We propose to develop an analytical method using capillary high-performance liquid chromatography at high temperature and pressure coupled to photoluminescence detection following electron transfer (UPLC-PFET) following online microdialysis sampling to enable fast serotonin neurotransmitter measurements in the brains of awake behaving animals. This method will provide substantial improvements in the speed and limits of detection over current methods (e.g., HPLC-EC, CE-LIF). It will be broadly applicable to other monoamine neurotransmitters and metabolites and peptides. The brain serotonin neurotransmitter system regulates many important psychophysiological functions including mood, anxiety states, cognition, reward-related behavior, motor activity and appetitive behavior. The presynaptic plasma membrane serotonin transporter (SERT) takes up serotonin from the extracellular space and plays a central role in regulating serotonergic neurotransmission. It is the primary target for the most widely prescribed class of antidepressant and anti-anxiety medications } the serotonin reuptake inhibitors (SRIs). In humans, reduced SERT expression driven by a commonly occurring gene variant has been associated with increases in anxiety-related personality traits. and susceptibility to stress-associated depression. To investigate effects of decreased SERT, we have produced mice that express reduced SERT. Decreased SERT in mice results in increased anxiety-like behavior similar to that observed in humans. Despite the key role that serotonin uptake plays, the kinetics of the uptake of endogenous serotonin has never been measured We will employ the optimized UPLC-PFET method developed here to measure these kinetics. PUBLIC HEALTH RELEVANCE: To understand the brain's internal communications, we need analytical methods that are absolutely selective and also fast. There are no general approaches that have both attributes. We will develop one, and apply it to a question about serotonin - the key player in anxiety, depression and related disorders.