Visual map development depends on the temporal pattern of binocular activity in mice

Abstract

Binocular competition is thought to drive eye-specific segregation in the developing visual system, potentially through Hebbian synaptic learning rules that are sensitive to correlations in afferent activity. Altering retinal activity can disrupt eye-specific segregation, but little is known about the temporal features of binocular activity that modulate visual map development. We used optogenetic techniques to directly manipulate retinal activity in vivo and identified a critical period before eye opening in mice when specific binocular features of retinal activity drive visual map development. Synchronous activation of both eyes disrupted segregation, whereas asynchronous stimulation enhanced segregation. The optogenetic stimulus applied was spatially homogenous; accordingly, retinotopy of ipsilateral projections was markedly perturbed, but contralateral retinotopy was unaffected or even improved. These results provide direct evidence that the synchrony and precise temporal pattern of binocular retinal activity during a critical period in development regulates eye-specific segregation and retinotopy in the developing visual system.

Figure 1. ChR2-expressing retinal ganglion cells (RGCs) are activated with high temporal precision with blue light both in vitro and in vivo

(A) Left panel: ChR2-expressing RGCs in Thy1-ChR2 transgenic mice are distributed across the entire retina; Right panel: Brn3b positive RGCs in red, ChR2-expressing cells in green. White arrows show examples of RGCs expressing both brn3b and ChR2. (B) Whole cell recording of a ChR2 RGC in response to 300 ms light stimuli at intensities of 0.65, 0.325 and 0.1625 mW/mm² (from top to bottom; mercury lamp filtered). Blue bars represent the presence of light stimuli; thickness represents the relative light intensity.(D) A few example channels from a multielectrode array (MEA) recording of whole mount retina in response to light stimuli (blue bars) of 1 s at 0.51 mW/mm² (left panel) and 5 ms at 3.18 mW/mm² (right panel). (E) –In vivo extracellular multiunit activity in superior colliculus neurons in response to 50 ms, 200 ms, 500 ms and 1s (top to bottom) light stimuli to the contralateral eye under 1% isoflurane anaesthesia. The light intensity is 0.64 mW/mm² immediately outside the cornea. (F) Raster plot of in vivo responses to 1s light stimuli. Error bars represent s.e.m. Animals were between P9-P11.

Figure 2. In vivo Ca²⁺ imaging demonstrates that a majority of superior colliculus neurons respond to optogentic stimuli before eye opening

(A) Experimental setup for in vivo calcium imaging. (B) Left panel is a bright field image of the craniotomy. White dashed line shows outlines for the right and left hemisphere of the superior colliculus (R-SC and L-SC, respectively). Right panel is the baseline OGB1AM fluorescent image. Each green square in the image is one region-of-interest (ROI). (C) Time lapse images of OGB1AM fluorescent signals in the superior colliculus due to synchronous LED stimulation of both eyes lasting 1 second starting at the second frame (200 ms interval between frames). Orientation of each frame is the same as in panel B. Color bar represents the normalized fluorescence change (ΔF/F from minimum to maximum). (D) Real-time calcium signals (raw traces) for individual ROIs (indicated by numbers) show that synchronous optogenetic stimulation of both eyes drives synchronous neuronal response in the superior colliculus of both hemispheres. One second long light stimuli occurred at the times indicated by blue shading. (E) Real-time raster plots for responses from all ROIs and the fraction of active ROIs to four consecutive light stimuli that occurred at the time of blue shading. (F) Normalized response frequency for each ROI in one hemisphere of the superior colliculus to stimulation of the ipsilateral (ipsi), contralateral (contra) or both eyes (synch) (n = 3 experiments for ipsi and contra, n = 5 experiments for synch). (G) Cumulative distribution of normalized response frequency for ipsi, contra and synch in (F). There is little response to stimulation of the ipsilateral eye itself, but strong response to stimulation of the contralateral eye or synchronous stimulation of both eyes.

