Experimental design. (A) Schematic of behavioral experimental setup comprises various behavioral tests, tFUS stimulation with multiple control groups, and video assessment with subsequent data analysis. Nocifensive reactions to heat, cold, and mechanical stimuli, as well as motor performance, were recorded and evaluated to investigate the effects of tFUS stimulation at different brain locations, including S1HL, insula, and thalamus. (B) Study design for EEG measurements measurements were designed to examine both intrinsic brain activity and heat stimulus–evoked brain activity along with tFUS stimulation. (C) Control experiments were conducted to determine the specificity of the modulatory effect to tFUS stimulation at a particular brain circuit, the effects of negative control (no tFUS stimulation, maintaining experimental procedures) and sham treatment (applying tFUS to a control brain structure near the targeted brain location) were examined. (D) tFUS parameters involved customizing the 128-element random array transducer to steer ultrasound focus. Targeted brain structures were subjected to tFUS with specific parameters, including a fundamental frequency of 1.5 MHz, TBD of 200 μs, PRF of 40 Hz and 3 kHz, ultrasound duration (UD) of 100 and 400 milliseconds, intersonication interval (ISoI) of 2 and 4 second, and total sonication time of 10 minutes, 20 minutes, and 1 hour.