The broad, long-term objectives of this technology-driven project are (1) to continue developing and (2) apply a powerful, new, simple, microfluidic device (and method for its use) for determining rates of processes in the extracellular space of brain in vivo. A specific focus is hydrolysis of neuropeptides in specific brain regions in awake, behaving animals. Method: The implantable device uses electroosmotic flow to direct solutions of substrates, with or without inhibitors, through brain tissue and direct unreacted substrate and products to an online measurement system or collect them for later analysis. Uniquely, this device and method uses natural substrates in vivo. The particular focus is ectopeptidases, the “ecto” implying cell-surface (membrane-bound) enzymes that hydrolyze peptides in the extracellular space. A typical experiment infuses a substrate peptide and an unhydrolyzable D-amino acid peptide analog with or without a selective inhibitor. The unhydrolyzable peptide represents initial substrate concentration. Michaelis-Menten rate parameters are derived from experimental data using a validated approach based on a simulation of the measurement. The simulation is required to correct for diffusion of substrate and products in the tissue. Health relatedness: There are many neuropeptides, and many critical brain functions such as learning and memory, pain perception, protection from injury due to anoxia, and many others that rely on neuropeptide concentrations in the extracellular space. But there is currently no means to directly measure the impact of the ubiquitous ectopeptidases on those concentrations. The proposed microfluidic device and the simulation provide the means to measure the rate of hydrolysis of active peptides. The proposed method permits quantitative modeling of peptide activity (e.g., Vmax) in the extracellular space in vivo. Specific Aims: There are two parallel, independent aims. One improves the device. The other applies the existing novel microfluidic device to a question about pain perception. (1) Improve upon the existing, fully functional device to make experimental measurements with more flexibility in choosing substate/inhibitor concentrations being passed through the tissue; to extend the device’s capability to reach deeper brain regions. (2) Apply an existing, fully functional microfluidic device to determine differences in specific ectopeptidase activities in the anterior cingulate cortex between rats experiencing mild chronic pain and controls.