Effect of D-serine on fear-regression of NMDA receptor in amygdala
OBJECTIVE: To investigate the changes of D-serine metabolizing enzymes in the amygdala of mice with conditional fear and regression and the effect of exogenous D-serine and NMDA receptor blocker MK-801 on fear regression and D-serine resistance. Possible mechanisms of anxiety. Methods (1) C57 mice were randomly divided into blank control group, conditional fear group, regression training group and regression test group, with 6 rats in each group. The control group did not give sound and electrical stimulation, and the other groups were conditionally feared. After the corresponding experimental steps, such as regression training and regression detection, the proteins extracted from the bilateral amygdala were taken for Western blot detection. (2) C57 mice were randomly divided into 3 groups: control group (F), D-serine group (F), MK-801 group (F), 8 rats in each group, respectively, intraperitoneal injection 1 hour before dissipation training Saline, D-serine 1000 mg / kg and MK - 801 0 1 mg / kg. (3) C57 mice were randomly divided into 3 groups: control group (X), D-serine group (X), MK-801 group (X), 8 rats in each group, respectively, injected into the amygdala area 1 h before dissipation training. Saline, D-serine 4 μg / side, MK - 801 0.1 μg / side. (4) Using the elevated plus maze to explore the effect of D-serine on anxiety behavior. Results (1) Compared with the blank control group, the expression of serine racemase in the amygdala of the regression training group and the regression test group decreased, and the expression of D-amino acid oxidase was up-regulated. (2) The D-serine group (F) was lower than the control group (F), and the MK-801 group (F) was higher than the control group (F) and D-serine group (F) (P < 0.05). . (3) The stupor behavior of the D-serine group (X) was less than that of the control group (X), and the MK-801 group (X) was more than the control group (X) and the D-serine group (X) (P < 0.05). ). (4) The percentage of open arms in the D-serine group was higher in the elevated plus maze than in the control group (P < 0.05). Conclusion The anxiolytic effect of D-serine is likely to be related to the activation of the fear of memory loss by NMDA receptors in the amygdala.
[Keywords] amygdala; D-serine; fear subsided
Anxiety disorders are characterized by extensive and persistent anxiety or recurrent episodes of panic disorder. Impaired fear is the main reason for the persistence of anxiety symptoms. NMDA receptors are subtypes of glutamate receptors, and numerous studies have shown that NMDA receptors play an important regulatory role in fear regression. L-serine is converted to D-serine by the action of serine racemase (SR), and D-amino acid oxidase (DAAO) is the main metabolic enzyme of D-serine. From June 2013 to September 2014, this study used the conditional fear and regression model to change the D-serine metabolism enzyme in the amygdala of mice and the fear of exogenous D-serine and NMDA receptor blocker MK-801. The effects of regression, a possible mechanism for studying the anxiolytic effects of D-serine.
1 Materials and methods
1.1 Experimental drugs D-serine, MK-801, anti-SR anti-body, anti-DAAO antibody, β-actin, and secondary antibody were purchased from Sigma.
1.2 Experimental animals and group C57BL/ 6J male mice (C57), 72 (provided by Beijing Vital Lihua Experimental Animal Technology Co., Ltd.), 9 to 10 weeks old, weighing 20 to 25 g, kept in a standard environment ( Ambient temperature 24 ~ 27 ° C, free access to food and water), light cycle 12 h. (1) Experiment 1: C57 mice were randomly divided into 4 groups: blank control group, conditional fear group, regression training group, and regression test group, with 6 rats in each group. The blank control group did not give sound and electrical stimulation, and the rest The corresponding experimental steps were performed according to 1.3. After the end, the proteins were extracted from the bilateral amygdala tissue for 1 h after the model and detected by Western blot. (2) Experiment 2: C57 mice were randomly divided into 3 groups: control group (F), D-serine group (F), MK-801 group (F), 8 rats in each group, administered 1 h before dispersal training. , intraperitoneal injection of normal saline, D-serine 1 000 mg / kg and MK-801 0.1 mg / kg. (3) Experiment 3: C57 mice were randomly divided into 3 groups: control group (X), D-serine group (X), MK-801 group (X), 8 rats in each group, administered 1 h before dispersal training. The amygdaloid nucleus was injected with normal saline, D-serine 4 μg / side, and MK -801 0.1 μg / side.
