Cellular Basis of Head Direction and Contextual Cues in the Insect Brain  Adrienn G. Varga, Roy E. Ritzmann  Current Biology  Volume 26, Issue 14, Pages 1816-1828 (July 2016) DOI: 10.1016/j.cub.2016.05.037 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Experimental Design to Test Head-Direction Coding in the Cockroach (A) Illustration of the experimental preparation that consisted of a raised rotating platform, cockroach restraint tube, and a single visual landmark, which contrasted with the otherwise landmark-void black recording arena. During recordings, physiological data were digitized through a headstage, and head angle was captured by a camera. (B) A 360° rotation (viz., one trial) consisted of 12 × 30° rotations with 10-s immobile periods between each one of them. Data were analyzed only during these immobile periods (∗activity during rotation was not analyzed). See also Figure S1. Current Biology 2016 26, 1816-1828DOI: (10.1016/j.cub.2016.05.037) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 CX Units Encode Head Direction by Changes in Firing Rate (A) Example unit response (A1) single- (SUA) and multi-unit activity (MUA) of tetrode channel (ch) 3 during 8 s of facing a non-preferred angle. Also shown are waveforms for channels 1–4 and the arrangement of the tetrode channels. (A2) Same as in (A1), but from when the animal is facing a preferred angle. (A3) Average firing rate/angle bin (8 s of stationary period each) of the single unit in (A1) and (A2) for the first three trials. The landmark (horizontal black bar) was maintained in control position, CW rotations. # and ˆ indicate the 8-s periods shown as the example traces above. (A4) Average firing rate ±SD across five trials following CW rotations. The unit was significantly modulated by head direction. p < 0.001 Rayleigh test. (B and C) Example of a narrowly tuned unit (B) and example of a broadly tuned unit (C). (B1 and C1) Circular representation of firing following clockwise rotations. Purple to green indicates the increase in firing rate as the animal rotates from non-preferred to preferred angles. Arch represents landmark position (65°–125°). The units were significantly modulated by head direction, p < 0.001 Rayleigh test. Red line represents R-vector (length and direction). (B2 and C2) Mean firing rate for each angle bin in five trials ±SD. (B3) and (C3) show the unit’s instantaneous firing rate in 1-s bins for each trial of the experiment. Red line, Gaussian filtered (Ϭ = 10 s) unit activity. See also Figures S2, S3, and S4. Current Biology 2016 26, 1816-1828DOI: (10.1016/j.cub.2016.05.037) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Tuning Characteristics of Angle-Modulated CX Units (A) Two-dimensional histogram illustrating the normalized firing rates of angle-modulated single units for every 30° bin. The normalized tuning curves were aligned at the peak. We utilized R values to indicate the spread or tuning of a cell. An R of 0–0.5 indicates that the cell is broadly tuned, whereas an R of 0.5–1 means the cell is narrowly tuned to an angle. Based on this criteria the normalized tuning curves were organized into four quartiles (Q1-Q4). The average normalized tuning curves ±SD are depicted in line graphs to the right. (B) Pie chart of R-value distribution showing that the majority of units were broadly tuned (R = 0–0.5) during all control trials. (C and D) R-value distribution separately for CW rotations (C1 and C2) and for CCW rotations only (D1 and D2). Rotation direction does not significantly impact angle tuning characteristics (p = 0.822; paired two-tailed t test). See also Figure S3. Current Biology 2016 26, 1816-1828DOI: (10.1016/j.cub.2016.05.037) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Visual Landmark Position Determines Head-Direction Coding (A) Head-direction coding tuned to allothetic cues, wherein angle-modulated units follow the shift in landmark position by shifting their peaks. Example unit’s mean firing rate ±SD over six trials. (B) Head-direction coding tuned to idiothetic cues, wherein angle-modulated units persist to encode the original peak. Example unit’s mean firing rate ±SD over four trials. (C) Bimodal responses during landmark rotation trials. These units developed a second peak in response to the new landmark position, while the original peak persisted to encode the peak from the control trials. Example unit’s mean firing rate ±SD over six trials. Black line, current landmark position. Gray line, previous landmark position. All examples were modulated by head direction, p < 0.05 Rayleigh test. Gray boxes show peaks (i.e., original, shifted, and expected mean vector positions). See also Figure S4. Current Biology 2016 26, 1816-1828DOI: (10.1016/j.cub.2016.05.037) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 CX Units Encode Head Direction in the Absence of Visual Landmarks or Any Visual Input (A) CX units are able to maintain already established directional activity in the absence of visual landmarks; p < 0.05 Rayleigh test. (B) Example unit that encoded head direction in a head-covered landmark naive animal; p < 0.05 Rayleigh test. The second graph shows the average firing rate of the same unit over four trials in 1-s bins. Red line, Gaussian filtered (Ϭ = 10 s) unit activity. See also Figure S4. Current Biology 2016 26, 1816-1828DOI: (10.1016/j.cub.2016.05.037) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Past Rotation Direction Affects CX Unit Firing Rate during the Stationary Epochs (A) Example unit not modulated by angle increased its firing rate following CW rotations. (B) Example unit that increased its firing rate following CCW rotations. The two units are from the same recording. p < 0.05, two-tailed paired t test. (C) A representative example of a CX unit that significantly encoded a preferred head direction and increased its relative firing rate during the stationary epochs following CW rotations. p < 0.05 for both Rayleigh test and two-tailed paired t test. (D) Mean firing rate across all trials and angle bins during stationary epochs following rotations in the preferred direction and in the non-preferred direction separately for units that did not respond to head direction and ones that significantly encoded head direction. The population mean ± SD is marked by the horizontal and vertical black lines. ∗∗∗p < 0.001, paired two-tailed t test. (E) Distribution of p values indicating the level of significance when firing rate following preferred rotation directions was compared to the firing rate following the non-preferred rotations. Current Biology 2016 26, 1816-1828DOI: (10.1016/j.cub.2016.05.037) Copyright © 2016 Elsevier Ltd Terms and Conditions