A brief description of relevant methodologies follows. More detailed methodological descriptions regarding our prosthetic stimulation circuitry and stimulation paradigms have been published [1
A. Animal preparation
The two squirrel monkeys were mature (> 700 grams) males. Each animal underwent three surgeries. Isoflurane was used to provide general anesthesia for all surgeries. During the first surgery an 11 mm diameter 3-turn frontal coil (Cooner Wire #AS632) was implanted on the surface of the sclera and a machined bolt-like structure (“headbolt”) was attached to the skull to allow painless head fixation. During the second surgery, one horizontal semicircular canal was plugged by inserting a small piece of fascia into the canal. A stimulating electrode was made from 150 μm diameter platinum wire (Cooner Wire #AS770-34) by stripping 100-200 μm of the Teflon insulation from the tip. This electrode was inserted into the canal toward the ampulla (away from the canal plug). The return electrode was made from the same platinum wire by stripping about 2 mm of the insulation from the tip and was placed superficially beneath the muscle near the ear. An electrical stimulator, specifically a current source, was connected to these electrodes. The stimulation provided during surgery was 600 pps on/off modulated (1 Hz) biphasic current pulses with a pulse width of 200 μs. The eye movements evoked by the stimulation were monitored visually while inserting the electrode toward the ampulla. The insertion depth and angle were adjusted to yield the desired eye movements (i.e., sufficient magnitude, primarily horizontal, little or no facial twitches, etc.). Then two stainless steel screws were inserted into bone near the electrode and dental cement was used to secure the electrode to the bone. The third surgery mimicked the second surgery but was performed on the remaining, previously unaltered, ear.
B. Experiment setup
A Robinson style coil system (CNC Engineering) was used to measure eye movements. The eye position sign convention was defined using the right-hand-rule: positive is to the left and down. Tests were conducted at least four weeks after the third surgery. All the tests were conducted while the animal was awake in the dark, with alertness maintained using low-dose d-amphetamine (0.3 mg/kg), with its head held so that the horizontal canals were roughly parallel with earth-horizontal. The animal was placed such that its eye was very near the center of the search coil frame. The output signals from the search coil system were sampled along with stimulation signals. The ADC card had 16-bit resolution, and the sample rate was 200 Hz for continuous stimulation tests and 18 kHz for individual pulse stimulation tests. (Details describing both tests are provided below.)
C. Electrical stimulation devices and stimulation parameters
The device for individual pulse stimulation had two independently controllable current sources that we developed. During testing, one current source was connected to the electrode implanted in the right horizontal semicircular canal, the other current source to the electrode in the left horizontal canal. A microcontroller (C8051F006, Silicon Laboratories, Inc.) output voltage pulses from its two D/A converters and the two current sources converted the voltage pulses to current pulses. The current pulses of the two channels were synchronized and the timing delay between the current pulses of the two channels was varied between 0 ms and 3 ms.
Three types of individual current pulses were used in this study. The first type was individual biphasic pulses composed of a 200 μs cathodic phase, a 200 μs rest phase, and a 200 μs anodic phase. The magnitudes of cathodic and anodic pulses were always the same. The second type was individual cathodic pulses having only a 200 μs cathodic phase. The third type was individual anodic pulses having only a 200 μs anodic phase. The individual cathodic and anodic pulses were provided by alternating cathodic and anodic pulses every 50 ms. For all three pulse types, the pulses were repeated every 100 ms. For each trial, the electrical stimulation was applied for about 100 seconds so roughly 1000 pulses were delivered.
For continuous stimulation, the bilateral stimulation was controlled by two microcontrollers; each controlled an independent current source that stimulated one electrode inserted in a horizontal canal. An analog signal was sampled by each of two A/D converters (12-bit) located on each of the two microcontrollers. This input signal was then high-pass filtered digitally (time constant 5 seconds) by each microcontroller and was used to modulate the pulse rates of the output current pulse. The modulation sensitivities (change in pulse rate per change in input analog signal) for the two channels were the same. The modulation directions were varied such that the pulse rate of the two channels was either in push/pull mode (pulse rate increased in one ear while it decreased in the other) or in common mode (pulse rate in the two ears always increased/decreased together). For the continuous modulated stimulation studies, the current pulses of the two channels were not synchronized on a pulse-to-pulse basis. The current pulses during continuous stimulation were the same biphasic pulses used for individual biphasic pulse stimulation (200 μs cathodic stimulation followed by 200 μs of rest followed by 200 μs of anodic stimulation). The signals used to modulate pulse rate were sinusoidal (0.01 Hz to 1 Hz).
All electrical stimulation experiments were conducted while the animal's head was stationary. Because the eye movements evoked by individual stimulation pulses were typically small, the largest possible current was used to elicit the largest possible eye movements. The maximal current was limited by stimulation of the facial nerve. The current levels were always maintained below the facial threshold, in part, because facial responses such as eye lid twitches may cause eye movements that interfere with the measurement of eye responses elicited by vestibular stimulation.
