| ObjectiveStress refers to the non-specific,systemic neuroendocrine immune response when the body is stimulated by internal and external environment.Anxiety disorder is a common mental disorder disease caused by stress.The increasing number of stress events and types nowadays leads to problems such as high morbidity,low diagnosis rate and low treatment rate,which seriously affect people’s living standards.Some individuals are susceptible to stressful environments leading to anxiety disorders,ie susceptibility;however,some individuals do not experience any physical or psychological changes under the same stress levels,ie,resilience.Resilience refers to the ability of individuals to quickly recover and adapt from stress and difficulty.In-depth study of neurobiology mechanisms related to stress resilience has important research value for disease prevention and provide a clue for potential drug targets.,The neural mechanisms related to stress resilience are mainly focuses on prefrontal cortex,cingulate cortex,amygdala,and insular cortex.Insular cortex(IC)connects the primary and secondary sensory areas structurally and functionally,receives and integrates information from other cortical and subcortical regions,and the increasingly important in emotional disease and resilience of insular cortex was highly valued.However,the role of the insular cortex in resilience has not been clarified.The specific regulation mechanism of the insular cortex in the animal model of anxiety resilience is urgently needed to be solved.There are many types of anxiety disorder model methods and the conclusions are inconsistent.Therefore,the objectives of this subject are:1.Establish a stable and effective stress susceptibility and resilience animal evaluation model of anxiety disorder 2.Explore the neurobiological mechanism of the insular cortex in stress susceptibility and resilience.Methods1.Single-prolonged stress(SPS),restraint stress(RS)and foot shock(FS)were used to establish anxiety disorder model.Behavioral comparison of stressors,different stress days,and different stress durations:Using the open field and elevated plus maze experiments to evaluate the anxiety-like behavior of mice,for establishing a simple,effective and stable stress resilience animal evaluation model for mechanism research.2.The stress resilience animal evaluation model was established by unpredictable foot shock.The behaviors of pre-stress,24h after stress and 4 weeks after stress were tested separately:The fear conditioning experiment was used to evaluate the re-experience of traumatic events.The open-field and light-dark shuttle experiments were used to evaluate the avoidance.The pre-pulse inhibition(PPI)was used to evaluate the high arousal.Finally,analysis behavior of 24h and 4 weeks after stress were distinguished with stress susceptibility and resilience mice.3.The planar microelectrode array technique(MED64)was used to detect the synaptic plasticity of the insular cortex neurons in the resilience group and the susceptible group,and to reveal the mechanism between the insular cortex and stress resilience and susceptibility.4.Excitatory and inhibitory adeno-associated virus were injected into the insular cortex of mice to activate or inhibit insular neurons by stereotactic virus injection technique,to observe the changes of anxiety-like behavior after activation or inhibition of insular cortical neurons;The behavior of the mice was test after intraperitoneal injection of its ligand clozapine-N-oxide(CNO):the anxiety-like behavior of the mice was evaluated by the open field and elevated plus maze test to observe the role of insular cortex in anxiety disorders.Results1.Screen an anxiety model by single prolonged stress,restraint stress and foot shockTo screen a simple,effective and stable animal model of anxiety disorder for subsequent stress resilience mechanism,the anxiety-like behaviors of mice SPS and RS 2h for 14 consecutive days;RS 2h,4h,6h,8h for 7 consecutive days;FS stress 14 days were compared separately.1.1 Effects of single prolonged stress and restraint stress for 14 days on anxiety-likebehavior in miceThe results showed that compared with the control group,the exploration time of the stress group in the central area of the open field decreased.In the elevated plus maze test,the staying time of the SPS group in the open arm increased.RS 2h,14days group had a decreasing trend,suggesting that SPS and RS 2h had no significant effect on anxiety-like behavior in mice for 14 consecutive days.1.2 Effects of restraint stress for 7days on anxiety-like behavior in miceThe results showed that compared with the control group,the staying time of the mice in the central area of the open field was decreased at RS 2h,6h,8h for 7 days group,and there was no significant change in the RS 4h group.In the elevated plus maze experiment,the residence time of RS 2h,4h and 6h,7-day group in the open arm decreased significantly,and the RS 8h group showed a decreasing trend,suggesting that RS 2h,4h,6h,8h,7 days may induce anxiety-like behavior in mice.1.3 Effects of foot shock stress for 14 days on anxiety-like behavior in miceThe results showed that compared with the control group,the staying time of the mice in the 14-day group of FS stress