Fig 1.
Gross anatomy of the locust brain, visual pathways, and relevant types of central complex neurons.
A. Bilateral pathways of light-sensitive neurons from the optic lobes converge onto a network in the central complex (frontal view). Stages of early visual processing include the lamina (LA), medulla (ME) and lobula (LO) of the optic lobe. Neuropils shaded red (green) are involved in an anterior (posterior) pathway of interneurons sensitive to sky compass signals. Additional pathways (yellow neuropils) might signal optic flow and / or represent features of the visual object-background scenery. DRLA (DRME), dorsal rim area of the lamina (medulla); ALO, anterior lobe of the lobula; AME, accessory medulla; AOTU, anterior optic tubercle; POTU, posterior optic tubercle; MBU (LBU) medial (lateral) bulb; LAL, lateral accessory lobe; together with the LAL, the MBU and LBU make up the lateral complex (LX). CBL (CBU) lower (upper) division of the central body; PB, protocerebral bridge; SMP, superior medial protocerebrum; CA, calyx of mushroom body. B. Relevant cell types of the central complex (frontal view). Tangential neurons invade all slices of the CBL (TL neurons) or some slices in the PB (TB1 neurons), as well as regions in a lateral complex (TL) or layers in a POTU (TB). Columnar neurons connect distinct slices of the PB to the CBU (CPU neurons) or CBL (CL neurons) of the central body and have additional arborizations in the lateral complexes. Scale bar, 100 μm.
Fig 2.
Experimental setup and visual stimulation.
A. Experimental setup with display showing stationary background clutter. CRT, cathode ray tube display; asterisk, fronto-dorsal corner mark of CRT; MM, micromanipulator for positioning of intracellular electrode; ELi, intracellular electrode; ELref, reference electrode; LOC, locust (viewed from dorsal); arrow, stimulated (left) eye. Note the slight tilt of the CRT and the mesh wire shielding which prevented inductive interference between display and measurement circuit. B. Region in the left antero-lateral visual field covered by the stimulus display. Arrow, position of stimulated (left) eye; asterisk, fronto-dorsal corner mark of CRT; LOC, locust (viewed from lateral). Dashed horizontal lines tagged H and L indicate high- and low-elevation trajectories for translations of single small-field squares. sr, br: commonly applied square- and bar-shaped stimulus; about 2°x1.5° and 2°x60°, respectively. Albeit both types of stimulus are shown together here, they were never presented simultaneously. B'. Terminology for directions of translational motion, illustrated according to the orientation of the diagram in B. Abbreviations are given in parentheses. Forward (backward) direction of translation is abbreviated "f" ("b") and high (low) elevation is abbreviated "H" ("L"). Thus, the term "b, L" ("f, H") refers to the backward (forward) translation of a 2°x1.5° square -29.5° (+25.5°) in elevation. See S1 Fig for commonly applied sequences of such simple stimuli. C, C'. Components (C) and time course (C') of a more complex stimulus sequence designed to mimic sudden forward small-field motion against a backward optic-background flow. The sequence begins with a stationary display of the "background clutter" pattern, followed by repeated backward translations of the entire pattern (indicated by horizontal arrows) in an optic-flow like manner at 70°/s (dashed horizontal line in C'). In the late phase of the sequence, an individual object in the background flow (short vertical arrows in C) pops out of it twice by changing its direction of motion, i.e. the object suddenly moved forward against the backward background flow (absolute velocity 80°/s; velocity relative to background flow -150°/s). The object, a black, rectangle square of about 2° visual angle azimuthal extent and 1.5° elevational extent, is identical to the one used in the simple stimulus regimes and moves at the same high elevation of 25.5° also applied in the latter. For further details on stimuli, see Materials and Methods.
Fig 3.
Background activity and responses to small-field motion in four types of central-complex neuron.
Subfigures show data from a CL1 neuron (A-D), TB1 neuron (A'-D'), CPU1 neuron (A''-D'') and CPU2 neuron (A'''-D''') of the central complex. A-A'''. Traces illustrating background activity. B-B'''. Spike count distribution and ISI histogram of background activity (trial duration for spike count 1 s). Asterisks and x-marks indicate the location of spike rates observed at the peak (or nadir) of the trial-averaged responses shown in D-D'''. C-C'''. Responses to presentations of a translating black, rectangular small-field square (about 2° visual angle azimuthal extent and 1.5° elevational extent) against a blank background showed fast adaptation to motion along the same trajectory. Stimulus types and periods, indicated below the recording traces, also hold for raster plots in D-D''', unless indicated otherwise. Stimulus abbreviations: f (b), forward (backward) small-field translation; H (L) trajectory with high (low) elevation. D-D'''. Raster plots and trial-averaged, Gaussian-smoothed PSTHs of additional responses. Spike rates occurring at the peak (or nadir) of the averaged response (marked by asterisks or x-marks) are located at the edges of the spike count distribution obtained from long-term background activity (see B-B'''). This indicates that responsiveness is robust across the trials shown here, but response amplitudes vary and individual responses resemble sections of background activity. In particular, arrows in A' and C' mark prominent bursts in background activity and in the phasic response period for the TB-neuron. In the two subtypes of CPU neuron (D'' and D'''), responses are independent from direction of motion. Dashed lines indicate periods of rebound-like increase in spike rate in D and a period of tonic increase in spike rate in C'. Bars, 1 s; 10 mV.
