Fig 1.
Madagascar hissing cockroach with implanted electrodes.
Active electrodes were inserted into antenna and cerci, and the ground electrode into the second abdominal segment. Inset: Ventral view X-ray image highlighting the position of wire electrode tips at the base of antennal sockets. The head is clearly visible in the center of the image.
Fig 2.
Motion tracking system used to measure stimulus response relationship.
Left: Madagascar roach implanted with electrodes sits atop floating polystyrene ball in tethered configuration. Rainbow-colored ribbon cable connects to stimulus electronics. Two orthogonal optical mice measure forward motion and rotation. Image retouched to remove background for clarity. Right: Illustration of optical mice and fictive path coordinate systems. The rotational axis of the trackball r is indicated in red.
Fig 3.
Illustration of automated analysis methods for forward and turning responses.
Top panel: Forward walking response. Raw data (red) is low-pass filtered (black) to aid identification of the beginning and termination of the forward response. Dotted green line and diamond mark the response initiation threshold and the beginning of the forward response, Ton. Dotted blue line and circle mark the termination threshold and time Tf (see Eq 3). Bottom panel: Turning response. Delivery of stimulus to the left antenna stimulus causes turn to the right (ω < 0). Timing of stimuli are indicated by solid horizontal bars (yellow = left antenna; blue and green = right and left cerci). Dotted gray lines: threshold crossings for identifying multiple phases of turning response. Successive pairs of green and blue markers indicate initiation and termination of a single phase of a turning response.
Table 1.
Summary of experiments and stimulus parameters.
All stimuli were square pulses with 50% duty cycle, with indicated polarity (B = bipolar, positive-first; M = monopolar), amplitude, frequency f, and duration Tstim. The number of insects tested is indicated by N.
Fig 4.
Example of trial-averaged responses of linear and angular velocity vs. time for stimuli delivered to both cerci and left antenna.
Turns to the right are indicated by ω < 0; turns to the left by ω > 0. Each column corresponds to a particular stimulus parameter combination. Solid and dotted curves: mean and median across all trials, respectively. Shaded area marks one standard deviation of trial-averaged responses. Each trial lasted for 15 s, and the the stimulus duration was 1.5 s. The subject was spontaneously walking during 1–3 V, 50 Hz trials, but not others. Repeated strong forward walking responses with turns in the proper direction were observed for all stimulus parameter combinations, except for 1 V, 200 Hz.
Fig 5.
The percent of test subjects responding to different stimulus types: Voltage controlled monopolar positive voltage pulses (A); charge-balanced voltage (B) and current controlled pulses (C); different durations with set voltage and frequency (D).
In (B) open marks indicate only that a clear forward response was observed, whereas closed markers additionally indicate turning in the proper (contraversive) direction.
Fig 6.
Comparison of sustained response with bipolar stimuli (left column) and habituated response (center column) with monopolar stimuli.
Additional path length (top row) and turning angle (bottom row) in response to successive trials are shown. Marker coding:{□, ⋄, ∘, ▿} = {1,2,3,4} V and {light gray, dark gray} = {50, 200} Hz. For ease of comparison, normalized responses (median ± S.D. computed for each stimulus type) vs. trial number for sustained (red, filled marks) and habituated (black, open marks) are shown in the right column.
Table 2.
Comparison of monopolar vs. bipolar voltage stimuli strong responders and habituated test subjects.
N = number of test subjects; Nhab = percent of subjects that exhibited habituated response; Nsr = mean ± S.D. of maximal number of strong responders; and η = Nsr/Nhab. Overall, bipolar stimuli were found to be much more effective.
Fig 7.
Locomotion response as a function of VCS (left) and CCS (center) bipolar stimulus amplitude, and stimulus duration (right).
Red and black color-code stimulus frequencies of 50 Hz and 200 Hz, respectively. Bulls eye markers indicate the median, and bars cover the 25–75th percentile. Variable amplitude and duration tests had constant Tstim = 1.5 s, while variable duration tests were done with constant 3V, 50 Hz.
Table 3.
Statistical tests for correlation of locomotion response metrics to stimulus amplitude or duration.
Variables in each column are: number of responses analyzed to 50 Hz and 200 Hz stimuli, respectively n50 and n200; stimulus frequency f; Pearson correlation coefficient r and associated p-value; and linear regression slope m, computed only when a significant correlation was identified (p < 0.06). Units of m are given in the physical units of the metric (e.g., cm) per 1 V for voltage pulses, per 50 μA for current pulses, per 1s for variable duration tests.
Fig 8.
Radius of curvature increases with of stimulus amplitude, but not duration.
Results are shown for bipolar VCS (left); CCS (center); and variable durations (right). Red and black color-code stimulus frequencies of 50 Hz and 200 Hz, respectively. Bulls eye markers indicate the median, and bars cover the 25–75th percentile. Variable amplitude tests had constant Tstim = 1.5 s, while variable duration tests were done with constant 3V, 50 Hz.
Fig 9.
Trial-averaged fictive paths for a single test subject in response to 8 different stimulus parameter combinations.
Path end point markers code for stimulus parameters: {∘, •, □, +} = 1–4V at 50 Hz; {⋄, ×, △, ▿} = 1–4V at 200 Hz. Stimuli are delivered to R antenna in left panel, and to the L antenna in right panel. The time scale is adjusted such that stimulus delivery occurs at t = 0 with all paths starting at the origin. Paths are color-coded for the the time at which a certain (x,y) coordinate was traversed.
Fig 10.
Trial-averaged fictive paths for 5 test subjects subject to various stimuli.
Scale bar is 10 cm in both dimensions. Color-coding corresponds to the time at which a roach was at a certain position (see color bar in Fig 9).
Fig 11.
Example of simultaneous voltage (gray) and current (blue) measurement during bipolar voltage pulse stimulation.
For the 3 V, 50 Hz waveform, the peak and time-averaged current amplitudes were 2.78 mA and 737 μA, respectively.
Table 4.
Comparison of turning angles to results reported in [4].
The mean turning angle and 25–75% range are given. For Stim 1, the range is estimated from data reported in [4].