decreased significantly in the central area of the open field;in the elevated plus maze test,the staying time of the mice in the 14-day group of FS stress decreased significantly in the open arm,suggesting that FS stress successfully induced anxiety-like behavior in mice for 14 days.The above behavioral results showed that FS stress for 14 days significantly increased the anxiety-like behavior of mice,so the 14-day model of FS stress was selected for screening of stress susceptibility and resilience animal.2.Establishment of an evaluation model for stress susceptibility and resilience animalThe evaluation criteria for susceptible and resilience animals were:the freezing time of the mice after stress in the fear conditioning experiment,the central zone time in the open field,the time of the bright zone,and the pre-pulse inhibition rate in the pre-pulse inhibition experiment was compared with the mean pre-stress behavior,and the more anxious mice were defined as positive mice.For example,the four positive symptoms behavioral are recorded as 4,the three positive symptoms behavioral are recorded as 3,and so on,and there are five kinds of behaviors of 4,3,2,1,and 0 respectively.Mice with 3 or more positive behaviors were defined as stress-susceptible mice(S,susceptible),and the rest were resilient mice(R,resilience).2.1 Effects of foot shock stress on body weight changes in miceCompared with the control group,the weight of the foot-shocked mice did not differ significantly during stress and after stress,but the trend of body weight increased slowly during stress,but decreased within 2 weeks after stress,and then slow growth in 3 weeks,and finally stabilized.It is suggested that the electric shock of the foot causes the weight of the mice to decrease more obviously in the short term after stress.2,2.Behavioral changes of mice in short term after stress2.2.1 Changes in trauma re-experience behaviorTo observe changes on re-experience behavior in short-term after stress,we examined the freezing times in context and cue tests in pre-stress and 24h post-stress.Compared with the pre-stress,24h post-stress mice had a significantly increased freezing time,but some of the mice had no significant difference,suggesting that these mice may not be sensitive to stress.Therefore,the unpredictable foot shock can significantly increase the trauma re-experience behavior after short-term stress in mice,but there exists stress susceptibility and non-susceptibility.2.2.2 Changes in avoidance behaviorTo observe the avoidance behavior in short-term after stress,we examined the time in the center zone of the open field and the time in the light box of the light-dark box shuttle experiment in pre-stress and 24h post-stress.Compared with pre-stress,the mice in the center zone and bright area were significantly reduced in 24h post-stress;in both experiments,some mice still had no significant difference,suggesting that these mice may not be sensitive to stress.Therefore,the unpredictable foot shock can significantly increase the avoidance after short-term stress in mice,but there is obvious stress susceptibility and non-susceptibility.2.2.3 Changes in high arousal behaviorPre-pulse inhibition experiments were used to detect changes in high arousal behavior in short-term after stress.Since the acoustic stimulation during the experimental test was high-intensity stress,to reduce the interference with subsequent experiments,we did not perform pre-pulse inhibition experiments pre-stress.Prior to the pre-pulse inhibition test,the experimental mice were first subjected to an input and output test to determine the threshold of the sound that produced the shock reflex.The results showed that 65～100dB of acoustic stimulation would not cause obvious shock reflex in mice,and the shock amplitude of 65-80dB stage was stable.When it was greater than 100dB,the shock reflection amplitude began to rise,and the plateau appeared at 105～110dB.In view of the above results,we used 105 dB as the stimulus intensity for shock reflex in subsequent tests,and used the acoustic stimulation of less than 80 dB(which did not cause shock reflex in mice)as the pre-pulse stimulation intensity.The results showed that the intensity of shock reflex increased with the increase of pre-pulse stimulation intensity at 24 h after stress,that is,the inhibition rate of shock reflex decreased gradually,which may be due to impaired sensory gating function in stressed mice.This results in a loss of pre-pulse inhibition leading to high arousal symptoms.The PPI value of each mouse was averaged at 80 dB of the average value of the pre-pulse stimulation intensity,and it was found that some mice still showed similar performance to the control group,that is,there was obvious stress susceptibility and non-susceptibility.2.3.Behavioral changes of mice in long term after stress2.3.1 Changes in trauma re-experience behaviorTo observe changes on re-experience behavior in long-term after stress,we examined the freezing times in context and cue tests in pre-stress and 4 weeks post-stress.Compared with pre-stress,4 weeks post-stress mice had a significantly increased freezing time,but some of the mice still had no significant difference,suggesting that these mice may not be sensitive