d, Peri-event time histograms of calcium event latencies relative to wave onset for same or opposite hemispheres. ROI event latencies: Same hemi, N = 103354; Opposite hemi, N = 97280. e, Fraction of presynaptic and postsynaptic waves having significant temporal (CaGr-Dx: 52/332 waves; OGB1-AM: 20/90 waves) and spatial (CaGr-Dx: 46/332 waves; OGB1-AM: 16/90 waves) correlations bilaterally. Error bars denote s.e.m. f, Cumulative distributions of the spatial similarity metric across hemispheres for observed wave pairs and random wave pairs.

Figure 4. Retinal waves propagate simultaneously in the SC and visual cortex

a, Experimental overview. b, Right visual cortex (R-V1) and SC (R-SC) craniotomies from a P9 mouse. c, OGB1-AM bulk loading in V1 and SC. d, Montage (dF/F) of spontaneous waves recorded in SC and V1 simultaneously. e, Raster plot from the recording in d. f, Boxplots show interval between successive wave onsets in SC and V1 between P6-P9. g, Activity maps showing the direction of wave propagation for 3 waves in V1 and SC indicated in e. Top left panel shows the approximate retinotopy of mouse primary visual cortex (V1) and SC. t, temporal, n, nasal, d, dorsal, v, ventral. h, Plot of SC retinal wave directions versus V1 wave directions normalized to SC coordinates, with linear regression (blue line) and 95% confidence intervals (grey shading).

Figure 5. Retinal wave driven activity in V1 and extrastriate visual areas

a, Experimental overview. b, Image of transcranial cortical GCaMP3 expression and OGB1-AM bulk loading in SC of a P6 Emx1-Cre:Ai38 mouse. c, Montage (dF/F) of a retinal wave recorded in SC and V1 simultaneously from same recording as in b. Arrows indicate direction and distance of wave travel, arrowheads indicate activations in V2. d, Topographic maps colorized by SC retinal wave front position reveal putative boundaries of V1 and secondary visual areas (Areas 18a and 18b) from same animal as in c. e, Cumulative distributions of calcium event frequencies before (control) and after ablation of contralateral (enuc-contra) or ipsilateral (enuc-ipsi) eye. SC, N = 2862 ROIs; V1, N = 3490 ROIs; extrastriate, 7717 ROIs. f, Peri-event time histograms of calcium event latencies relative to SC wave onset. SC, N = 52,284 events; V1, N = 38,244 events; extrastriate, N = 49,363 events. g, Max dF/F intensity projections from 10 min recordings before and after contralateral eye enucleation. From same recording as in d.

Figure 6. Retinal waves depend on cholinergic neurotransmission

a, Experimental overview. b, Image of transcranial cortical GCaMP3 expression and RGC axon GCaMP3 expression in SC in a P4 Rx-Cre:Ai38 mouse. c, Topographic maps colorized by SC retinal wave front position for a single retinal wave simultaneously propagating in SC and V1. d, Montage (dF/F) of a single retinal wave from same recording as in c. e, Montage (dF/F) showing typical activity patterns in visual cortex after contralateral injection of 1 mM epibatidine, from same experiment as d. f, Box plots showing retinal wave frequency in 10 min recordings before (control) and after epibatidine injection into contralateral (contra epi; p = 2.2e-12 vs control, pairwise-t-test) or ipsilateral (ipsi epi; , p = 7.3e-08 vs control) eye in 6 mice between P2-P5.