Does glutathione improve behavior in depressed mice after stroke?

 Post-stroke depression (PSD) is a common complication of stroke, with approximately one-third of stroke survivors suffering from PSD, which can lead to slower recovery and higher rates of disability [1]. Currently, tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs) are the mainstay of treatment for PSD[2-3] , but the pathogenesis of PSD is not fully understood. PSD and major depression disorder (MDD) patients share some similarities, with symptoms such as depressed mood, unstable mood, and loss of interest, and reduced 5-hydroxytryptamine, dopamine, and norepinephrine are the most important pathologic changes in PSD and MDD patients[5] , but the lack of 5-hydroxytryptamine, dopamine, and norepinephrine is a major pathological change in PSD[7] . - 7], but there is a lack of research on the characteristics of brain lesions in both PSD and MDD animal models. Exploring the common pathogenic mechanism of PSD and MDD can help to study the common changes in the development of depression in different scenarios, and provide in-depth understanding of the pathogenesis of depression, as well as provide a basis for finding potential therapeutic targets.

 

The medial prefrontal cortex (mPFC) is one of the core brain regions for depression research, and previous studies have suggested that α-amino-3-hydroxy-5-methyl-4-isohmzopropionic acid receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR), and prefrontal and cingulate cortex (NMDAR) have the same function as the medial prefrontal cortex (MFC), and the prefrontal and cingulate cortex (NMDAR) have the same function as the medial prefrontal cortex. receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR), and frontal limbic circuits including prefrontal lobe and cingulate cortex are potentially associated with mPFC and depression[8-10] . In the present study, we established a PSD mouse model[11] and a chronic social defeat depression (CSDS) mouse model[12] to simulate PSD and MDD, and analyzed the changes of neurotransmitters, amino acids and other metabolites in the mPFC brain area of mice in each group through neurotransmitter-targeted metabolism, and explored the exogenous replenishment of key metabolites, such as glutathione (NMDAR), glutathione (NMDAR) and cingulate cortex, in the brain area of mPFC in each group[13] . We also investigated whether the exogenous key metabolite glutathione (GSH) could improve the depressive behaviors of mice, which would provide new insights for further elucidation of the pathogenesis of depression and the development of therapeutic methods.

 

1 Materials and Methods

1 . 1 Laboratory animals

The experimental animals used in this study were 8~10 weeks old male C57BL/6J mice with body mass of 22~25 g and 3~6 months old male CD-1 mice with body mass of 45~55 g, which were purchased from Chongqing Lepit Technology Company. All mice were housed in the SPF-grade animal room of the Second Affiliated Hospital of the Army Medical University at room temperature (22±2 ℃), with sufficient diet and 12 h/12 h of light/darkness. The experimental mice were numbered one by one, and then randomly divided into three groups, namely, the no-treatment control (SHAM) group, the PSD group, and the CSDS group using the random number table method, with 20 male C57BL/6J mice in each group (8~10 weeks), and 20 male C57BL/6J mice in each group (8~10 weeks).  Each group consisted of 20 male C57BL/6J mice (8-10 weeks old). All animal experiments were conducted in accordance with the Animal Experimentation Law and the Ethical Review Guidelines for Medical Animal Welfare of the Army Military Medical University.

 

1 . 2 Reagents and Instruments

Anti-glutathione peroxidase 4 (GPX4) antibody (Abcam, UK), mouse anti-GFAP antibody (Abcam, UK), goat anti-CD31 antibody (Abcam, UK), mouse anti-MAP2 antibody (Abcam, UK), goat anti-rabbit antibody (Invitrogen, USA), donkey anti-MAP2 antibody (Invitrogen, UK), rabbit anti-MAP2 antibody (Invitrogen, USA), donkey anti-MAP2 antibody (Invitrogen, UK), donkey anti-MAP2 antibody (Invitrogen, USA) Invitrogen), donkey anti-goat antibody (Invitrogen, USA), donkey anti-rabbit antibody (Invitrogen, USA), donkey anti-mouse antibody (Invitrogen, USA); ACQUITRON, ACQUITRON, ACQUITRON, ACQUITRON, ACQUITRON, ACQUITRON, ACQUITRON.    ACQUITY Premier Ultra High Performance Liquid Chromatograph (Waters, USA), Frozen Slicer (Leica, Germany), Confocal Microscope (Olympus, Japan).

