Effects of acute emotional stress on behavior and brain neurogranin phosphorylation in rats

Abstract To investigate the effects of acute emotional stress on the behavior of rats in the open field , and the relationship between the changes of neurogranin (NG) and the effects of stress behavior. To stimulate the acute uncertainty bottles, establish animal models of emotional stress. Were randomly divided into 40 male SD rats emotional stress group 1 (ES1, emotional stress and receiving the open field test), emotional stress group 2 (ES2, only accept emotional stress), the normal control group 1 (C1, No emotional stress , but received the open field test ) and the normal control group (C2, no emotional stress , no open field test ) (n=10) . Behavioral changes after stress in rats were assessed by open field behavior and elevated plus maze task . Western blotting (Western blotting) was used to determine NG content and phosphorylation level in hippocampus and forebrain cortex . Knot

The results showed that : (1) The level of activity in the ES1 group increased after stress , compared with the C1 group , the difference was significant , p<0.01. (2) The NG phosphorylation level of hippocampus and forebrain cortex in ES1 group was higher than C1. and C2 groups, the difference was statistically significant, both p <0 05;. phosphorylation levels forebrain NG, ES2 group than C1, there is a significant difference for p <0 05;. (3 ) hippocampus There is a significant correlation between the level of NG phosphorylation and horizontal activity. It is suggested that acute emotional stress can lead to obvious behavioral changes in animals such as anxiety , which may be related to changes in NG phosphorylation levels in the brain . Horizontal activity may be a sensitive activity indicator reflecting acute emotional stress . Hippocampal NG phosphorylation may be a more sensitive biology to predict anxiety or depression caused by acute emotional stress.

index.

Keywords acute stress , emotional stress , hippocampus , forebrain cortex , behavior , neurogranin

1 Introduction

To date , little is known about the brain mechanisms of stress-induced behavioral changes , including the molecular molecules involved, pathways of action, and signaling pathways. Previous studies have focused on changes in neurotransmitters and neuromodulators and function, but also useful c - fos explore related brain regions as a probe, but there are still many mysteries in the understanding of brain mechanisms of stress behavior change. In recent years , many scholars at home and abroad believe that the change of synaptic plasticity is the biological "mark" left by stress in the central nervous system , which can cause changes in brain function and behavioral activities. During stress, the central mechanism of synaptic plasticity change is an important central mechanisms involved in emotional and behavioral disorders, including changes in structural plasticity of synaptic function and synaptic plasticity mechanisms. The former refers to the reduction of the number of neurons in the relevant brain regions and the organic damage of the morphological structure such as cell loss and dendritic spine atrophy . The hippocampus and forebrain cortex are the most important brain structures involved in stress. The latter refers to defects in protein signaling pathways, synaptic transmission dysfunction, etc. , and most attention is paid to changes in central NR -dependent LTP .

Because central neuron-specific proteins and their phosphorylation reactions play an important role in maintaining synaptic structure and function , including synaptic growth, development, compensatory changes, and functional transmission. Therefore, changes in content and level of phosphorylation of specific proteins of certain central neurons under stress, possibly by changing the structure and function of synaptic plasticity involved in the mechanism, the central mechanisms involved in stress-induced behavioral effects. Recent studies have shown that certain proteins expressed in central neurons such as Heat Shock Protein 70 (HSP-70) , brain-derived neurotrophic factor (BDNF) , and membrane growth-related proteins (thepresynaptic43-kDa growth) - Associated protein , GAP-43), which is closely related to stress and certain mental disorders , has led researchers to focus on the possible role of synaptic-specific proteins in the central mechanisms of stress response.

Neurogranin (NG) is a neuron-specific protein that is highly expressed in brain structures such as cortex, hippocampus, and amygdala , which are closely related to stress, mood, and behavior . It is an important substrate protein for postsynaptic PKC . . Found, NG the LTP involved in the central NR dependent, many important proteins signal PKC, PKA and CaMK â…¡ other pathways, synaptic strength by changing the dielectric guide, part of the common pathway configured to induce expression of synaptic plasticity, in projection It has a key role in the plasticity of touch. Further, by phosphorylation level changes of central NR NG produce a significant effect of LTP-dependent reaction sensitivity to certain stressors, and is closely related to learning and memory. Therefore , it may involve synaptic structures and functional plasticity mechanisms of stress-induced behavioral disorders. Studies have found that chronic physiological stress and emotional stress can lead to abnormal animal behavior and a significant decrease in NG content , and abnormal behavior is associated with a significant level of NG content. It indicates that the decrease of NG content after chronic stress is a relatively sensitive biological indicator for predicting the occurrence of behavioral disorder , which may involve the central mechanism of behavioral disorder caused by chronic stress. The results of the above studies suggest that NG may give water to animals as a 9: 00 to 9: 10 and 21: 00 to 21: 10 , then remove the water bottle and not give water for the rest of the time. The stress test was started after the regular feeding period , and the animals in the ES group were given empty during the regular feeding time.

