Abstract
Probing the spatiotemporal release dynamics of multiple neurochemicals simultaneously in vivo is essential for understanding the molecular basis of brain function and behavior. Microdialysis enables multiplexed neurochemical analysis when combined with advanced molecular tools such as mass spectrometry, but existing microdialysis probes typically exhibit low recovery rates and provide temporal resolution of several minutes, which is insufficient to capture rapid neurochemical release events. Moreover, it remains challenging to investigate the release of multiple neurochemicals upon cell type–specific neuromodulation with a single probe. Here, we present an opto-microperfusion platform that integrates time-sequential microfluidics with cell type–specific optogenetic neuromodulation and push–pull microsampling capabilities. We demonstrated subminute temporal resolution in vitro and near-minute temporal resolution in vivo, with in vivo sampling intervals limited by downstream analytical requirements. The platform achieved high recovery rates for both small molecules and neuropeptides, including 76% for serotonin, 69% for dopamine, and approximately 14% for neuropeptide Y, representing a significant improvement over conventional microdialysis probes. In vivo studies in rats demonstrated reliable microsampling of multiple neurochemicals with minimal sample-to-sample variation. Studies in mice demonstrated the simultaneous detection of the release dynamics of up to 23 different neurochemicals following optogenetic stimulation when coupled with mass spectrometry. Together, this multimodal neural probe paves the way for investigating the molecular mechanisms underlying behavior and neurological disorders.