 

1 . 3 Animal modeling and administration

1 . 3 . 1 Establishment of the PSD mouse model     

The left side of the mPFC was injected with ET-1. After anesthetizing the mice with isoflurane, the mice were clipped and fixed in a stereotaxic device, and the cranial vault was completely exposed after the scalp was cut open with ophthalmic scissors, and the cranial plane was adjusted according to the Bregma point and Lambda point. The cranial drill was used to remove the skull and expose the meninges, and a glass electrode was used to aspirate ET-1 solution (2 μg/μL) to localize the mPFC brain area at anterior fontanel site 1: 2.0 mm anterior to fontanel, 0.5 mm to the left of the middle suture, and a depth of 2.4 mm under the skull; anterior fontanel site 2: 1.5 mm anterior to fontanel, 0.5 mm to the left of the middle suture, and a depth of 2.4 mm under the skull. 2. 4 mm; fontanel site 2: 1.5 mm anterior to fontanel, 0.5 mm to the left of the middle suture, subcranial depth 2.4 mm. 5 mm, subcranial depth 2.6 mm; all injections were left-sided. 1 μL of ET-1 solution was injected into the left side of the mPFC at a rate of 100 nL/min. After the injection, the needle was stopped for 10 min and then slowly withdrawn, and erythromycin ophthalmic ointment was applied after the suture was completed to prevent infection.

 

1 . 3 . 2 Establishment of the CSDS mouse model     

CD-1 mice with an aggressive tendency were first selected, i.e., non-modeled C57BL/6J mice were placed in the cage of CD-1 mice for 3 d before the formal experiment. CD-1 mice were selected if they met the following conditions: the first attack on a C57BL/6J mouse was within 60 s, and they attacked more than 5 times, and each attack lasted for more than 5 s. For the formal experiment, CD-1 attack mice and modeled C57BL/6J mice were kept in the same cage for 10 d, and the two mice were separated by a transparent partition every day. In the formal experiment, CD-1 attacking mice and model C57BL/6J mice were kept in the same cage for 10 d. The two mice were separated by a transparent partition, and the C57BL/6J mice were placed on the side of the CD-1 mice for 5 min every day, and then returned to the other side of the partition to maintain the sensory stimulation, and then behavioral assays were conducted on the C57BL/6J mice after 10 d. The C57BL/6J mice were then tested for behavior. The behavior of C57BL/6J mice was examined 10 d later.

 

1 . 3 . 3 Mode of administration        

GSH was dissolved in saline into a solution of 25 mg/mL and injected intraperitoneally at a dose of 100 mg/(kg - d) for 1 week in the experimental group of PSD and CSDS mice, while the control group was injected intraperitoneally with the same dose of saline (Vehicle) daily in the PSD and CSDS mice. 1 . 4 Behavioral tests

 

1 . 4 . 1 Absentee field experiments       

In a quiet environment, the mice were placed in the center of the bottom of the absconding box (box height 30 cm, bottom length 50 cm, width 50 cm, the bottom was divided into 64 squares), while video recording and timing were carried out, and the observation duration was 5 min. The observation indexes were: total distance, average speed, time to enter the central area, time to enter the peripheral area, latency, resting time, total time, etc. Finally, the data were analyzed by Super Animal Behavior Software.

 

1 . 4 . 2 Elevated Cross Experiment       

The mice were placed into the center grid of the elevated cross maze in a quiet environment, and their activities were recorded within 5 min. The observation indexes were: total distance traveled, number of entries into the open arm, stopping time of the open arm, number of entries into the closed arm, stopping time of the closed arm, total time, etc. The data were analyzed by Super Animal Behavior Software. Finally, the data were analyzed by Super Animal Behavior Software.

 

1 . 4. 3 Tail-hanging experiments       

The mice were suspended upside down in a 20 cm × 25 cm × 25 cm box with their heads about 5 cm from the bottom of the box, and the suspension time was 6 min, with the first 2 min as the acclimatization period, and the last 4 min as the time the mice remained stationary. The experiment was conducted at a fixed time every day. Data were collected to compare the differences between different groups.

