Observation of Acetylcholinesterase in Stress-induced Depression

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Observation of Acetylcholinesterase in Stress-induced Depression Phenotypes by Two-photon Fluorescence Imaging in the Mouse Brain Xin Wang, Ping Li, Qi Ding, Chuanchen Wu, Wen Zhang, and Bo Tang J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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Journal of the American Chemical Society

Observation of Acetylcholinesterase in Stress-induced Depression Phenotypes by Two-photon Fluorescence Imaging in the Mouse Brain Xin Wang, Ping Li,* Qi Ding, Chuanchen Wu, Wen Zhang, and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Institutes of Biomedical Sciences, Shandong Normal University, Jinan 250014, People’s Republic of China. KEYWORDS Two-photon fluorescence imaging; depression; acetylcholinesterase; oxidative stress; brain

ABSTRACT: Oxidative stress in depression is a prime cause of neurotransmitter metabolism dysfunction in the brain. Acetylcholinesterase (AChE), a key hydrolase in the cholinergic system, directly determines the degradation of neurotransmitters. However, due to the complexity of the brain and lack of appropriate in situ imaging tools, the mechanism underlying the changes in AChE activity in depression remains unclear. Hence, we generated 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 depressionrelated behaviors. This work provides a new and rewarding perspective to elucidate the role of oxidative stress regulating AChE in the pathology of depression.

Introduction Depression is the most common mood disorder and contributes substantially to the burden of disease and disability.1 Unfortunately, the current knowledge regarding the pathophysiology of depression remains rudimentary.2-3 A growing body of work indicates that depression is characterized by oxidative damage to relevant biological components.4-6 Above all, oxidative stress in depression is supposed to be the primary cause of neurotransmitter metabolism dysfunction in the brain.7 Acetylcholinesterase (AChE) is the key hydrolase in the cholinergic system, cleaving acetylcholine (ACh) and rendering ACh inactive.8 Apparently, aberrant fluctuation of AChE directly influences the metabolism of ACh, thereby disrupting neurotransmission in the brain. Consequently, emotions and behaviors are inevitably affected,9 and worse yet, depressive disorders may occur. Therefore, defining these changes in AChE activity is essential for better understanding the molecular mechanism underlying depression. However, the changes in AChE activity and the regulation of AChE dynamics involved in depression-like behaviors remain unclear. For instance, the Soreq group found that stress could facilitate a three-fold increase in AChE activity over a long period.10-11 Similarly, mice overexpressing AChE exhibited heightened depression-like behavior.12 In contrast, other studies have shown that inhibitors of AChE enhance the depressive phenotype.13-14

To solve this puzzle, a reliable method for real-time monitoring of AChE in vivo is essential. Due to its excellent compatibility, durability and high spatiotemporal resolution, fluorescence imaging is becoming an ideal approach for investigating biological molecules.15-16 Recently, some specific fluorescent probes have been developed to study AChE in vitro.17-21 However, additional substances are needed in these methods, rendering unsuitable and not sufficiently precise for direct detection of AChE in vivo, especially in live brains. Twophoton (TP) fluorescence imaging technology can achieve deeper tissue penetration, lower background fluorescence, higher spatiotemporal resolution and less specimen photodamage than one-photon (OP) fluorescence imaging, which should be more suitable for imaging of bioactive molecules in live systems.22-25 In particular, due to their stable properties, low cytotoxicity and blood brain barrier (BBB) permeability, molecular fluorescent probes are the best choice for in vivo TP imaging in brains.26-27 However, to date, there has been no report on real-time visualization of the fluctuations in AChE activity in live animals, especially using TP imaging. Therefore, development of a small molecule TP fluorescent probe for detecting AChE activity in the brains of mice with depression-like behaviors is urgently needed. To address this issue, we created a TP fluorescent probe (2-(4-((dimethylcarbamoyl)oxy)styryl)-1-ethyl-3,3-

