Focused ultrasound (FUS) is a novel noninvasive modality for neuromodulation in the central nervous system with a high spatial resolution and the ability to target deep brain structures. Rodent studies have shown motor responses following FUS but mostly failed to demonstrate consistent and target-specific effects. Here, we used a high-frequency (4 MHz) transducer with a small focal area to investigate the target-specificity of FUS neuromodulation. Mice were implanted with a large cranial window consisting of an ultrasound-transparent Polymethylpentene membrane. For the experiment, awake mice were head-fixed and allowed to move freely on a floating platform, while being monitored with a camera (60 fps). 8 pulses of FUS (3.0 MPa, 100 ms) were administered to the right/left hindlimb motor cortex (-1.0 mm AP and +/-1.0 mm ML from Bregma) and the right/left posterior Thalamus (-2.5 mm AP and +/-1.5 mm ML from Bregma). The motor responses were classified by semi-automated tracking with Deeplabcut. Our preliminary results (n = 1, 8 trials per target) show a specifically contralateral motor response for both targets, while off-target stimulation had no effect (Figure 1). In conclusion, we demonstrate that FUS neuromodulation to the motor cortex and posterior thalamus can induce target-specific contralateral motor responses in awake mice.Figure 1. Lateralized motor responses following FUS to motor cortex and thalamus. Boxplots (a,b,c) show mean relative displacement of hindlimbs from 200 ms following the FUS stimulus. d shows opposite directions of movement of the tail base following right/left thalamic FUS. Statistical test: independent t-test (a,b,c).