The light illuminates the slope of the hill, which defines the gradient of the shift in neuronal activity and the resulting shift in saccade endpoint. Behavioral changes give us some idea of the spatial spread of the optogenetic influence, but saccades are defined by the activity of a population of neurons that can span millimeters in the SC (1.4 mm estimated for SC burst cells; Munoz and Wurtz, 1995). Also, from Figure 2E, the laser illumination from the optrode could be affecting neurons over a millimeter away. A better estimate

of the extent of sensitized neurons can be derived from the suppression of neuronal responses, Bcl 2 inhibitor the detection of which is limited to few hundred microns from the optrode. We pursued this in two monkeys, OZ and RO, making an effort to sample SC sites beyond the presumed center of the injection. We used the established map of saccade directions and amplitudes across the intermediate layers of the SC to determine each optrode location, and characterized the degree to which we could affect neuronal responses with light at that location. We

calculated the maximum reduction in response at each optrode site and produced a map by interpolating PF-02341066 molecular weight between the irregularly spaced data. Figure 4 shows the maps for the two monkeys. The area of the injection in OZ (Figure 4A) was clearly delineated at the lateral edge of the interpolated region where response differences diminished (whiter areas). We estimated the spread of the effect by fitting a two-dimensional Gaussian curve to the responses. The Gaussian was free to vary its center, orientation, height, and extent. We show the extent of the Gaussian (±1 SD along each axis) as the dashed ellipse Fossariinae in Figure 4A. The region measured approximately 2,700 μm long by 2,100 μm across. For monkey RO (Figure 4B) the red ellipse indicates the actual extent of transfected neurons obtained from the histological

evaluation described below. Figure 4C shows how we constructed a three-dimensional representation of the injection in monkey RO from histological sections. Our histological methods are explained in detail in Supplemental Experimental Procedures. For each of 11 coronal sections separated by 250 μm, we obtained the total number of neurons by counting the cells expressing the NeuN stain. We then counted which neurons expressed GFP, indicating transfection with the ArchT construct (see Figure S3). The marginal histograms in Figure 4C are simply the running means of the proportion of transfected cells in each section. Over the 11 sections, we used the extent of each running mean to establish the anterior-posterior and medial-lateral spread of the transfection. The central wireframe structure schematizes the three-dimensional reconstruction of the spread of the virus in monkey RO.