Figure 3. Synchronous but not asynchronous stimulation of both eyes disrupts eye-specific segregation in the superior colliculus

(A) Both eyes of Thy1-ChR2 mice were stimulated on a 12h stimulation - 12h feeding cycle for two-three days starting at P9. Panels show parasagittal sections through the superior colliculus. Ipsilateral axons (red arrow) normally terminate in clusters in the rostral superior colliculus just inferior to the contralateral layer (SGS, dotted line). Synchronous stimulation (middle column) caused axons from the ipsilateral eye (grey scale signal) to form abnormal clusters in the contralateral (SGS) layer; asynchronous stimulation (right column) didn’t affect eye segregation in comparison to unstimulated controls (left column). (B) Quantification of stimulation and control experiments by measuring the fraction of the contralateral (SGS) layer which is occupied by ipsilateral pixels. P9 (n = 3) and P14 (n = 4) results from unmanipulated Thy1-ChR2 mice; Ctrl (n = 5) is Thy1-ChR2 mice which were manipulated daily the same as experimental mice, but were not optically stimulated; Async (n = 8) and Sync (n = 6) were asynchronously and synchronously stimulated Thy1-ChR2 mice; One eye Stim (n = 2) were Thy1-ChR2 mice with the ipsilateral eye only stimulated; WT Sync: synchronously stimulated wild-type mice lacking ChR2 (n = 5); P14 Sync (n = 3): synchronously stimulated Thy1-ChR2 mice starting at P14. (C) Results from experiments in which stimuli with the same frequency (0.2 Hz or 5 sec between stimuli) but varying durations (1 s, n = 4; 2 s, n = 3; 2.5 s, n = 5) show that eye segregation got worse as the duration of synchronous activity increased. * p < 0.05, ** p <0.005. Error bars represent s.e.m.. R: rostral; D: Dorsal. L: Left eye; R: Right eye.

Figure 4. As much as 100 msec asynchronous binocular stimulation disturbs eye segregation

(A) Bursts of 5 msec light pulses were used to stimulate both eyes in Thy1-ChR2 mice with a timing difference of 0 s (n = 4), 20 ms (n = 3), 50 ms (n = 4), 100 ms (n = 4) and 200 ms (n = 5) between the two eyes. (B) The delay of RGC spiking responses to the onset of the 5 ms light stimuli with a range of different light intensities in vitro is less than 10 ms, and not very variable, particularly at higher light intensities. Error bars represent S.D.. (C) Eye segregation in the superior colliculus was not disturbed with asynchronous stimuli with a 200 ms difference between the eyes, but got worse as the temporal difference between the two eyes decreased. (D) The total number of ipsilateral clusters in the contralateral SGS layer per animal increased as the optogenetic stimulation became increasingly synchronous. n.s., not significant (p > 0.05); * p < 0.05, ** p < 0.005. Error bars represent s.e.m..

Figure 5. Synchronous stimulation disrupted eye segregation and asynchronous stimulation improved eye segregation during development in AAV-ChR2 treated mice

(A) Injection of AAV-DIO-ChR2-mCherry virus into retinas of P0-P1 Rx-Cre mice induced the expression of ChR2 in RGCs 5 days later. Viral exposure induced ChR2 expression in neurons that express CRE. Example shows ChR2 expression in RGCs in the ventral-temporal retina (binocular zone) of Rx-CRE mice 5 days after intravitreous injection of the AAV-DIO-ChR2-mCherry virus. (B) Example Multielectrode Array (MEA) recording from a whole mount retina in response to 1s light stimuli of 2.55 mW/mm² in these mice. Blue bars represent the light stimuli. (C) Synchronous stimulation of both eyes (Sync, n = 4) when eye-specific segregation is just emerging (P5-P6) disturbed segregation in the superior colliculus, whereas asynchronous stimulation (Async, n = 6) improved segregation in comparison to control animals (Ctrl, n = 4). Gray boxes in the upper images indicated the area in the high–magnification images below. * p < 0.05, *** p < 0.001. Error bars represent s.e.m..

