For calcium dye loading, Oregon green Bapta-1 AM (OGB) was injected into the dLGN of C57/Bl6 mice (Figure 1A). To test for direction selectivity in the dLGN, we presented drifting square-wave gratings of 12 equally

spaced directions at a speed known to stimulate DSRGCs (Weng et al., 2005; Kim et al., 2008, 2010; Huberman et al., 2009; Yonehara et al., 2009) (i.e., 25 deg/s, 0.01 cycle per degree). Five repeats of each stimulus and a blank gray stimulus were presented in random order to the animal while visually evoked calcium responses were recorded in up to dozens of neurons simultaneously at a known depth in the dLGN, reflecting the underlying changes in firing rate of each neuron (Figures 1C–1E and 2). This method allows even rare neuron subtypes find more to be detected, and each neuron’s precise location to be mapped STI571 ic50 anatomically

within the dLGN. Many neurons responded robustly and reliably to at least one direction of the drifting grating, characterized by a time-locked increase in fluorescence to the period of the drifting grating (n = 353, ΔF/F amplitude at F1 or F2 > 2.5% and circular T2 test p < 0.05; see Figure S1 available online). We used the modulation of the fluorescence signal at the temporal frequency of the grating (0.25 Hz, F1) or at twice the temporal frequency of the grating (0.5 Hz, F2) as the measure of neuronal responsiveness. The F1 modulation corresponds to either the onset (On) or offset (Off) of each bar of light passing through a cell's receptive field, while the F2 modulation corresponds to both the onset and offset (On-Off) of each bar of light. Importantly, since the OGB signal attenuates higher frequencies, a large, detected F2 modulation represents an even stronger than recorded modulation, increasing confidence in On-Off designations. Likewise, an apparently low F2 modulation leaves characterization

of On-Off ambiguous or not possible. We computed the direction-selectivity index (DSI) and axis-selectivity index (ASI) of each responsive neuron in our sample. Neurons next with high DSI values (DSI > 0.5) responded preferentially to a single direction of the grating. Neurons with high ASI values (ASI > 0.5) responded preferentially to gratings drifting along a single axis of motion, responding selectively to gratings drifting in either opposing direction along a motion axis at a single orientation. The majority of neurons were not selective for motion in a particular direction or along a particular axis (n = 320/353, Figure 2B, DSI < 0.5 and ASI < 0.5). These responses are consistent with the circular direction tuning curves typical of dLGN neurons (Hubel and Wiesel, 1961). These findings demonstrate that the superficial dLGN is far from a purely DS layer. Conversely, 18 of the visually responsive cells in the data set were strongly and consistently direction selective (example cells Figures 2C, 2D, and 3A, DSI > 0.5, Hotelling T2 test, p < 0.05).