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dimethyl-3H-indol-1-ium, MCYN) for in situ detection of AChE activity, which was inspired by neostigmine, an effective AChE inhibitor. 28-30 In the structure of neostigmine, the carbamate can bind covalently to a serine residue in the active site of AChE, and then cause carbamoylation of the active site serine in AChE. This specific binding leads to the deactivation of AChE, which is employed to be the recognition principle to AChE in our probe. In this design, AChE can specifically recognize the dimethyl carbamate of MCYN; in the meantime, MCYN interacts with the hydroxyl group of the serine at the hydrolytic center of AChE via covalent bonds. Therefore, dimethyl carbamate was chosen as the recognition group in this probe. Meanwhile, merocyanine was selected as the fluorophore due to its extraordinary optical properties. In particular, the nitrogen cation (N+) of merocyanine can bind

to the anionic site of the hydrolytic center of AChE, thus improving the efficiency of hydrolysis by decreasing the distance between MCYN and AChE. The carbonyl group in MCYN is directly attached to the oxygen atom and weakens the push-pull electronic effect of merocyanine; therefore, MCYN exhibits weak fluorescence.31-32 When the hydrolytic center of AChE recognizes the dimethyl carbamate in MCYN, the ester bond is cleaved, and MCYN is transformed to MCYO. In MCYO, the push-pull electron effect heightens, resulting in fluorescence enhancement. Based on the above idea, we synthesized MCYN and examined the response of MCYN to AChE. MCYN was also used in the brains of mice with depression phenotypes to image the activity of AChE. Furthermore, in association with a TP fluorescence probe of O2•−, we explored modulation of oxidative stress on AChE.

Scheme 1. Synthesis and recognition mechanism of MCYN. (A) Synthesis steps of MCYN: 1. Reflux in toluene for 16 h. 2. Reflux in ethanol for 48 h. 3. Stir at room temperature for 3 days. (B) Recognition mechanism of MCYN and structure of MCYO.

Experimental Section Apparatus and Reagents Details regarding the apparatus and reagents used in this study can be found in the Supporting Information. Synthesis Compound a was synthesized according to our previous report.33 Compound MCYO: Compound a (0.301 g, 1 mmol) and Phydroxybenzaldehyde (0.122 g, 1 mmol) were dissolved in ethanol, and the mixture was stirred under reflux at 80 °C. After 48 h, the mixture was concentrated under vacuum, and then an orange product was obtained. Compound MCYN: Compound MCYO (0.393 g, 1 mmol) and cesium carbonate (0.326 g, 1 mmol) were dissolved in dichloromethane, and the mixture was stirred under

nitrogen at room temperature. After 30 min, dimethylcarbamoyl chloride was added to the mixture (200 μL). Then, the mixture was stirred for 3 days, and additional dimethylcarbamoyl chloride was added twice each day (200 μL). The mixture was purified by preparative thin layer chromatography on silica gel GF254 with dichloromethane/methanol (10:1) as the eluent, and a yellow product was obtained. 1H NMR (400 MHz, d4CH3OH) δ (ppm): δ 8.50 (d, 1H), 8.14 (d, 2H), 7.89 (t, 1H), 7.79 (t, 1H), 7.65 (m, 3H), 7.36 (d, 2H), 4.76 (q, 2H), 3.15 (s, 3H), 3.03 (s, 3H), 1.87 (s, 6H), 1.60 (t, 3H); 13C NMR (100 MHz, d4-CH3OH) δ(ppm): 182.06, 155.80, 154.35, 153.96, 153.90, 144.00, 140.42, 131.58, 131.43, 129.79, 129.29, 122.81, 122.53, 122.17, 114.76, 111.50, 52.71, 42.34, 35.56, 35.43, 25.02, 22.33, 12.71. HRMS (ESI) m/z calcd. for C23H27N2O2+ [M]+: 363.2067, found: 363. 2147. After reaction with excess AChE, MCYN transformed to MCYO.


