(A) Experimental design. When the mice learned the visually-guided eye movement task, we performed two photon calcium imaging to investigate directional preference of the MOs neurons in one hemisphere (‘Before’ condition). Then optogenetic suppression was applied to the MOs in the contralateral hemisphere. While the mice were trained for several days, they regained the ability to make contraversive eye movements. We again performed two-photon calcium imaging (‘After’ condition) to obtain the neuronal response for the same neurons imaged in the ‘Before’ condition. (B, C) Training-induced changes in direction selectivity of the MOs neurons in a representative imaging session. In the ‘Before’ condition (B), many neurons preferred the contraversive direction (magenta polygons), but in the ‘After’ condition (C), these neurons got sparser, and one neuron selective for ipsiversive direction (cyan polygon) showed up. White polygons indicate movement-related cells, and gray polygons non-significant cells. Black squares: example neurons shown in D, E. (D, E) Examples of two neurons (indicated in B, C) where direction selectivity changed between the ‘Before’ and ‘After’ conditions. Average fluorescence changes for trials with ipsiversive movements (cyan traces: Before, n = 30; After, n = 31 trials) and contraversive movements (magenta traces: Before, n = 35; After, n = 32 trials). (F) Training for several days induced difference in selectivity index for MOs neurons (six mice). White dots in the scatter plot (n = 225) are neurons showing significant movement-related activity, and black dots (n = 190) are neurons showing significant selectivity on top of the movement-related activity (total of 415 neurons are shown). The x-axis indicates selectivity index for the Before condition and the y-axis for the After condition. The histograms for the selectivity index are shown along the x-axis (Before) and the y-axis (After). Black bars are neurons showing significant direction preference either ‘before’ or ‘after’ conditions (n = 190 neurons). On top of black bars, magenta and cyan bars are overlaid for neurons showing preference for contraversive and ipsiversive directions (‘before’ condition: contraversive, magenta, n = 78 neurons, ipsiversive, cyan, n = 18 neurons, ‘after’ condition: contraversive, magenta, n = 40 neurons, ipsiversive, cyan, n = 69 neurons). Change in the selectivity index is shown in a histogram in the top right corner. White bars indicate the movement-related neurons (n = 225) and black bars the direction-selective neurons (n = 190).