Focused Ultrasound Stimulation Protocol

A single element ultrasound transducer (H115-MR, diameter 64 mm, Sonic Concept, Bothell, WA, USA) with 51.74 mm focal depth was used with a coupling cone filled with degassed water and sealed with a latex membrane (Durex). The resonance frequency of the ultrasonic wave was set at 250 kHz with 30 ms bursts of ultrasound generated every 100 ms, controlled through a digital function generator (Handyscope HS5, TiePie engineering, Sneek, The Netherlands). The stimulation lasted for 40 s. A 75-Watt amplifier (75A250A, Amplifier Research, Souderton, PA) was used to deliver the required power to the transducer. A TiePie probe (Handyscope HS5, TiePie engineering, Sneek, The Netherlands) connected to an oscilloscope was used to monitor the voltage delivered. The recorded peak-to-peak voltage was kept constant throughout the stimulation. Voltage values per session ranged from 130 to 142 V, corresponding to 1.17 to 1.35 MPa as measured in water with an in house heterodyne interferometer (Constans et al., 2017 (link)). Based on numerical simulations (see Acoustic and thermal modelling below for more details), the maximum peak pressure (P_max) and I_sppa at the acoustic focus point were estimated to be 0.88 MPa and 24.1 W/cm² for the SMA target, and 1.01 MPa and 31.7 W/cm² for the FPC target (I_spta: 7.2 W/cm² and 9.5 W/cm² for SMA and FPC, respectively). Each of the areas targeted in experiments 1–4 lie close to the midline. Therefore, we applied a single train over the midline stimulating the target region in both hemispheres simultaneously.
In order to direct TUS to the target region, we guided the stimulation using a frameless stereotaxic neuronavigation system (Rogue Research, Montreal, CA; RRID:SCR_009539) set up for each animal individually by registering a T1-weighted MR image to the animal’s head. Positions of both the ultrasound transducer and the head of the animal were tracked continuously with infrared reflectors to inform online and accurate positioning of the transducer over the targeted brain region: SMA in experiment 1, (Montreal Neurological Institute (MNI) X, Y, and Z coordinates in mm [0.1 2 19]); FPC in experiment 2 [0.6 24 10]; FPC in experiment 3 [-0.7 24 11]; pre-SMA in experiment 4 [0.2 11 17]. The ultrasound transducer/coupling cone montage was placed directly onto previously shaved skin prepared with conductive gel (SignaGel Electrode; Parker Laboratories Inc.) to ensure ultrasonic coupling between the transducer and the animal's scalp. In the non-stimulation condition (control), all procedures (anaesthesia, pre-scan preparation, fMRI scan acquisition and timing), with the exception of actual TUS, matched the TUS sessions.