Bottle stimulation induces emotional stress , and the stimulation is given irregularly , but once a day ; stress lasts for 3 days. Groups C1 and C2 did not receive any treatment , were kept in cages , free to drink and feed. All animals were weighed four times at the beginning of the adaptation period, at the end of the adaptation period, at the end of the regular feeding period, and on the third day of stress to examine the degree of stress.

2. 3 instruments and reagents

First antibody anti- NG total protein monoclonal antibody, phosphorylated NG polyclonal antibody, β -actin monoclonal antibody, second antibody horseradish peroxidase-labeled goat anti-rabbit IgG antibody, goat anti-mouse IgG antibody, BCA ( The bicinchoninic acid protein detection kit, whole cell lysate (buffer) , and nitrocellulose filter (NC) are all products of Sigma , and the enhanced chemiluminescence system (ECL) kit is purchased from Pierce, USA. The Gel. Doc Gel Imaging Semi-Quantitative Analysis System was purchased from Bio-Rad .

2. 4 behavioral testing

Using the open field test and the elevated plus maze task procedure , animals in the ES1 and C1 groups performed two behavioral tests at the end of the adaptation period and at the end of the stress period.

2.4.1 open field test the animals were placed high 50cm, 180cm diameter, are black and the bottom periphery of a circular open field, the illuminance of 60lux, soundproof chamber. The observer used a camera system in the behavioral laboratory to record the behavior of the animal in the open field for 5 minutes , including horizontal activity distance, erection, modification, exploration, sluggishness and defecation. Among them, the horizontal activity distance and the inquiry obtained the data through the behavior tracking analysis system , and the erect, sluggish, and modified behaviors are counted according to the behavior camera statistics. The amount of defecation per animal is expressed as the number of particles of defecation after the end of the open field test. 2. 4. 2 The elevated cross maze task device is 50cm high , the open boom is 110cm × 10cm × 10cm ( length × height × width ), and the closed boom is 110cm × 50cm × 10cm ( length × height × width ) . The test was conducted every day from 10 : 00 to 14 : 00h . At the beginning of the test , the rat was placed

To the middle platform of the maze, open face with a boom maze.

The test time was 5 min and 4 behavior indicators were recorded . (1) Entering fans

The total number of open arms of the palace , (2) when staying in the open labyrinth

Between , (3) the total number of girders entering the labyrinth , (4) closed in the labyrinth

The dwell time in the boom.

2. Determination of NG in hippocampus and forebrain cortex

After the next day, and the group of C1 ES1 rats after stress testing behavioral experiment, the rats were decapitated immediately quickly peeled hippocampus and frontal cortex of the same volume of tissue in the frontal cortex tissue is a rat brain The midline of the map is determined by the anterior lateral prefrontal cortical tissue ( see the M1 and M2 regions shown in Figure 1 ) . The tissue is then quickly cooled with liquid nitrogen.

Determination of the content of NG and the hippocampus and frontal cortex phosphorylation levels by Western blot hybridization (Westernblotting). Add tissue to the buffer homogenate. After homogenization, add BCA solution, shake well , incubate in a 37 °C water bath for 30 minutes , carry out preliminary quantification of total protein in hippocampus and forebrain cortex by protein spectrophotometer , calculate each sample by electrophoresis according to the total protein content of the sample. The amount of buffer to be added. Homogenate sample buffer was added, taking a 15% polyacrylamide gel electrophoresis and transferred to NC membrane, NC membrane in blocking solution (10% skimmed milk powder, dissolved in TTBS for) rt IH closed, with Tris / Tween buffer saline ( Trisbuffered Tween- 20sa2line, TTBS) Wash the membrane for 10 min (3 times. Add anti- NG total protein monoclonal antibody for 3 h at room temperature , then wash the membrane with TTBS for 10 min (3 times. TTBS solution diluted goat anti-rabbit IgG antibody (1: 4000) hatch

Incubation , room temperature , shaking for 1 h, the same membrane was washed 3 times. ECL fluorescent labels were added and the film was exposed to light. Thereafter , the internal reference protein β -Actin and phosphorylated NG on the same NC membrane were hybridized. Bringing NG , Phosphorylated NG and β -Actin Protein Strips on Film by Gel. Doc Gel Imaging Semi-Quantitative Analysis System

3 results

3. 1 weight

At the beginning of the adaptation period, at the end of the adaptation period, at the end of the regular feeding period and at the end of the stress period , there was no significant difference in the overall weight of the four groups of animals (p>0105) . However, during the stress period ( from the end of the regular feeding period to the end of the stress period ), the animals in the ES1 and ES2 groups grew very slowly.

3. 2 market behavior test

Before the stress , there was no significant difference in the horizontal activity distance, erect order, modification behavior, sluggish behavior and defecation volume between the two groups in the open field test (p>0.05) . After the stress, the ES1 group exhibited increased activity level, compared with the C1 group, there was a significant (p <0. 01) difference. The difference in the remaining behavioral indicators was not significant.