Fig 4.
Stimulus-specific adaptation of responses to small-field motion in CL-, TB- and CPU neurons.
A. Responses to small-field motion are marked by rapid adaptation between the first and second presentation of a single moving small-field square. Box- and bubble plots show differences in spike count between stimulus time-windows and background activity in a period of 450 ms preceding stimulation, based on N responses obtained from n neurons. Notches in box plots indicate 95% confidence interval of the median and circular plot markers in bubble plots are scaled to the frequency of observations (see scale, bottom right). Some outliers were truncated for the sake of better visualization. m, median; asterisks: level of statistical significance (Bonferroni-corrected) of difference in median as calculated by Wilcoxon rank-sum tests (* pcorr<0.05, ** pcorr<0.01, *** pcorr<0.001). TB1 cells showed excitatory responses to the first stimulus in a sequence, while all other types of neuron showed inhibition. Note the increased spike rate of CL1 cells during presentation of the second stimulus, which is related to rebound-spiking after the inhibitory response to the first stimulus (see Fig 3D). Data include many cases where first and second stimulus differed in direction of motion (see Table 1). Nevertheless, pronounced adaptation occurs between the first and second stimulus. B. In many cases, adaptation could be reversed by switching the elevation of a horizontal trajectory, as illustrated here for population data of CL1 neurons sufficient in sample size for statistical testing.
Table 1.
Statistics of data distributions shown in Fig 4A.
Table 2.
Statistics of data distributions shown in Fig 4B.
Fig 5.
Context dependent responses to moving objects.
The courses of N responses to a respective stimulus sequence are visualized for selected neurons by normalized instantaneous interspike intervals (norm. ISI), averaged across trials when N > 1. For a TB1 neuron, raster plots were preferred. Values of 0 or 1 indicate highest consistency in response course across trials. A-A'''. Each subplot represents N responses to a specific stimulus sequence, obtained from cells identified by capital letters within each cell type (compare to subfigure B). A-A'': Horizontal trajectories; forward (f) or backward (b) motion at high (H) or low (L) elevation. In A'', the third and fourth stimulus consist of two objects that move in the same direction but at different elevations. A''', Responses to upward (u) and downward (d) motion along vertical trajectories at different azimuths (-38°, 7.5° and 54°). Here, the transitions from d to u' and d' to u'' correspond to switches in the azimuth of the trajectory, whereas the particular sequence of azimuths was randomized in each trial. As a consequence, the lower subplot shows data pooled across 3 different chronological orders of azimuths. For both vertical and horizontal trajectories, it is the switch in trajectory that triggers responses, not the particular position of the trajectory or changes of the direction of motion. Bars, 1 s. B. Responses of the same types of neuron are also suited to detect a distinct object that changes its direction of motion (pop out) against a moving background of identical and very similar objects. For detailed description of the stimulus sequence, see Fig 2.
Fig 6.
Modulation of responses to moving small-field objects by variable background activity.
A. Raster plots show selected responses to a translating small-field square (dashed line: stimulus window) which are modulated by relatively low states (left subplot) and relatively high states (right subplot) of precedent background activity (BA). The relevant period of background activity may roughly cover the last 500 ms (grey shading) preceding the stimulus. B. Correlation analysis in CPU neurons. Spike counts within the first (second) 700 ms-window during stimulus presentation were plotted against those in the 700 ms-window of background activity directly preceding the stimulus. Data points close to or above the bisecting line correspond to trials that lacked the inhibitory response typical for CPU neurons; strongest responses are reflected by data points closest to the x-axis. Data were obtained from 3 CPU1- and 4 CPU2 neurons and cover 62 responses to the first stimulus in sequences of single small-field squares translating against a blank background; different plot marks correspond to different neurons. Plot marks are not scaled to the frequency of observations, thus identical values from the same experiment appear as a single data point. Dotted lines show linear regressions. C. Response amplitudes compared to different states of background activity. Box plots show distributions of relative response amplitudes (changes in spike rate relative to different levels of background activity). These were calculated for responses to the first stimulus in a respective sequence of small-field squares translating against a blank background (N responses from n neurons). For estimation of relative response amplitudes, we subtracted the spike rates of different levels of background activity (1s bins), obtained from the same respective cell throughout the entire experiment. These levels include the median and two additional quantiles of the background activity's spike count distribution that lie beyond the median in the same side of the distribution as expected for responses. In case of the inhibitory responses in CL- and CPU neurons, the 25% and 2.5% quantile of the spike rate distribution were used. For the excitatory responses of TB neurons, the 75% and 97.5% quantile provide relevant normalization. Indicated above each box-plot is the result of a two-sided sign test for zero median (* pcorr<0.05, ** pcorr<0.01, *** pcorr<0.001, ns not significant). Note that a non-significant test result here does not imply that responses did not involve a significant change in spike rate as compared to the local background activity that actually preceded the responses (see Fig 4A). While responses are relatively robust compared to the median levels of background activity (left columns of subplots), the additional response-type specific normalizations (middle and right columns) reveal that more extreme levels of background activity may result in masking of responses, as the difference to zero change decreases substantially and may become non-significant. Notches in box plots indicate 95% confidence interval of the median. Some outliers were truncated for the sake of better visualization.
Table 3.
Statistics of data distributions shown in Fig 6c.