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Journal of the American Chemical Society MCYO was purified by preparative thin layer chromatography on silica gel GF254 with dichloromethane/methanol (10:1) as the eluent, and an orange product was obtained. 1H NMR (400 MHz, d4CH3OH) δ (ppm): δ 8.22 (d, 1H), 7.84 (d, 2H), 7.58 (d, 1H), 7.49 (m, 2H), 7.40 (t, 1H), 7.05 (d, 1H), 6.69(d,2H), 4.41 (q, 2H), 1.73 (s, 6H), 1.43 (t, 3H); 13C NMR (100 MHz, d4CH3OH) δ(ppm): 180.10, 174.93, 156.25, 144.12, 142.49, 136.37, 130.36, 130.02, 128.87, 125.19, 123.97, 120.81, 117.06, 114.10, 104.80, 52.48, 41.84, 27.47, 20.42, 13.45. HRMS (ESI) m/z calcd. for C20H22NO+ [M]+: 292.1696, found: 292.1745. Results and Discussion Photophysical Properties and Selectivity of MCYN The synthesis and recognition mechanism of MCYN are shown in Scheme 1. As depicted in Figure S1, the absorption spectrum of MCYN exhibited a band at approximately 520 nm. In the presence of 20.0 U/mL AChE, the intensity of this absorption band increased considerably. We next examined the fluorescence properties of MCYN under simulated physiological conditions (40 mM PBS, pH 7.4). As shown in Figure 1A and Figure S4-S6, upon excitation at 520 nm (OP) or 800 nm (TP), MCYN emitted a faint OP fluorescence at

560 nm. In the presence of 20.0 U/mL AChE, a distinguishable fluorescence at 560 nm was observed. The fluorescence quantum yield Фf increased from 0.04 to 0.19, and two-photon absorption cross-section from 3 GM to 21 GM. These changes in the photophysical properties confirm that the carbonyl group of MCYN can tune the charge redistribution, causing fluorescence enhancement. To further confirm the changes in fluorescence are ascribed to the transformation from MCYN to MCYO, we examined the photophysical properties of the pure MCYO. The maximal absorption peak of MCYO is also located at 520 nm (Figure S2). Its OP fluorescence emission peak is centered at 560 nm (Figure S3) and the fluorescence quantum yield is 0.21, which is basically accordant with the probe in the presence of AChE. These results demonstrate that changes of fluorescence in the proposed method can be based on the transformation from MCYN to MCYO. Furthermore, according to a computational study performed at the B3LYP/6-31G (d, p) level, the charge on the oxygen atom decreased from -0.554 to -0.686 (Figure S7). This indicates that the push-pull electronic effect in MCYO is stronger than that in MCYN, resulting in marked fluorescence intensity. All these data reveal that MCYN can sense changes in AChE activity.

Figure 1. Photophysical properties and selectivity of MCYN. (A) OP emission spectra of MCYN (20.0 μM) before (black line) and after addition (blue line) of AChE (20.0 U/mL) upon excitation at 520 nm. (B) Changes in the fluorescence emission spectra of MCYN (20.0 μM) in the presence of various concentrations of AChE (0-20.0 U/mL). (C) Fluorescence responses of MCYN (20.0 μM) to different competing species: Na+ (1.0 mM), K+ (1.0 mM), Zn2+ (1.0 mM), Mg2+ (1.0 mM), Ca2+ (1.0 mM), glycine (100.0 μM), alanine (100.0 μM), cysteine (100.0 μM), lysine (100.0 μM), arginine (100.0 μM), butyrylcholine esterase (10.0 U/mL), pepsin (10.0 U/mL), BSA (10.0 mg/mL) and AChE (10.0 U/mL). (D) Inhibitory activity of MCYN against neostigmine (10-9-10-3 M). All of the spectra were acquired in 40 mM PBS (pH 7.4) at λex = 520 nm (OP) and λem = 560 nm (OP) by an FLS-980 Edinburgh fluorescence spectrometer. Similar results were observed in five independent experiments. The data are expressed as the mean ± S.D.

Next, we tested the AChE activity dependence of the fluorescence intensity of MCYN. Figure 1B showed that the fluorescence of the probe recovered stepwise, which was

dependent on changes in AChE activity. The linear regression equation was F = 99.58[AChE](U/mL) + 1192.05, with a correlation coefficient of 0.990 in the range of 1.0 -



