Functional Brain Imaging and Targeted Lesion Studies Using Manganese-Enhanced MRI and Focused Ultrasound in Non-Conventional Model Species.

BACKGROUND: Linking behavior to neuroanatomy in non-conventional species is hindered by a lack of tools like stereotaxic atlases, antibodies, or transgenic lines. Traditional methods like electrophysiology or surgical lesions are often impractical for aquatic species or organisms with continuously growing brains. Consequently, neuroethology is frequently limited to a few classical models, creating a need for a versatile, non-invasive framework to map and validate brain function across diverse taxa. NEW METHOD: We present an integrated protocol combining Manganese-Enhanced MRI (MEMRI) and MR-guided High-Intensity Focused Ultrasound (HIFU) into a single cohesive workflow. MEMRI provides non-invasive, sub-millimeter mapping of neuronal activity during behavior, identifying candidate regions of interest, while MR-guided HIFU enables precise, surgery-free thermal ablation of targeted regions to test their causal necessity for behavior. Demonstrated in the convict cichlid (Amatitlania nigrofasciata), this adaptable protocol requires only an MRI scanner and ultrasound transducer, bypassing the need for a stereotaxic atlas to guide targeting. RESULTS AND CONCLUSION: MEMRI identified significant manganese accumulation in the inferior lobe of fish performing a puzzle-box task, suggesting its role in object manipulation. Validation via MRI-guided HIFU thermal lesions showed that inferior lobe damage caused massive behavioral deficits, whereas cerebellar lesions had minimal impact. These results confirm the MEMRI-HIFU framework as a powerful toolkit for linking brain structure to behavior in non-model organisms. COMPARISON WITH EXISTING METHODS: Unlike immediate early gene mapping, MEMRI requires no species-specific antibodies and is non-terminal. Compared to surgical lesions, HIFU reaches deep targets through the intact skull without needing a stereotaxic atlas.