1.3 Establishment of conditional fear and regression training model The experimental device is a small iron column organic plastic container with conductive bottom (provided by Shanghai Xinsoft Information Technology Co., Ltd., model XR-XC404). The first day of the experiment was to form a fear learning memory, and the mice were placed in a container for 3 min. Then, four groups of training were performed in succession. Each group consisted of: 30 s sound stimulation (80 dB), and the last 2 s of the sound stimulation was current stimulation (0.5 mA), with each group being separated by 30 s. On the second day, the fear memory was dissipated and the mouse was placed in another organic plastic container with a black and white strip. Then four sets of training were performed in succession, each consisting of: 30 s sound stimulation (80 dB) with an interval of 30 s. The third day of the experiment was a fear memory dissipation test, and the container and stimulation were the same as on day 2. Observe the stupor behavior of the mice. At the end of each mouse experiment, the experimental equipment was washed with 70% ethanol.
1.4 Western blot was used to detect the protein extracted from the amygdala region. The mouse was decapitated, and the coronal slice was cut with a vibrating slicer. The thickness was 400 μm. The cells were incubated for 1 to 1.5 h in an artificially cerebrospinal fluid. . The amygdala was separated into a EP tube under a stereo microscope, and the protein was extracted and determined by BCA protein content. Load at 30 μg protein.
1.5 After the amygdala was placed in a cannula with 10% chloral hydrate anesthetized mice, the head was fixed with a stereotactic device. Previously used as the base point, according to the brain area coordinates of the amygdala (1.5 mm backward, 3.4 mm in the midline, 3.5 mm down), the needle was inserted into the required brain according to the depth of the positioning coordinates. Area, fix the needle and close it, put it in the cage and rest for 4 to 5 days.
1.6 The elevated cross maze experiment placed the mouse in the central region of the maze 3 h after the regression training test. Record the movement of the mice within 5 min, and analyze the indicators: enter the open arm time / total time. After the end of the experiment, the elevated cross maze instrument was cleaned with 70% ethanol (the experimental instrument was supplied by Shanghai Xinsoft Information Technology Co., Ltd., model: XR-XG201).
1.7 Statistical Methods One-way ANOVA was performed using SPSS 18.0 statistical software. The pairwise comparison uses the LSD-t test.
3 Discussion One of the most important features of anxiety disorder is that fear memory persists and is difficult to forget. The amygdala is the core area that regulates animal emotions. Numerous studies have shown that the amygdala plays a decisive role in the development of anxiety disorders. The amygdala participates in the formation, maintenance, and dissipation of fear memory. D-serine is abundant in some mammalian brain regions such as hippocampus and amygdala. Studies have shown that D-serine is an important co-activator of endogenous regulation of NMDA receptors. It affects the regressive memory by acting on the NMDA receptor glycine binding site to increase NMDA receptor activity. Some scholars have found that animals trained by conditional reflex fear have behavior similar to those of patients with anxiety disorders. They are classic animal models for studying anxiety and learning and memory. The elevated plus maze is a new method for anxiety research with reliable results. By studying the conditional fear and regressing the changes of D-serine metabolizing enzymes in the amygdala of mice, we can indirectly understand the changes of D-serine concentration in the amygdala. In this experiment, the expression of SR in the amygdala of the mice in the regression training group (day 2) and the regression test group (day 3) decreased, and the DAAO level increased, indicating that the formation of fear memory affects the metabolism of D-serine in the amygdala. . However, the expression of SR in the amygdala of the conditional fear group (day 1) was not significantly different from that of the control group, which may be related to the early fear memory that has not affected the protein synthesis of SR in the amygdala.
Impaired fear is the main reason for the persistence of anxiety symptoms. Promoting the dissipation of fear memory is an important target for drug research. Therefore, we investigated whether the anti-anxiety effect of D-serine is achieved by activating NMDA receptors to promote fear memory dissipation. By intraperitoneal injection of exogenous D-serine or NMDA receptor blockade, the fear regression test and elevated plus maze were used to observe the changes of fear or anxiety behavior in mice. The study found that the intraperitoneal injection of exogenous D-serine rats was significant. Less than the control group, the intramuscular injection of MK-801 blocker rats was higher than the control group. In the elevated plus maze, the percentage of time that the D-serine group entered the open arm was higher than that of the control group, while the percentage of time that the MK-801 group entered the open arm was lower than that of the control group. The above results indicate that D-serine can promote fear regression, and its anti-anxiety effect is achieved by activating NMDA receptors to promote fear memory regression.
Recent studies have shown that the amygdala is involved in the regulation of the formation, consolidation and dissipation of fear memory. Therefore, we further study whether the anti-anxiety effect of D-serine is achieved by activating the NMDA receptor in the amygdala region. The cannula was placed through the amygdala, and microinjection of exogenous D-serine or NMDA receptor blocked MK-801. The fear regression test was used to observe the change of fear or anxiety behavior in mice. The study found that the micro-injection of the exogenous D-serine group in the amygdala was less than that in the control group, while the NMDA receptor blocker MK-801 group had higher stupidity than the control group. It is suggested that the anti-anxiety effect of D-serine is related to the activation of NMDA receptor in the amygdala region to promote fear memory regression.
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