20.0 U/mL. The detection limit was calculated to be 0.36 U/mL, which is lower than the physiological activity (approximately 5.0 U/mL). The kinetic parameters, such as the Michaelis constant (Km) and maximum initial reaction rate (Vmax), for the enzymatic cleavage reaction of MCYN were found to be 141 μM and 3.11 μM/min (Figure S8), respectively. These results indicate that MCYN could appropriately function in the presence of AChE under physiological conditions. To evaluate the specificity of MCYN for AChE, we investigated the response of MCYN in competition assays with other biological molecules, such as some hydrolases (butyrylcholine esterase, pepsin), BSA, amino acids (glycine, alanine, cysteine, lysine, and arginine), metal ions (Na+, K+, Zn2+, Mg2+, and Ca2+), glucose, and formulations of various pH values. As shown in Figure 1C, the fluorescence signals demonstrated high selectivity for AChE. In addition, varying the pH from 6.5 to 8.5 had little effect on the fluorescence of MCYN (Figure S9). In the meantime, distinct fluorescence enhancement of MCYN after the reaction with AChE was observed at approximately pH 7.4, as the enzyme usually functions well under normal physiological conditions. Time course studies showed that the fluorescence intensity plateaued in approximately 30 min and remained virtually unchanged for another 30 min (Figure S10). Taken together, these results confirm that MCYN is suitable for the highly sensitive detection of AChE in complex biological contexts with excellent selectivity. Based on the recognition capability of MCYN toward AChE, we examined whether MCYN could be used to screen AChE inhibitors.34 For this study, we selected neostigmine, an effective inhibitor that is currently employed clinically.

In the inhibition assay (Figure 1D), solutions of AChE and the inhibitor at varying concentrations were incubated at 25℃ for 15 min, and then MCYN was added. Subsequently, the mixtures were allowed to stand for 15 min to make hydrolysis occur. The inhibitory ability of an inhibitor is described by the IC50 value. Neostigmine was found to inhibit AChE at an IC50 value of 11.3 nM, which is approximately consistent with the previous report.35 Thus, the MCYN described herein can be used as a convenient tool for screening AChE inhibitors. AChE Imaging in Live Cells with MCYN To determine whether MCYN could function in live systems, we initially utilized MCYN to visualize AChE in live neural cells. Previous reports have suggested that the change in AChE activity could be causally related to apoptosis.36-37 For this reason, we planned to imaging AChE activity in PC12 cells during apoptosis using MCYN. To elicit apoptosis, we selected phorbol-12-myristate-13-acetate (PMA) inducing cell apoptosis by activating protein kinase C (PKC).38 PC12 cells were pretreated with 1.0 μg/mL PMA for 60 min. To confirm apoptosis caused by PMA, these cells were analyzed by the Annexin V-FITC Apoptosis Detection Kit via flow cytometry (Figure S11). The images of PC12 cells treated with PMA (Figure 2C) showed much brighter fluorescence (approximately 2.5-fold) than the untreated cells (Figure 2A). To confirm the enhancement of fluorescence is ascribed to the increase in AChE activity, we used inhibitor neostigmine to incubate PC12 cells. As expected, weak fluorescence was observed within these cells (Figure 2B). The results suggest that the increased fluorescence could be due to the overactivity of AChE upon apoptosis.

Figure 2. TP fluorescence images of PC12 cells. (A) PC12 cells pretreated with MCYN (20.0 μM) for 20 min. (B) PC12 cells pretreated with neostigmine (10.0 μM) for 30 min, followed by preincubation with 20 μM MCYN. (C) PC12 cells pretreated with 1.0 μg/ mL PMA for 60 min, followed by incubation with MCYN (20.0 μM) for an additional 20 min. (D) PC12 cells treated with small interfering RNA (siRNA 5'-CCGUGUUGGUAUCUAUGAATT-3':) for silencing of the AChE gene, followed by incubation with MCYN (20.0 μM). (E) Relative fluorescence intensities of all PC12 cells in group A-D. The data are expressed as the mean ± S.D. ***P < 0.001 for C compared with A. Insert: Western blot of AChE in group A-D. Similar results were obtained in three independent experiments. Images were acquired using 800 nm for TP excitation. TP fluorescence emission window: 480-650 nm. Scale bar = 20 μm.

