Simultaneous Fluorescence Imaging Of Two Biologically Active Molecules In The Brains Of Mice With Depression

Depression is a kind of mental illness that does great harm to human beings.It has the characteristics of high morbidity,high mortality and high disability rate,which causes heavy burden to families and society.However,there is still a lack of effective methods to diagnose and treat depression,mainly due to the lack of understanding of the pathogenesis of depression.Bioactive molecules,such as reactive oxygen species?ROS?,metal ions,H⁺and reducing substances,play an important role in regulating the balance of intracellular redox system and the transmission of neurotransmitters,which are closely related to the occurrence and development of diseases.Interactions of active molecules and relationships in concentration variation form molecule communication.For example,zinc has been linked to a variety of brain diseases such as Alzheimer's disease and depression.H⁺can regulate the neurons'function by regulating various voltage-gated and ligand-gated ion channels,such as N-methyl-D-aspartate?NMDA?receptor.Moreover,Zn²⁺and H⁺are both regulatory binding ions of the NMDA receptor,which are closely related to the activity of the NMDA receptor.Therefore,research on the synergistic changes of Zn²⁺and H⁺levels will promote the understanding and treatment of depression.which is closely correlated with the occurrence and development of major diseases.Cysteine?Cys?is involved in the synthesis of the antioxidant glutathione?GSH?and plays a key role in protecting cells from oxidative damage.Superoxide anion radical(O₂^·-)is the precursor of other ROS.The excessive accumulation of O₂^·-in the body induces apoptosis and causes many diseases.Therefore,it is of great significance for the diagnosis and treatment of depression to simultaneously monitor the changes of Cys and O₂^·-levels and explore the relative levels of reduction and oxidation states in cells.Fluorescence imaging technology has the advantages of high spatial,temporal resolution and real-time in-situ imaging,which has become a robust method to investigate biological events in living cells and in vivo.In recent years,fluorescence probes for the detection of ROS,metal ions,H⁺and reducible substances in living cells and in vivo have been widely reported.However,due to the complexity of the brain's chemical composition and the existence of blood-brain barrier,there are relatively few specific probes to monitor the synergistic changes of active substance concentration in the living mouse brain.Therefore,it is urgent to develop a new fluorescent probe to monitor the synergistic changes of two active molecules in living brain tissues in real time,and to further explore the relationship between active molecules and depression,is of great significance for the early diagnosis of depression patients.In order to solve the problem of simultaneous imaging of two active small molecules in the brain of living mice,we carried out the following two aspects of work:1.We designed and synthesized a fluorescent probe DNP for simultaneous imaging of Zn²⁺and H⁺in the living brain.DNP was covalently linked by coumarin 2,2'-dimethylpyridinamine?DPA?as Zn²⁺indicator,naphthalene fluorescein as H⁺indicator,and then connected by piperazine with amide bond.DPA quenches the coumarin fluorescence through photoinduced electron transfer?PET?.Encountering Zn²⁺,PET was blocked,and bright blue fluorescence was generated at 460 nm after excitation at 390nm.At the same time,by adding H⁺,the naphthalene fluorescein group in the probe changed from an open quinone form to a closed spironolactone structure,so that under 610nm excitation,the red fluorescence at 680 nm was reduced.Using the blue/red dual fluorescence signal of DNP,we observed a simultaneous increase in Zn²⁺and H⁺in PC12 cells under oxidative stress.Notably,the first in-vivo imaging showed a decrease in both Zn²⁺and pH in the brains of mice with depression-like behavior.Further results suggest that NMDA receptors may be the cause of simultaneous fluctuations in Zn²⁺and H⁺during depression.In conclusion,this work provides strong direct evidence for revealing the relationship between Zn²⁺,H⁺and depression,thereby deepening our understanding of the pathogenesis of depression.2.A fluorescent probe COP was designed and synthesized for the simultaneous imaging of Cys and O₂^·-in the living brain.COP uses phenyl chlorothionocarbonate as the specific recognition group for Cys,and caffeic acid is the specific recognition group for O₂^·-.Due to the strong electron absorption effect of carbon-sulfur double bond in the probe,the electron feeding ability of amino group is reduced,resulting in the weakened electron absorption effect of partial cyanine and reduced fluorescence.After the reaction with Cys,the carbon-sulfur double bond was destroyed,and the electron supply and absorption effect of partial cyanine was restored,which enhanced the fluorescence.At the same time,when the probe reacts with O₂^·-,the electron donating structure of caffeic acid residue catechol changes into the electron absorbing structure of o-benzoquinone,and the charge distribution of the probe molecule changes,resulting in the fluorescence change.The probe has the characteristics of fast response,strong selectivity and high sensitivity.The probe COP was expected to achieve rapid,real-time,in-situ imaging detection of Cys and O₂^·-in vivo,which would be of great significance to explore the relationship between Cys and O₂^·-and depression.