 

1 . 4 . 4 Sugar water preference experiment       

The sugar-water preference test is a useful indicator of pleasure loss in depressed mice. The mice were housed individually, and during the acclimatization period, two bottles of 1% sucrose water were placed in the cage for 24 h, and then one of the bottles was replaced with pure water for 24 h. After acclimatization, the mice were deprived of both water and food for 24 h, and then the mice were placed in cages with one bottle of 100 mL of 100% sucrose water and one bottle of 100 mL of pure water for 24 h. During the period of acclimatization, the position of the bottles of water was randomly changed to avoid positional preference, and their consumption of sugar-water was recorded at the end of the 24 h period. The consumption of sugar water was recorded 24 h later. The formula for calculating sugar water consumption was (sugar water consumption/total fluid intake) × 100%.

 

1 . 5 Sample collection and neurotransmitter-targeted metabolome sequencing

Three groups of mice were anesthetized with isoflurane, then perfused with 50 mL of PBS through the heart, and the left side of the mPFC brain area was removed from the brain tissue. Nine mice were taken from each group, and since the mass of mPFC from a single mouse was too small to meet the detection requirements, the mPFC from every three mice were mixed together for targeted metabolome assay. The metabolites were detected using an ACQUITY Premier (Waters) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC HSS T3 (100 mm×2.1 mm, 1.5 mm, 1.5 mm, 1.5 mm, 1.5 mm, 1.5 mm). 1 mm, 1.8 μm) liquid chromatograph. The target compounds were separated on a Waters ACQUITY UPLC HSS T3 (100 mm×2.1 mm, 1.8 μm) liquid chromatographic column. The liquid chromatographic phase A was 0.1% formic acid and 1 mmol/L ammonium acetate solution, and the phase B was acetonitrile. The column temperature was set at 40 ℃, the sample plate was set at 10 ℃, and the injection volume was 2 μL. The mass spectrometry analysis was performed in multiple reaction monitoring (MRM) mode. The characterization and quantification of the target compounds were performed by SCIEX Analyst Work Station Software (Version 1.6.3) and DATA DRIVER. 6.3) and DATA DRIVEN FLOW (Version 1.0.1). 0.1).

 

1 . 6 Selection of Potential Differential Metabolites and Analysis of Pathway Enrichment

The metabolite results were analyzed by principle component analysis (PCA) and correlation analysis to compare the differences between groups, and quantitative analysis was performed to screen the differential metabolites, in which P<0.05 was considered as sufficient for differential metabolites. P<0.05 was regarded as the metabolite that met the criteria for differential metabolism, and the metabolic pathway enrichment of differential metabolites was carried out by using the Kyoto encyclopedia of genes and genomes (KEGG) database.

 

1 . 7 GSH/GSSG, MDA assays

According to the instruction manual of Biyuntian GSH/GSSG and MDA Detection Kit, mPFC samples were extracted from the brain tissues of mice in PSD, CSDS and SHAM groups, homogenized, and the concentrations of GSH/GSSG and MDA were determined by colorimetric method.

 

1 . 8 GPX4 immunofluorescence staining and semi-quantitative analysis of the

Fresh brain tissue was extracted and frozen sections of 30 μm thickness were prepared, washed with PBS and incubated with 5% goat serum and 0.3% Triton×100 for 1 h at room temperature. The primary antibody (rabbit anti-GPX4, Abcam, 1:200) was incubated at 4 ℃ overnight, and the sections were washed with PBS on the second day. The secondary antibody (goat anti-rabbit, Invitrogen, 1:1,000) was incubated for 1 h at room temperature and protected from light, and the nuclei were stained with DAPI for 10 min, and then the sections were washed again with PBS and sealed, and then fluorescence images were obtained by observation with a confocal microscope. Three to five randomly selected areas in the brain region of each mouse mPFC were observed, and the GPX4 and DAPI positive cells were counted by using Cell Counter in the Image J software plug-in, the DAPI number was regarded as the total number of cells in the area, and the GPX4 number/DAPI number was the proportion of GPX4 positive cells in the area, and the average value was taken as the result of analyzing the results of the three to five random areas in each mouse. The average of 3~5 randomized regions of each mouse was analyzed as the proportion of GPX4-positive cells in the mPFC brain region of that mouse.