To further demonstrate the selective fluorescence response of MCYN to intracellular AChE, we performed AChE gene silencing in PC12 cells using small interfering RNA (siRNA). Successful gene silencing was verified by Western blotting. Compared with the control cells, these cells treated with siRNA exhibited obvious reduction in fluorescence (Figure 2D), confirming that AChE should be responsible for the fluorescence signal. Moreover, the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the cytotoxicity of MCYN (Figure S12). From MTT assay, IC50 was calculated to be 0.53 mM. This means MCYN possesses good biocompatibility at concentrations less than 0.53

mM.39-40 These observations imply that MCYN can selectively and sensitively detect fluctuations in AChE activity in live cells. Previous studies have suggested that high concentrations of glutamate can cause oxidative stress in the neural system.41-43 Therefore, we incubated PC12 cells with 10 mM glutamate to explore how AChE activity varied. As expected, the fluorescence of these cells upon the treatment of glutamate (Figure 3C) intensified significantly compared with that of the control cells (Figure 3A). This result strongly indicated that AChE activity increased under stimulation of high concentration of glutamate. To confirm that the increase in fluorescence intensity came from high


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Journal of the American Chemical Society AChE activity, we added an inhibitor of AChE (Figure 3B), neostigmine, into PC12 cells treated with excess glutamate, and then examined the fluorescence. The cells incubated with neostigmine clearly showed lower fluorescence than cells without neostigmine. Moreover, the PC12 cells

subjected to AChE gene silencing did not exhibit brighter fluorescence, indicating that the activity of AChE dwindled (Figure 3D). These data provide sufficient evidence that AChE activity increases under oxidative stress.

Figure 3. TP fluorescence images of PC12 cells. (A) PC12 cells pretreated with MCYN (20.0 μM) for 20 min. (B) PC12 cells pretreated with neostigmine (10.0 μM) before stimulation with 10 mM glutamate for 24 h, followed by incubation with MCYN (20.0 μM) for an additional 20 min. (C) PC12 cells pretreated with MCYN (20.0 μM) for an additional 20 min after preincubation with 10 mM glutamate for 24 h. (D) AChE gene silencing of PC12 cells treated with 10 mM glutamate for 24 h, followed by incubation with MCYN (20.0 μM). (E) Relative fluorescence intensities of all PC12 cells in panels A-D. The data are expressed as the mean ± S.D. ***P < 0.001 for C compared with A. Similar results were obtained in three independent experiments. Images were captured using 800 nm for TP excitation. TP fluorescence emission window: 480-650 nm. Scale bar = 20 μm.

Figure 4. In situ TP fluorescence imaging in the brains of mice. (A) Stress: mice exposed to 14 consecutive days of chronic-restraint stress. (B) Control: mice not subjected to chronic-restraint stress. (C) Sketch of three different TP fluorescence imaging areas. (D) Relative fluorescence intensities of mice in A and B. The data are expressed as the mean ± S.D. ***P < 0.001 for stress group compared with the control. 7 mice in each group. TP images, at a 20× magnification, of mice labeled with 0.15 mg kg-1 MCYN via intraperitoneal injection. Fluorescence emission window: 480-650 nm. Scale bar = 50 μm.

Imaging of AChE in Brains of Mice Following Stress Motivated by the satisfactory performance of MCYN in cellular imaging applications and good in vivo biocompatibility (Figure S15), we attempted to further investigate the performance of MCYN as a fluorescent probe for small animal bioimaging in vivo, especially in the brain. Current studies suggest that human depression generally results from stress.44 Therefore, we planned to develop the mouse model of depression following chronic stress, and imaging the fluctuation of AChE in the brain. First, we exposed mice to 14 consecutive days of chronic-restraint stress and then chose the mice with depression-like behaviors (Figure S13).45 Subsequently, we measured the levels of AChE in the brains of mice with depression phenotypes. The brains of the mice following stress (Figure 4A) exhibited distinct fluorescence enhancement (approximately 4-fold) compared to that of the control mice