Figure 6. Synchronous stimulation disrupted and asynchronous stimulation improved eye-specific segregation in ChR2;β2^−/− mice

(A) Eye segregation for synchronously stimulated ChR2;β2^−/− mice (Sync, n = 5) is worse than in β2^−/− controls (Ctrl, n = 4). Asynchronous stimulation in ChR2;β2^−/− (Async, n = 3) substantially improved eye segregation in comparison to β2^−/− controls. (B) As expected, synchronous (Sync, n = 6) and asynchronous (Async, n = 4) light stimulation had a similar effect in ChR2;β2^+/− mice as in Thy1-ChR2 mice (Figure 3B) (ChR2;β2^+/− control, n = 3). n.s., not significant (p > 0.05), ** p < 0.005, *** p < 0.001. Error bars represent s.e.m..

Figure 7. Synchronous stimulation disrupts eye segregation in the dLGN

Synchronous stimulation caused an increase in the overlap (white – bottom row) between ipsilateral (red) and contralateral (green) axons in both (A) Thy1-ChR2 and (B) ChR2;β2^−/− mice. Asynchronous stimulation didn’t affect eye segregation. n = 5 for Thy1-ChR2 Ctrl, n = 3 for Thy1-ChR2 Syn, n = 4 for Thy1-ChR2 Async; n = 4 for ChR2;β2^−/− Ctrl, n = 3 for ChR2;β2^−/− Syn, n = 4 for ChR2;β2^−/− Async. * p < 0.05, ** p < 0.005. Error bars represent s.e.m..

Figure 8. Chronic stimulation of ChR2-expressing RGCs disrupts retinotopy of ipsilateral RGCs, but improves retinotopy of contralateral RGCs

(A) Distribution of ipsilateral axon arbor clusters along the rostral-caudal axis of the superior colliculus in Thy1-ChR2 experiments. The ipsilateral domains of both control (Ctrl, n = 28 sections) and asynchronously stimulated animals (Async, same as Figure 3, n = 28 sections) are located only in the rostral superior colliculus just inferior to the SGS layer (dark grey arrows and histogram). Ipsilateral clusters in the SGS caused by synchronous stimulation are distributed throughout the rostral-caudal axis of the superior colliculus (Sync, n = 52 sections, light grey arrows and histogram). Normal ipsilateral domains in the SO layer just inferior to the SGS are located similarly in the control, synchronous and asynchronously stimulated mice (Sync, n = 24 sections, dark grey arrows and histogram). Each “x” represents the relative position of one cluster, which is calculated by the distance to the caudal end of the SGS (a) divided by the total length from the caudal to the rostral end of the SGS (a+b). Light and dark grey arrows indicate the ipsilateral clusters in the SGS (light grey) and the SO layer just inferior to the SGS (dark grey) where ipsilateral axons normally terminate. (B, C) Whole mount superior colliculus images (dorsal view; outlined by white dotted line) of control (Ctrl) and whole-eye stimulated (Stim) Thy1-ChR2 mice with focal injections of DiI into the dorsal or ventral-temporal (VT) retinas. (D) Chronic optogenetic stimulation resulted in smaller target zones in the superior colliculus for dorsal projections (Dorsal, n = 22, p < 0.05) and a similar trend for ventral-temporal projections (VT; n = 17) in comparison to Ctrl mice (Dorsal Ctrl, n = 8; VT Ctrl, n = 14) in Thy1-ChR2 animals. (E, F) Whole mount superior colliculus images of control (Ctrl) and whole-eye stimulated (Stim) ChR2;β2^−/− mice with focal injections of DiI into the dorsal or ventral-temporal retinas. (G) Similar to Thy1-ChR2 mice, ChR2;β2^−/− mice also had smaller target zones in the superior colliculus for both dorsal (Dorsal, n = 11, p < 0.05) and ventral-temporal (VT, n = 12, p < 0.001) projections after chronic optical stimulation (Dorsal Ctrl n = 5, VT Ctrl n = 5). n.s., not significant (p > 0.05); * p < 0.05, *** p < 0.001. Error bars represent s.e.m.. C is caudal, L is lateral.