 

1 . 9 Statistical analysis

Statistical analysis was performed using GraphPad Prism 8, count data were expressed as x- ±s, data were normally distributed and tested for chi-square, one-way ANOVA was used to assess the differences between the groups, and non-parametric tests were used if the variance was not homogeneous, and P<0.05 was considered statistically significant. P<0.05 was regarded as statistically significant.

 

2 Results

2.1 Medial prefrontal cortex injection of ET-1 has the same effect as CSDS in inducing depressive behavior in mice.

After modeling (Figure 1A, B), the mice were subjected to behavioral tests, such as the open field test and the elevated cross test to evaluate the anxiety level of the mice, the sugar-water preference test to detect the lack of pleasure, and the tail-hanging test to detect the desperation behavior of the mice. The results showed that compared with the SHAM group, the PSD and CSDS mice showed a significant decrease in the time in the central area of the open field and the time in the open arm of the elevated cross (P<0.001, Figure 1C and D), and a significant decrease in the proportion of sugar and water preference (P<0.001, Figure 1E), and a significant decrease in the proportion of sugar and water preference (P<0.001, Figure 1E). 001 , Figure 1E) , and the time of tail-hanging immobilization was significantly increased (P<0 . 001 , Figure 1F) . These results indicated that localized infarction of the medial prefrontal cortex and CSDS induced anxiety and depression-like behaviors in mice.

 

2.2 Decreased expression of neurotransmitters, amino acids, and other metabolites in the mPFC brain of PSD and CSDS mice

In order to clarify the metabolite changes in the brain tissues of PSD and CSDS mice, mPFC was sequenced for neurotransmitter-targeted metabolomics in the three groups. PCA results showed that the PSD and CSDS groups exhibited significant differences in overall metabolite levels relative to the SHAM group (Figure 2A). Heatmap results showed that the expression of several metabolites, including norepinephrine (NE), GSH, and spermidine (Spd), decreased in both depressed mouse models (Figure 2B). Correlation analysis showed that the correlation between metabolites such as epinephrine (E), γ-amino- butyric acid (GABA), GSH, and 3,4-dihydroxyphenylacetic acid (DOPAC) gradually weakened as shown in the figure (Figure 2C). 2C). The above results showed that the levels of neurotransmitters, amino acids and other metabolites in PSD and CSDS mice were significantly different from those in control mice, and mainly showed a decreasing trend.

 

2. 3 Glutathione, L-asparagine, and L-lysine were significantly reduced in mPFC of both PSD and CSDS mice

In order to explore the changes of individual metabolites, the metabolome sequencing results were analyzed by radargram analysis and intergroup variation analysis. The radargram results showed that GSH, L-asparagine (Asn), NE, L-lysine (Lys), L-serine (Ser), Spd, spermine (Spm) and L-valine (Val) were the eight metabolites with the most significant changes (P < 0.05, Figure 3A).

The results of intergroup analysis showed that the three metabolites of GSH, Asn and Lys were significantly decreased in both PSD and CSDS groups (P<0.05, Figure 3B); the six metabolites of ornithine (Orn), Ser, Spd, Spm, threonine (Thr) and NE were specifically decreased in PSD, and the four metabolites of L-alanine (Ala), glycine (Gly), Val, and L-glutamine (Gln) were decreased in PSD, and the four metabolites of L-glutamine (Gly), Val, and L-glutamine (Gln) were decreased in PSD and CSDS groups. L-alanine (Ala), glycine (Gly), Val, and L-glutamine (Gln) metabolites were less specific in CSDS (P < 0.05, Figure 3C). 05, Figure 3C). The results showed that the similarity of the altered metabolites in mPFC of PSD and CSDS mice was the significantly decreased expression of GSH, Asn and Lys, which may be potential targets for depression treatment.

 

2.4 Reduced GSH is a key metabolite in the mPFC brain of PSD and CSDS mice.

In order to explore the changes of metabolic pathways in the two depression models, pathway enrichment analysis was performed on the differential metabolites, which were mainly enriched in glutathione and β-amino acid metabolism pathways in the PSD group (Figure 4A), and glutathione, alanine, aspartate, and glutamate metabolism pathways in the CSDS group (Figure 4B), and the differential metabolites screened by the two models showed high enrichment of glutathione metabolic pathway. Both models showed a high enrichment of glutathione metabolism pathway. Reduced and oxidized GSH in the mPFC brain of mice in each group were measured by using GSH/GSSG kits, and the results showed that both PSD and CSDS showed a significant reduction in reduced GSH (P<0.05, Figure 4C ~ E). 05 , Figure 4C ~ E), suggesting that the absence of reduced GSH is a potential pathogenic factor for depression.