D. Methods specific to modulated continuous stimulation
Six experimental conditions were utilized for these investigations. Two of these conditions were rotation experiments conducted without electrical stimulation:
For conditions 3 through 6, the electrical stimulation was first turned on with the pulse rate held constant at 200 pps for 30 minutes. Then the pulse rate was modulated sinusoidally - with the pulse rate ranging from 133 pps to 267 pps (around the baseline of 200 pps). This pulse rate modulation was set to be equivalent to the modulation provided in our earlier study [5
] for a rotation amplitude 80 °/s (modulation sensitivity of 0.9 pps/°/s at rest, time constant 5 seconds). Therefore, we will calculate VOR gains assuming a rotation of 80°/s. The amplitudes of the current pulses to the right ear and left ear were both set at 125 μA.
For each of the six experimental conditions listed above, the animal was tested using seven modulation (or rotation) frequencies, they were 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1 Hz. Each trial included between 2 and 20 sinusoidal cycles (fewer cycles were used for low frequency tests).
Data analysis was described in details in our previous publication [5
]. In brief, the eye velocity was computed from eye position data recorded originally during tests, and then the fast phases were removed to keep only the slow phase velocities (SPV). For each sinusoidal cycle, the SPV data, as well as the modulation signal (for electrical stimulation tests) or rotation velocity (for rotation tests), were fit to both sine and cosine waves using linear regression. The average of the sine and cosine components were calculated across cycles for a given trial and the mean amplitude
and phase (ϕ
= arctan(As / Ac
)) were then calculated from these average values. The standard error (SE) of the mean was found by calculating the covariance ellipse for the sine and cosine components. The VOR gain was defined as the amplitude of SPV divided by 80°/s, which was the peak angular velocity (or the peak angular velocity simulated via the prosthesis). The phase of VOR was defined relative to the modulation signal for electrical stimulation trials or rotational velocity for motion trials. For each experiment, a first-order high-pass transfer function was fit to the average data across each of the seven frequencies to compute the fitted transfer function gain constant and time constant.
E. Methods specific to single pulse stimulation
The relationship between eVOR evoked by unilateral and bilateral individual pulse stimulation was investigated under three conditions:
- It was reported that the amplitude of the VOR evoked by acoustic clicks  and rotation  varies with eye position. Therefore, we examined the effect of eye position on responses evoked by bilateral and unilateral electrical stimulation;
- The VOR evoked by unilateral electrical stimulation increases with the strength of stimulation [1, 5]. To investigate how the VOR changes with bilateral electrical stimulation with current level, we designed two experiments:
- We investigated the effect of timing delays between the pulses to the right ear and the pulses to the left ear.
show examples of the horizontal eye movements elicited by individual biphasic pulses applied to the right ear at a current magnitude of 300 μA. The eye movements evoked by the stimulation pulses summed with a small (circa 2-3°/s), but consistent, nystagmus. In , the small electrically-evoked responses are barely noticeable. expands the data inside the small rectangle in shows the corresponding biphasic current pulses. The background noise is clearly noticeable in . At lower current levels (e.g., 60 μA), the magnitudes of the responses were smaller (the magnitude of the response was roughly proportional to stimulation current pulse level) such that the response magnitude became comparable to the background noise. To increase the signal-noise ratio, the evoked responses were processed as follows.
Fig. 1 (A) Horizontal eye responses of monkey “G” evoked by individual biphasic pulse stimulation (300 μA) of the right ear lateral semicircular canal. The evoked responses “piggybacked” on a small (circa 2-3°/s) (more ...)
First the falling edge of each cathodic phase of the current pulse was found. The data between 10 ms prior to that edge and 90 ms after that edge was defined as a cycle. Then the peak-to-peak magnitude of each cycle of eye movement was calculated and the median of these peak-to-peak values times 1.3 was used as a threshold. To reject cycles that included fast phases or saccades, any cycle with a peak-to-peak magnitude smaller than the threshold was labeled as a “good cycle” for further processing. The threshold was chosen through trial and error to include as much data as possible while maintaining good data quality. The top trace in (the gray trace) shows the result of averaging of “good cycles” for the data shown in (only for data in the -1 degree eye position group, see section III.B.1 for detail). This trace is the averaging of 10 cycles of data and only the part of data from 10 ms prior to and 30 ms after the falling edge of the cathodic phase is shown. indicates the corresponding biphasic pulse.
As shown in , the nystagmus yields a leftward (positive) change in eye position for this animal. This complicates measures of the amplitude and latency of the response. To minimize the influence of the nystagmus, the data 10 ms prior to the falling edge of the cathodic pulse were fit with a line segment, and the slope of this line was projected for the remaining 90 ms of each data cycle. Then this slope was subtracted from the data. The bottom trace in (the black trace) shows the response after this nystagmic slope removal.
To compute the amplitude of the response, we first found the time of the peak response and defined the average of the data within 1 ms (0.5 ms prior and after the peak time) as the average peak response. The average taken 10 ms prior to the falling edge of the cathodic phase was defined as the base value. The amplitude of the response evoked by stimulation was defined as the difference between the peak and base.