Multimodal imaging of gene expression, morphology, and activity of the same neuron

Elucidating the relationships among in vivo activity, brain-wide projection, and gene expression is critical for understanding neuronal functions, but characterizing these modalities for the same neuron remains technically challenging. Here, we developed a trimodal platform combining in vivo Ca²⁺ imaging, morphological reconstruction of single neurons in cleared whole brains, and post hoc imaging-based in situ transcriptomic profiling in thick brain sections. We applied this platform to the mouse primary visual cortex (VISp) and obtained trimodal profiles for 141 intratelencephalic (IT) and pyramidal tract (PT) neurons. We found that regional axonal arborization, soma location, transcriptomic signatures, and subcellular RNA localization emerged as informative predictors for distinguishing neurons preferentially responsive to different visual stimuli. Importantly, morphological and transcriptomic features are complementary and, when integrated, can better predict neuronal function. Thus, this trimodal platform enables a comprehensive understanding of the relationships among gene expression, morphological diversity, and functional properties of single neurons.