 

2. 5 Cellular iron death induced by reduced GSH in the mPFC brain region of PSD and CSDS mice

An important function of GSH in cells is to scavenge peroxides and inhibit iron death. mPFC mPFC iron death indexes were detected by using MDA kit and GPX4 immunofluorescence staining. The results showed that the proportion of GPX4-positive cells in PSD and CSDS mice decreased by about 20% compared with the control group (P<0.01, Figure 5A, B). The results showed that the proportion of GPX4-positive cells in PSD and CSDS mice decreased by about 20 percentage points compared with the control group (P<0.01, Figure 5A, B), and the MDA content increased by about 1-fold compared with the control group (P<0.01, Figure 5C). 01 , Figure 5C), suggesting that cellular iron death occurred in the mPFC brain region of PSD and CSDS mice.

 

In order to determine what types of cells in the mPFC of PSD and CSDS mice were subjected to iron death, GPX4 was stained with GFAP (astrocyte marker), CD31 (endothelial cell marker), and MAP2 (neuron marker) by immunofluorescence double staining, respectively. The results showed that the proportions of GPX4-positive astrocytes, GPX4-positive endothelial cells and GPX4-positive neuronal cells in the mPFC brain area of mice in the PSD and CSDS groups were significantly reduced compared with those in the SHAM group (P < 0.05, Figure 6), indicating that the proportion of mPFC brain area in mPFC brain was significantly lower than that in the SHAM group. 05, Figure 6), indicating that the ability of the corresponding cells to inhibit iron death after modeling was reduced. Among them, astrocytes had the highest GPX4-positive rate of 50% to 60% before modeling, and the proportion of GPX4-positive astrocytes decreased the most in PSD and CSDS mice, suggesting that astrocytes may be the main cell type responsible for iron death in mPFC of PSD and CSDS mice.

 

2.6 Exogenous GSH supplementation improves depressive behaviors in PSD and CSDS mice

To explore whether GSH can be used as a therapeutic target for depression, the present study was conducted in PSD and CSDS mice for 7 days. The results showed that GSH supplementation significantly increased the open-arm time in the elevated experiment and the central area time in the open field experiment in PSD and CSDS mice compared with the injection of equal dose of physiological saline (P<0.05, Figure 7A ~ D), and decreased the time in the tailing experiment. 05, Figure 7A~D), and decreased the immobilization time in the tail suspension experiment (P<0.05, Figure 7E, F). 05, Figure 7E and F). This indicates that GSH supplementation can effectively improve the depressive behaviors of PSD and CSDS mice.

 

3 Discussion

There are various hypotheses on the pathogenesis of depression, including the monoamine hypothesis, hypothalamic-pituitary-adrenal axis hypothesis, inflammation hypothesis, neuroplasticity hypothesis, structural and functional changes in the brain, and genetic and environmental hypotheses, etc. [6 , 13], and it has been found that the neurological circuits involved in prefrontal, hippocampal, amygdaloid, and vomeronasal nuclei play important roles in the onset of depression [14]. mPFC is one of the key nuclei that processes remote information inputs from cortical and subcortical areas and projects them to almost all sensory cortex, motor cortex, and subcortical areas. The mPFC is also one of the key nuclei that processes inputs from remote cortical and subcortical areas and projects them to almost all sensory cortex, motor cortex and subcortical areas, where they play an important role in controlling behavior and regulating cognition and emotion[15] . In the present study, we used the mouse model of local infarction caused by mPFC injection of ET-1 to simulate PSD, which is easy to fabricate and has a high success rate, and the CSDS mouse to simulate MDD.

 

Behavioral results showed that both models exhibited depressive behaviors. Using these two models to study the common changes between PSD and MDD will allow us to explore the therapeutic targets of depression in greater depth.

