Channel-specific differential effects of bacterial mechanosensitive channels for ultrasound neuromodulation in precision sonogenetics

<b>Rationale:</b> Ultrasound neuromodulation offers promising therapeutic potential, but its effectiveness is limited by imprecise targeting of neural circuits. Engineering mechanosensitive ion channels can enhance ultrasound sensitivity, providing a more precise approach for targeted neuromodulation. This study aimed to compare three bacterial mechanosensitive channels (MscL-G22S, MscL-G22N, and MscS) for mediating ultrasound-responsive hippocampal activity to identify optimal candidates for precision sonogenetics applications. <b>Methods:</b> We expressed MscL-G22S, MscL-G22N, and MscS in the rat hippocampus using AAV vectors and applied focused ultrasound stimulation at various intensities while recording local field potentials. Neural oscillatory patterns, ultrasound-evoked potentials, behavioral outcomes, immunohistology, and transcriptomic analyses were conducted to assess response consistency, efficacy, and biosafety. <b>Results:</b> Each channel conferred distinct neuromodulatory signatures: MscL-G22S exhibited remarkable ultrasound sensitivity with non-monotonic intensity-response amplification of evoked potentials (2.3-fold increase at maximum intensity), and accelerated response timing (latency reduction). Notably, MscL-G22N showed weaker ultrasound responses despite having a lower mechanical threshold than G22S, suggesting ultrasound sensitivity depends on factors beyond mechanical gating thresholds. Conversely, MscS displayed diminished responses at higher intensities. No statistically significant differences were detected in behavior assessments and histology evaluations. All channels maintained normal anxiety indices, spatial memory, and neuronal morphology, though MscS selectively increased depressive-like behaviors. Transcriptomic analysis revealed that MscS demonstrated exceptional genomic compatibility with minimal off-target gene alterations (9 vs. >400 in MscL variants). <b>Conclusion:</b> This characterization provides insights for potential precision sonogenetics applications: MscS offers a biosafety-optimized option with minimal genomic footprint, whereas MscL-G22S enables modulation of neural oscillations. These findings contribute to the development of customized neuromodulation approaches for targeting pathological circuits in neurological disorders.