Tracing Superoxide Anion in Serotonergic Neurons of Living Mouse Brains with Depression by Small-Molecule Fluorescence Probes

In brains, the serotonergic neurons are the unique resource of the neurotransmitter serotonin, which plays a pivotal role in the physiology of the brain. The dysfunction of serotonergic neurons caused by oxidative stress in the brain is closely related to the occurrence and development of various mental diseases, such as depression. As the biomarker of oxidative stress, the superoxide anion radical (O₂^•-) can cause oxidative damage to proteins, nucleic acids and lipids, disturbing the function of neurons and brains. A serotonin transporter (SERT) specifically expresses in serotonergic neurons, which is the biomarker of serotonergic neurons. Thus, we created two novel small molecular fluorescent probes (PA-CA and HT-CA) for imaging O₂^•- in serotonergic neurons of living brains of mice based on specific targeting groups of SERT. Both PA-CA and HT-CA exert excellent SERT-targetable and glorious selectivity for O₂^•-. Those two probes could monitor the boost of O₂^•- in living hsert-HEK293 cells that specifically express SERT under oxidative stress. With two-photon fluorescence imaging, we revealed for the first time that O₂^•- is significantly increased in serotonergic neurons in living brains of mice with depression. More importantly, proteomic analyses suggested that O₂^•- could oxidize cysteine and histidine in the active site of SERT, which is involved in the development of depression. This work provides new materials for living brain imaging and offers new strategy for unraveling the pathophysiology of depression.