| Objective: High stress usually leads to the release of various hormones in the peripheral nervous system and brain.These stress hormones not only prepare individuals for the serious consequences of dangerous situations,but also induce long-term adverse reactions of fear adaptation.Stress-related disorders are persistent anxiety and fear disorders caused by severe traumatic events,which are mainly manifested by high arousal and excessive fear generalization.However,it is found in human studies that fear generalization usually shows a generalization gradient,but in animal studies,fear generalization uses single stimulus research.Therefore,the purpose of this study is to establish a feasible fear generalization model that can respond to the stimulus gradient by using different stress intensities,to explore whether the continuous development of high arousal and fear generalization symptoms caused by different stress intensities can be alleviated by intervening in the memory consolidation process and fear regression process,and to further explore the dosage and effect of propranolol in fear regression learning.Methods:1.In Experiment 1,an animal model of fear generalization that responds to stimulus gradients was established.The auditory fear conditioning paradigm was used.The electric shock intensity was designed between groups and the time interval was designed within groups.The Freezing level of animals was used as the behavioral index of fear response.Three different intensities of electric shocks(0 m A,0.5 m A×2s×3,1.5 m A×2s×3)and lowfrequency sound(2.8 k Hz,75±2 d B)were used to conduct joint matching training 3 times in environment A to form fear memory.In the new environment,pure tones of different frequencies(0.7/1.2/6/10 k Hz)were used as generalization stimuli to test mice for generalization.Fear expression and fear generalization tests were performed 1 day after stress,hyperarousal was measured using heat pain,white noise test and open field test 4days after stress,and then 4 weeks after stress,three different Electric shocks of different strengths(0 m A,0.5 m A×2s×3,1.5 m A×2s×3)mice were tested for fear expression,fear generalization and hyperarousal response.2.Experiment 2 perfected the fear generalization model based on Experiment 1,using four different intensities of electric shocks(0 m A,0.3 m A×1s×3,0.5 m A×1s×3,1.0m A×1s×3,1.0 m A×2s×3,3)Combined matching training with high-frequency sound(9k Hz,75±2 d B)in environment A for 3 times to form fear memory,in the new environment,pure tones of different frequencies(1.3/2.5/4.8 k Hz)were used as generalization stimuli for small Rats were tested for generalization.The fear generalization test was performed 1day after the stress,and the hyperarousal was measured using the white noise test and the open field test,and then 2 weeks after the stress,the fear generalization and hyperarousal response test.3.Experiment 3 used pharmacological intervention in the memory regulation process to block the effect of fear memory on fear generalization and hyperarousal response.Based on the animal model of Experiment 2,the shock intensity and dosage were designed between groups,and different doses of protein inhibitors were used to block fear memory.The mice were injected with the protein inhibitor actinomycin(30,60,120 mg/kg)30minutes before the training,and the mice were tested for fear generalization 2 days after the stress,followed by hyperarousal with white noise and open field Measurement.4.Experiment 4 Effects of extinction training on fear generalization and hyperarousal responses.The electric shock intensity and time were designed between groups,respectively high stress(1.0 m A×1s×5)group short-term extinction group and long-term extinction group,low stress(0.4 m A×1s×5)group short-term extinction group and longterm extinction group.The medium-frequency sound(6 k Hz,75±2 d B)was used as the conditioned stimulus,2.4 k Hz and 4.8 k Hz were used as the generalized stimulus,and the electric shock and the sound were matched five times.Generalization and white noise test,two consecutive days of fear extinction training after an interval of 24 hours,and then fear generalization test again,while the high and low stress short-term extinction group performed fear spontaneous recovery test 26 days after stress,high and low stress The long-term extinction group underwent fear renewal,reconstruction and generalization tests23 days after stress.5.In Experiment 5,the effects of norepinephrine antagonists intervening in the process of fear memory extinction on hyperarousal response and fear generalization under different stress intensities.On the basis of Experiment 4,the electric shock intensity was designed between groups.The fear generalization test was performed one day after the stress,and two extinction trainings were performed after an interval of 24 hours.Norepinephrine β-receptor antagonist was injected 30 minutes before each extinction Propranolol,followed by fear generalization and hyperarousal tests.Results:1.In the establishment of animal models of fear generalization,both stress intensities of 0.5 m A×2s×3 and 1.5 m A×2s×3 can induce the generalization of high-frequency sounds and the discrimination of low-frequency sounds(p<0.001),High fear responses to novel sound stimuli continued to develop and were maintained at 5 days post-stress versus 27 days post-stress,and there was a marginally significant difference in open field total movement distance at 30 days post-stress(p=0.0548).2.In the animal model of fear generalization,the three stress intensities of 1.0m A×2s×3,1.0 m A×1s×3,and 0.5 m A×1s×3 can induce the generalization of fear in mice,and with the increase of electric shock intensity,the generalization of fear The generalization curve gradually flattened,indicating that with the increase of stress intensity,fear generalization increased(p<0.05).Under the three stress intensities,the fear response to novel stimuli increased significantly on the second day after the stress(p<0.0001),and there was no significant decrease after two weeks.This result shows that the hyperarousal response caused by high stress continues develop.3.In the fear extinction training,the 0.4 m A×1s×5 and 1.0 m A×1s×5 stress groups,the fear response to the conditioned stimulus sound increased in a time-dependent manner,and the resolution of the sound decreased with the passage of time;The short-term extinction group had obvious hyperarousal response 1 day after stress,which decreased after extinction training,but showed hyperarousal and spontaneous recovery of fear 26 days after stress;the long-term extinction group disappeared 17 days after stress,and hyperarousal Response was not significantly reduced.4.Injection of normal saline or propranolol 20 minutes before extinction training,5mg/kg propranolol in the 1.0 m A×1s×5 stress group reduced fear expression during extinction training,but it did not affect the acquisition and retention of extinction memory,10 mg/kg propranolol did not reduce fear expression during extinction training,but it impaired extinction memory.10 mg/kg propranolol at 1.0 m A × 1 s × 5 stress reduced fear expression during extinction training,but it did not affect the acquisition and retention of extinction memory.Conclusion:1.High stress can cause animals to generalize to sounds of different frequencies,and the higher the stress intensity,the higher the degree of fear generalization,and the timedependent increase of fear generalization.2.High stress causes hyperarousal response and continues to develop for a long time.3.Protein synthesis inhibitors cannot impair memory consolidation process.4.Extinction training can reduce fear generalization,but it does not reduce hyperarousal response.5.Although propranolol cannot promote extinction learning,it can reduce fear expression in an inverted U-shaped dose effect.This study uses different sound frequencies to establish an animal model of fear generalization with a stimulus gradient under high stress conditions,simulating the fear generalization gradient found in human studies,which provides a new idea for establishing a new animal model of fear generalization.In addition,this study further explored the dosage of propranolol,and provided an experimental reference for the application and treatment of propranolol in the process of exposure therapy,and suggested that propranolol may have Helping to adjunct exposure-based cognitive-behavioral therapy,appropriate doses of propranolol during exposure can reduce undue stress in patients with high levels of phobic responses and thus make treatment more tolerable. |
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