Fluorescence Imaging Of Depression Related Active Molecules In Cells And In Vivo

Depression with core symptoms of depressed mood,anhedonia,irritability,difficulties in concentrating,and abnormalities in appetite and sleep,is one of the most prevalent and disabling mental disorders.Depression has ranked as the leading cause of the global burden of disease and disability.To effectively prevent and treat depression,it's essential to thoroughly understand molecular mechanisms of depression.However,its pathophysiology is still rudimentary due to the complexity in etiology of depression.Previous findings suggest that bioactive molecules in brains,including neurotransmitters,corresponding synthetic or hydrolytic enzymes,reactive oxygen species?ROS?,reactive nitrogen species?RNS?,metal ions and so on,play a crucial role in the occurrence and development of depression.Therefore,it is of significant for revealing the mechanism of depression by accurately detecting the bioactive molecules including neurotransmitter,hydrolase/synthetase,metal ions,and uncovering their regulatory effects on neuron functions and depression behaviors in mice.At present,fluorescence imaging technology possessed excellent advantages such as high resolution,less damage and simple operation,which is widely used for the in situ real-time detection of biologically active molecules in living cells and in vivo.In particular,the two-photon fluorescence imaging method is more suitable for in situ visualization of living organisms due to its long-wavelength excitation,low background fluorescence interference,deep tissue penetration,and light optical damage.However,up to now,few fluorescent probes have been developed to recognize depression-related active molecules in neurons and in brains with high selectivity,especially two-photon fluorescent probes.Small molecule fluorescent probes possessed the advantages of stable performance,easy to cross blood-brain barrier,relatively simple operation and good biocompatibility have become the ideal tools for two-photon in vivo imaging of depression-related active molecules.Therefore,it is urgent to develop ideal organic small molecular two-photon fluorescence probes to accurately detect depression-related biologically active molecules in neurons of living brain and further explore related signaling pathways by two-photon fluorescence imaging technology.Based on the above reasons,this dissertation aims at the bottleneck problem of detecting depression-related biologically active molecules in living brain neurons,and develops a series of stable,biocompatible and fast-responding two-photon fluorescent probes.High sensitivity and specificity of two-photon fluorescence imaging of reactive oxygen species?hydroxyl radicals,superoxide anion radicals?,neurotransmitter hydrolase?acetylcholinesterase?and metal ions(Zn²⁺)in neurons of living brain were realized.The changes of those depression related biologically active molecules in vivo were systematically studied,and the related signaling pathways involved by the active molecules were preliminarily explored.This work provides important information and ideal imaging probes for revealing the molecular mechanism of occurrence and development during depression.The main results of this dissertation were shown as follows:1.We fabricated a two-photon fluorescence probe?MCYN?for real-time visualization of AChE with excellent sensitivity and selectivity.AChE can specifically recognize and cleave the carbamic acid ester bond in MCYN,and MCYN emits bright fluorescence at 560 nm by two-photon excitation at 800 nm.By utilizing MCYN to monitor AChE,we discovered a significant increase in AChE activity in the brains of mice with depression phenotypes.Notably,with the assistance of a two-photon fluorescence imaging probe of the superoxide anion radical(O₂^·-),in vivo visualization for the first time revealed the positive correlation between AChE and O₂^·-levels associated with depressive behaviors.This finding suggests that oxidative stress may induce AChE overactivation,leading to depression-related behaviors.This work provides a new and rewarding perspective to elucidate the role of oxidative stress regulating AChE in the pathology of depression.2.We created a two-photon fluorescence probe MD-B with high selectivity and sensitivity for imaging·OH in mouse brain based on intramolecular charge transfer?ICT?.This probe achieves exceptional selectivity towards·OH via the one-electron oxidation of 3-methyl-pyrazolone as a new specific recognition site.Using two-photon imaging technology,MD-B can monitor the burst of·OH in human astrocytes stimulated by high concentration of glutamate.With the help of the lipophilicity trifluoromethyl group,MD-B could traverse the blood brain barrier?BBB?and map·OH in mouse brain,thereby revealing that the increased·OH is positively correlated with the severity of depression phenotypes.Proteomic analysis further demonstrated that the excess·OH could lead to inactivate deacetylase SIRT1,leading to the occurrence and development of depression phenotypes.This work can help researchers to fully understand the molecular mechanism of depression and offer crucial information for antidepressant treatments.3.We generated a two-photon fluorescent probe for simultaneous detection of Zn²⁺and O₂^·-in the brains of mice with depression.Encountering Zn²⁺,the probe evoked bright fluorescence at560 nm upon excitation at 800 nm?TP?or 400 nm?OP?.Meanwhile,the fluorescence at 480 nm was increased in the presence of O₂^·-based on the same excitation.With the dual distinguishable fluorescence signal,the probe realized the synchronous detection of Zn²⁺and O₂^·-.The probe also possessed high selectivity for Zn²⁺or O₂^·-.The cell imaging showed that Zn²⁺and O₂^·-simultaneously increased under oxidative stress induced by high concentration of glutamate.Notably,in vivo imaging for the first time synchronously revealed the decreased Zn²⁺and the increased O₂^·-in brains of mice with depression-like behaviors.With the help of a fluorescence probe for SIRT1,we revealed that the burst of O₂^·-could cause oxidative damage to SIRT1 which would release Zn²⁺,leading the occurrence and development of depression.This work provides a new strategy for diagnosis and treatment of depression.4.We developed a two-photon fluorescent probe for quantitative detection of·OH in mitochondria with high selectivity and sensitivity.Upon excitation at 800 nm?TP?or 400 nm?OP?,the probe emitted slight fluorescence at 450 nm.In the presence of·OH,the fluorescence at 450nm decreased,and the fluorescence at 560 nm is significantly enhanced,realizing the ratio detection of·OH.By introducing a lipid-soluble triphenylphosphine structure with positive charge,the probe can accurately aggregate into mitochondria and detect the changes of·OH.Co-localization experiments showed that the probe possessed excellent ability of mitochondrial localization.Notably,the probe can quantitatively visualize·OH in mitochondria of living mouse brain.In addition,the SIRT3 Elisa kit analysis further revealed that excessive·OH produced in mitochondria during oxidative stress may aggravate depression by inhibiting the activity of SIRT3.This work can help us to further understand the ROS-mediated molecular mechanism of depression and offer crucial information for antidepressant treatments.