(Figure 4B). This enhanced fluorescence should be attributed to increased AChE activity. These findings demonstrate that elevation of AChE activity in the brains of mice with depression-like behaviors was induced by stress. In the current work, we confirmed at the cellular level that the activity of AChE increases under oxidative stress. As shown in previous studies, the level of superoxide anion (O2•−), as the first reactive oxygen species produced during oxidative stress, directly reflects the degree of oxidative damage.46-47 To explore the role of O2•− in the regulation of AChE activity, we simultaneously measured the flux of O2•− and change on AChE activity in PC12 cells under oxidative stress. We also used caffeic acid ethyl ester (CA, a scavenger of O2•− and a TP fluorescence indicator of O2•−), to reduce O2•− and monitor the associated changes. In these imaging experiments, there were a blue fluorescence signal for O2•−



and a green fluorescence signal for AChE. First, we treated the cells with 10 mM glutamate for 24 h (Figure 5A) and then incubated these cells with both 20 μM MCYN and 10 μM CA. Next, we observed the changes in O2•− levels and AChE activity. The TP fluorescence images showed that the cells stimulated by glutamate exhibited brighter blue and green fluorescence than the control. This means that the increase in AChE activity was accompanied by increased O2•− levels. Interestingly, when CA was added into the cells pretreated with glutamate, green fluorescence was witnessed at distinctly low level. The decrease in fluorescence should be because of reduction in AChE activity. Considering that CA can scavenge O2•−, the decrease in AChE activity was attributed to remission of oxidative stress.

From the above results, we uncovered increases in both the AChE activity and O2•− level inside the nerve cell upon oxidative stress. When we used caffeic acid ethyl ester scavenging O2•− to relieve the oxidative stress, dramatic decrease in AChE activity was observed (Figure 5). These results demonstrate that the O2•− may be a critical node modulate the activity of AChE during oxidative stress. Importantly, we also revealed that the elevation of AChE activity in the brains of mice with depression-like behaviors induced by chronic stress that can trigger oxidative stress (Figure 4), indicating AChE plays a pivotal role in the pathophysiology of depression. Altogether, our findings highlight that the AChE appears to be a bridge to link oxidative stress to depression, providing strong evidence for the hypothetical role of oxidative stress in depression.

Figure 5. Simultaneous imaging of O2•− and AChE. (A) The blue fluorescence signal indicates O2•−, and the green signal indicates AChE. First column (control): control cells without any treatment but pretreated with MCYN (20.0 μM) and CA (10.0 μM) for 20 min. Second column (Glu): PC12 cells treated with 10 mM glutamate for 24 h, followed by incubation with 20.0 μM MCYN and 10.0 μM CA. Third column (Glu+CA): PC12 cells treated with 10 mM glutamate and10.0 μM CA for 24 h and then incubated with 20.0 μM MCYN for an additional 20 min. First row: blue channels. Second row: green channels. Third row: differential interference contrast (DIC) images. (B) Relative fluorescence intensities of all PC12 cells in A. The data are expressed as the mean ± S.D. ***P < 0.001 for control. Similar results were observed in three independent experiments. Images were acquired using 800 nm for TP excitation. TP fluorescence emission windows: blue: 410-460 nm, green: 480-650 nm. Scale bar = 20 μm.

Conclusion In summary, by focusing on the role of oxidative stress in the modulation of AChE activity, we describe a novel TP fluorescent probe for in situ and real-time detection of AChE activity in live cells and in vivo unprecedentedly, especially in brains. This new probe exhibited exclusive selectivity for AChE, eliminating interference from other biologically relevant substances. In situ visualization verified an increase in AChE activity in PC12 cells under oxidative stress. Notably, the probe could be used to successfully monitor AChE levels in the brains of mice following chronic-

restraint stress for the first time by TP fluorescence imaging, revealing a positive correlation between AChE and the depression phenotype. Furthermore, with the aid of a TP fluorescent indicator of O2•−, we also demonstrated that oxidative stress factors, especially O2•−, may be critical nodes that modulate AChE activity, leading to depressionlike symptoms. This work provides a robust tool for elucidating the roles of AChE in the pathophysiology of depression, and also offers crucial information for effective prevention and treatment of depression.

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Journal of the American Chemical Society Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.” Additional experimental data, including synthesis, characterization, photophysical properties, cytotoxicity, and experimental details.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID Bo Tang: 0000-0002-8712-7025

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21535004, 91753111, 21675105, 21475079, 21390411) and the Key Research and Development Program of Shandong Province (2018YFJH0502), National Major Scientific and Technological Special Project for “Significant New Drugs Development” (2017ZX09301030004) and the Natural Science Foundation of Shandong Province of China (ZR2017ZC0225).

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Observation of Acetylcholinesterase in Stress-induced Depression

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