to stress.Therefore,there is obvious stress susceptibility and non-susceptibility in long-term after stress.2.3.2 Changes in avoidance behaviorTo observe the avoidance behavior in long-term after stress,we examined the time in the center zone of the open field and the time in the light box of the light-dark box shuttle experiment in pre-stress and 4 weeks post-stress.Compared with pre-stress,the mice in the center zone and bright area were significantly reduced at 4 weeks after stress;in both experiments,some mice still had no significant difference,suggesting that these mice may not be sensitive to stress.Therefore,there is obvious stress susceptibility and non-susceptibility in long-term after stress.2.3.3 Changes in high arousal behaviorPre-pulse inhibition experiments were used to detect changes in high arousal behavior in long-term after stress.The results showed that the intensity of shock reflex increased with the increase of pre-pulse stimulation intensity at 4 weeks after stress,that is,the inhibition rate of shock reflex decreased gradually,suggesting that the mice had high arousal symptoms 4 weeks after stress.Compared with 24 h after stress group,the PPI values of mice at each decibel level were higher after 4 weeks of stress,indicating that the pre-pulse inhibition loss of mice at 4 weeks after stress was not severe after 24 h of stress,suggesting that after 4 weeks of stress,the high arousal symptoms of most mice may have recovered.The PPI value of each mouse was averaged at 80 dB of the average value of the pre-pulse stimulation intensity,and it was found that some mice still showed similar performance to the control group,that is,there was obvious stress susceptibility and non-susceptibility.2.4 Distinguish with stress susceptibility and resilience animalAccording to the evaluation criteria of susceptible and resilience animals,the susceptibility and resilience of the mice after 24 hours of stress and 4w after stress were differentiated,and the susceptibility and resilience transformation of each mouse from 24h to 4w after stress were observed.Divided into four categories:S-S,R-S,S-R,R-R,each accounting for 56%,10%,26%,8%of the total number of stressed animals,the mechanism of these susceptible and resilience mice was tested next.(S,susceptible)(R,resilience)3.Mechanism of insular cortex in stress susceptibility and resilience3.1 Changes in plasma corticosterone in stress susceptible and resilience miceThe plasma corticosterone of susceptible group,resilient group and control group were detected by radioimmunoassay.It was found that there was no significant change in the corticosterone of the model mice,suggesting that plasma corticosterone has returned to normal levels in the four weeks after stress.3.2 Changes in synaptic plasticity in the insular cortex of susceptible and resilient miceThe synaptic plasticity of mouse insular cortex neurons was detected by planar microelectrode array technique(MED64).It was found that compared with the control group,the insular cortex LTP of the susceptible group was significantly decreased,but there was no significant change in resilience mice,suggesting that the synaptic plasticity of the insular cortex in stress-susceptible mice was damaged,resilience mice showed no significant change.suggesting that LTP damage in the insular cortex may be one of the mechanisms leading to stress susceptibility.3.3 Effects of exciting or inhibiting insular neurons on anxiety-like behavior in miceTo further clarify the effect of changes in insular cortical neuron activity on anxiety-like behavior in mice,we used stereotactic techniques to accurately inject the virus into the insular cortex to achieve excitation or inhibition of insular cortex neuron activity.The results showed that compared with the control group,the number and time of exploration in the central area of the open field of the excitatory insular neurons increased,and the inhibitory insular neurons group were significantly reduced.In the elevated plus maze,the number of open arms and the open arm time decreased in both groups,suggesting that inhibition of the activity of the insular neurons can induce an anxiety-like behavior in mice.Conclusions(1)Differentiating the stress-susceptible and resilience mice in the foot shock model,a stable evaluation model of stress resilience animals was established,and the synaptic plasticity damage of the insular cortex of stress-susceptible mice was found.There was no significant change in resilience mice,suggesting that synaptic plasticity damage in the insular cortex may be one of the mechanisms of stress susceptibility;(2)Inhibition of insular cortex neurons can induce the trend of anxiety-like behavior in mice,suggesting that the decreasing activity of insular cortex neurons may induce anxiety behavior.In summary,this study established a stress resilience animal evaluation model,and found that the insular cortex plays a role in regulating the onset of anxiety disorder.The damage of synaptic plasticity may be one of the mechanisms of stress susceptibility in the body.And this study provides a basis for studying the disease in depth and biological mechanisms to find new therapeutic targets. |
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