 

Brain tissue metabolites can reflect the physiological state of the brain, and metabolites such as neurotransmitters, lipids, and inflammatory factors all have an important influence on the occurrence of depression[16- 17] . Existing studies do not have a unified understanding of the specific changes of various metabolites in depression, and the expression levels of metabolites may be different in different studies due to the influence of different disease states and samples[18] . In the present study, we obtained the expression of 38 neurotransmitters and amino acids through neurotransmitter-targeted metabolomic sequencing in the mPFC of key brain regions in two models of depression and control mice, and found that the expression of 13 metabolites was significantly decreased in the PSD and CSDS groups, and that the metabolites with significant differences included NE, a classical neurotransmitter used in depression, as well as GSH and Gly, which play a role in cellular activities. The significantly different metabolites included NE, a classic neurotransmitter in depression research, as well as amino acids such as GSH and Gly, which play important roles in cellular activities. Pathway enrichment analysis revealed that the different metabolites in the PSD and CSDS groups were significantly enriched in the glutathione metabolic pathway, and further testing showed that both PSD and CSDS mice had a reduced prototype glutathione phenotype, suggesting that abnormal metabolism of GSH plays an important role in the pathogenesis of depression, and that the study of this common alteration of reduced glutathione may help to analyze the underlying pathogenic causes of depression. The study of this common alteration in reduced glutathione may help to analyze the underlying causes of depression.

 

GSH is widely distributed in the body and has important roles in antioxidant, oxygen radical scavenging [19] and immune system maintenance [20]. In recent years, the role of GSH in scavenging intracellular peroxides and inhibiting iron death has received more and more attention. Cellular iron death is triggered by the depletion of GSH, the decrease of glutathione peroxidase 4 (GPX4) activity, the inability of lipid oxides to be scavenged through the GPX4-catalyzed reaction, and the accumulation of reactive oxygen species[21] . In the present study, we found that GSH was significantly reduced in the mPFC brain region of PSD and CSDS mice, suggesting that the occurrence of depression may be related to oxidative stress and iron death of cells in the mPFC brain region. Previous studies have found that antidepressant drugs can improve depressive behaviors in mice by inhibiting iron death, and DANG et al.[22] found that edaravone could alleviate depressive behaviors in CSDS mice through the Sirt1/Nrf2/HO-1/Gpx4 axis, and YANG et al.[23] found that dihuangyinzi could inhibit depressive behaviors in PSD mice through the P53/SLC7A11/Gpx4 pathway, and that dihuangyinzi could inhibit depressive behavior in the frontal cells of PSD mice through the P53/SLC7A11/GPX4 pathway. YANG et al. [23] found that Dihuang yinzi could inhibit frontal cell iron death through the P53/SLC7A11/GPX4 pathway in PSD rats.

 

In the present study, we examined the iron death markers MDA and GPX4, and the results showed that there were obvious iron deaths in the mPFC brain region of PSD and CSDS mice compared with the control group, suggesting that there was an increase in iron deaths induced by GSH reduction in the key brain regions of mice in the two depression models, and we also found that astrocytes might be the main type of cells involved in the occurrence of iron deaths, which might be related to the fact that astrocytes are responsible for the function of brain substance metabolism. This may be related to the function of astrocytes in brain substance metabolism, and the iron death induced by GSH reduction in PSD and CSDS mice has an important effect on astrocytes. Meanwhile, the possibility of iron death in other cells, such as microglia, pericytes, oligodendrocytes, etc., cannot be ruled out, which needs to be verified by subsequent experiments. The results of the present study suggest that exogenous supplementation of GSH can improve the depression-like behavior of PSD and CSDS mice, which provides a theoretical basis for the treatment of depression by targeting GSH.

 

In summary, the present study analyzed the metabolite changes in the mPFC brain region of PSD and CSDS mice by metabolomics, and found that GSH metabolism abnormality plays a key role in the development of depression, which provides a theoretical basis for the search of therapeutic targets for depression. However, there are some limitations in this study: the sample size needs to be enlarged to increase the reliability of the experimental results; the results of this study show that cellular iron death triggered by the reduction of GSH plays an important role in the development of depression, but the reason for the reduction of GSH needs to be explored in further experiments. In the future, we will further analyze the similarities and differences between PSD and MDD, and explore the changes in brain cells and functions caused by GSH reduction, so as to lay the foundation for further research on the etiology of depression and the search for therapeutic targets.

 

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