Optimizing Sensory Stimulation Protocol

After constructing BrainWAVE stimulators, testing was performed to determine whether the devices generate appropriate stimulus intensity, timing, and other signal properties. Light illuminance and audio volume were measured with a light meter and decibel meter, respectively, with the distance between the sensor and meter approximating the distance from the sensory to the subjects’ eyes and ears (Extended Data Table 3-1). For mouse studies, light intensity was set at ∼150 lux and sound intensity at 60–65 dB (Garza et al., 2020 (link); Martorell et al., 2019 (link)). For human studies, we adjusted stimulus intensity for each subject based on tolerance, with the levels ranging from 0 to 1400 lux for brightness and 0–80 dBA for sound (He et al., 2021 (link)). We measured the frequency and duty cycle of the audio and visual stimuli in real-time using an oscilloscope connected to the analog output ports of the light and decibel meters (Fig. 3A). Alternatively, the timing of the light and sound stimulus may be measured with a photodiode and a microphone connected to an oscilloscope, or the stimulus may be recorded on a laptop and analyzed on a computer. Audio and visual signals were measured simultaneously to compare their duty cycle, frequency, and phase timing.
To modulate neural activity, we generated sensory signals at specific frequencies depending on the experimental design. Visual γ flicker (40 Hz) was produced using a 5.17-V, 40-Hz square wave with a 50% duty cycle (Fig. 3B). The voltage must be greater than 4 V to operate the MOSFET. Auditory γ flicker was produced with a pure sinusoid tone signal that was modulated by a 40 Hz square wave with a 50% duty cycle for audiovisual stimulation, and a 4% duty cycle for audio-only stimulation (Fig. 3C). The pure tone used was adjusted to fall within the center of the hearing range of the species tested: 10 kHz for mice and 7 or 8 kHz for humans (Heffner and Heffner, 2007 ). We used a 4% duty cycle for audio-only stimulation to more closely match the timing of clicks in studies on auditory steady-state responses evoked with 40-Hz click trains (Galambos et al., 1981 (link); Stapells et al., 1984 (link); Osipova et al., 2006 (link); Ma et al., 2013 (link); Thuné et al., 2016 (link)). Other frequencies of sensory were generated in a similar manner typically with a 50% duty cycle. Randomized stimulation was used to compare periodic to aperiodic flicker stimulation and had varying duty cycles (from 33% to 99%). Audio and visual signals were typically synchronized with similar duty cycles, but offset signals or different duty cycles may be desired in some cases (Fig. 3D–G,I).