Direct Observation of ON and OFF Pathways in the Drosophila Visual System  James A. Strother, Aljoscha Nern, Michael B. Reiser  Current Biology  Volume 24, Issue 9, Pages 976-983 (May 2014) DOI: 10.1016/j.cub.2014.03.017 Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 1 The Layered Structure of the Drosophila Medulla Is Revealed by Two-Photon Calcium Imaging (A) Medulla neurons expressing the fluorescent calcium indicator GCaMP5G were imaged in head-fixed flies using two-photon microscopy. Visual stimuli were presented using a high-speed projector system. (B) In this schematic of the fly visual system, representative columnar neuron types are drawn with the position of the cell body indicated by a disc and the main arborizations indicated by short bars (photoreceptors in red, lamina monopolar cells in blue, medulla intrinsic neurons and transmedullary neurons in green, and T cells in orange; depicted neurons are R1–R6, R7, L1, L2, L3, L4, Mi1, Mi4, Tm1, Tm3, T3, T4, and T5). Each of these cell types is present within every column of the fly visual system. (C) Responses to visual stimuli were recorded in the medulla of flies that simultaneously express the calcium indicator in the lamina monopolar cells L1, L2, L3, and L4 (detailed in Figures S1A and S1B). The medulla has a layered structure, and the schematic on the left indicates the approximate position of each layer and the layers in which each neuron type arborizes. The calcium response to a 5° disc changing from a high to low light intensity is displayed in the center image, with white arrows indicating the position of each layer. The retinotopic nature of the response to the visual stimulus is accentuated by subtracting the response just prior to the change in the visual stimulus and is displayed in the right image (mean response of 1 s after light change minus mean response of 1 s before change). (D) Responses to visual stimuli were also recorded in the medulla of flies that simultaneously express the calcium indicator in all neurons (detailed in Figures S1C and S1D). All results are displayed as in (C), although here all layers of the medulla are shown rather than just the first five. See also Figure S1 and Movie S1. Current Biology 2014 24, 976-983DOI: (10.1016/j.cub.2014.03.017) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 2 The Lamina Monopolar Cell and Pan-Neuronal Calcium Responses Reveal Anatomically Segregated Pathways for Processing of Light-On and Light-Off (A) Representative images of the LMC and pan-neuronal calcium responses to a flickering disc stimulus. The flickering disc was 30° in diameter, flickered between high and low light levels at a frequency of 0.05 Hz, and was centered over the receptive field of the imaged neurons. The response to a light increment is shown in the images on the left, and the response to a light decrement is shown in the images on the right. The proximal and distal borders of the medulla are indicated by dashed white lines, and the position of each layer of the medulla is indicated along the left edge the images (e.g., M1 is the first layer). (B) Time history of calcium responses to the same flickering disc stimulus. Collected images were rotated and scaled to a common coordinate system, and the mean response of each row of pixels, for each fly, was examined as a function of time. Activity is presented as F/F¯, or the instantaneous fluorescence normalized by the average fluorescence over the experimental protocol (motivation for this metric is provided in Supplemental Experimental Procedures). Results shown are the median values from multiple individuals (LMC, n = 8; pan-neuronal, n = 7). Positions within the medulla are given on the vertical axis and correspond approximately to the positions of (A), whereas time points are displayed on the horizontal axis. The white and black bars on the horizontal axis indicate the periods during which the disc was on and off, respectively. (C) Time series for calcium responses at selected positions within the medulla. Results are calculated as in (B), error bars indicate the SEM, and solid black lines below each curve represent a F/F¯ value of zero. The white and gray backgrounds indicate the periods during which the disc was on and off, respectively. For LMC images, each layer was readily identifiable and is shown separately. For pan-neuronal images, layers M4/5 and M8/9 could not be readily distinguished based on either anatomy or activity and consequently are labeled as aggregates. Statistical analysis of these time series demonstrates that LMC responses are less rectified than pan-neuronal responses (see Figure S2C). (D) Principal components of LMC and pan-neuronal calcium responses to a flickering disc stimulus. The principal components analysis used the individual pixels of all trials as variates and each time point as an observation, such that the calculated components simultaneously represent both the LMC and pan-neuronal data sets. The contribution of each principal component to the predicted F/F¯ is shown as a function of position in the medulla and time. Positions within the medulla are given on the vertical axis (e.g., M1 is the first layer), whereas time points are displayed on the horizontal axis. The white and black bars on the horizontal axis indicate the periods during which the disc was on and off, respectively. The percentage of the explainable variance contributed by each principal component is presented below each panel, and significant differences in these percentages between the LMC and pan-neuronal data sets are indicated with asterisks (two-sided Mann-Whitney U test, ∗p < 0.05, ∗∗p < 0.01; ns, not significant). (E) Predicted response to flickering disc stimulus based on first three principal components. Compare to (B). (F) Schematic of the “L1 pathway.” Several processing pathways in the optic lobe have been previously proposed based on the identification of neurons with correlated arborization patterns. The layers involved in the “L1 pathway” proposed by Bausenwein et al. [14] are indicated with gray shading in the medulla schematic. The representative neurons of Figure 1B are reproduced here, and two central neurons belonging to the pathway, L1 and Mi1, are marked on the schematic and shown as Golgi stains. The mean signal energy in PC2 is shown to the right of the schematic as a function of position. (G) Schematic of “L2 pathway.” The displayed schematic indicates the layers involved in the “L2 pathway” proposed in Bausenwein et al. [14], which includes the L2 and Tm1 neurons. The mean signal energy in PC3 is shown to the right of the schematic as a function of position. See also Figure S2. Current Biology 2014 24, 976-983DOI: (10.1016/j.cub.2014.03.017) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 3 The Calcium Responses in Output Layers of the Medulla Are Filtered Relative to Responses in the Input Layers (A) Representative images of the LMC and pan-neuronal calcium responses to a disc flickering at slow (0.33 Hz) and fast (9 Hz) frequencies. The flickering disc was 30° in diameter, flickered between high and low light levels, and was centered over the receptive field of the imaged neurons. The displayed image is the mean fluorescence during the stimulus, averaged over multiple flicker periods. The proximal and distal borders of the medulla are indicated by dashed white lines, and the position of each layer of the medulla is indicated along the left edge the images. (B) Calcium responses to the same flickering disc stimulus over a range of frequencies. Displayed values are mean F/F¯ during the stimulus, averaged over multiple flicker periods, and represent the median value from multiple individuals (LMC, n = 8; pan-neuronal, n = 7). Positions within the medulla are given on the vertical axis and correspond approximately to the positions of (A). (C) Frequency response of F/F¯ for selected positions within the medulla. Results are calculated as in (B), error bars indicate the SEM, and solid black lines below each curve represent a F/F¯ value of zero. As in previous figures, for the pan-neuronal data set M4/5 and M8/9 are labeled as aggregates. (D) Scaled frequency response of calcium responses for selected positions. To enable comparisons of the shape of the frequency response curves, results from (C) were scaled so that the maximum and minimum values have the same vertical positions on all subpanels. (E) Schematic of Hassenstein-Reichardt correlator (HRC). The cups represent photoreceptor inputs. Red lines indicate signals prior to the temporal delay element, and blue lines indicate signals after the delay element. The frequency response of the predelay signals extends to high frequencies (where it is limited by phototransduction and LMC processing), whereas the frequency response of the postdelay signals is low-pass filtered, as indicated by schematic frequency response curves below the diagram. See also Figure S3 and Movie S2. Current Biology 2014 24, 976-983DOI: (10.1016/j.cub.2014.03.017) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 4 Individual Neuron Types within the L1 and L2 Pathways Show Rectification and Temporal Filtering (A) Representative images of calcium responses to a flickering disc stimulus. The response to a flickering disc stimulus was examined in flies that specifically express a calcium indicator in either Mi1 or Tm1 neurons (see Figures S4A and S4B). The stimulus protocol and analysis of images was identical to that performed for LMC and pan-neuronal imaging (see Figures 2 and 3). Images represent the response of Mi1 to a light decrement and of Tm1 to a light increment. The pan-neuronal response to a light increment is reproduced from Figure 2A for comparison. (B) Time history of Mi1, Tm1, and pan-neuronal calcium responses to the same flickering disc stimulus. Results shown are the median values from multiple individuals (Mi1, n = 8; Tm1, n = 7, pan-neuronal reproduced from Figure 2C), error bars indicate the SEM, and solid black lines below each curve represent a F/F¯ value of zero. The white and black bars on the horizontal axis indicate the periods during which the disc was on and off, respectively. (C) Frequency response of Mi1, Tm1, and pan-neuronal F/F¯ to flickering disc stimulus. Results shown are the mean F/F¯ during the stimulus period, averaged over multiple flicker periods. Presented values represent the median values from multiple individuals (Mi1, n = 8; Tm1, n = 7), and error bars indicate the SEM. (D) Scaled frequency response of Mi1, Tm1, and pan-neuronal calcium responses. To enable comparisons of the shape of the frequency response curves, results from (C) were scaled so that the minimum and maximum values have the same vertical positions on all subpanels. See also Figure S4. Current Biology 2014 24, 976-983DOI: (10.1016/j.cub.2014.03.017) Copyright © 2014 Elsevier Ltd Terms and Conditions