| [Objective]Post-traumatic stress disorder(PTSD)is a mental disorder with the highest incidence after experiencing or witnessing a traumatic event.The outbreak and spread of COVID-19 has triggered global turmoil,causing severe panic and great psychological stress among the public.In the post-epidemic era,the prevention and treatment of PTSD will become a major global public health issue.However,due to the unclear pathogenesis of PTSD,the current treatment of PTSD drugs still have major shortcomings such as low effectiveness and serious adverse reactions.Therefore,revealing the new pathogenic mechanism of PTSD and discovering the potential targets of prevention and treatment drugs are hot issues in this research field.According to the characteristics and core symptoms of PTSD,the irretrievable fear memory induced by traumatic stimulus is an important behavioral basis for high alertness,high arousal and cognitive impairment.Therefore,the extinction of fear memory is an important inducement of PTSD and a breakthrough point to study the pathogenesis of PTSD.Recent studies suggest that the regression regulation of fear emotion is an extrication process under the effect of positive happy emotion,so the regression of fear memory needs the participation and regulation of reward system.The mesolimbic cortex dopamine system is an important neural structure and material basis for the body to seek benefits and avoid harm,and plays a positive emotional regulation role.Previous studies have found significant abnormalities in the DA system in both PTSD patients and PTSD model animals compared with the control group.However,it is not clear whether the above changes in the brain DA system are related to PTSD behavior and through what molecular mechanisms affect the occurrence and development of PTSD.Revealing this scientific problem will provide new mechanisms and potential molecular intervention targets for the prevention and treatment of PTSD.[Method]First of all,taking D3R knockout model mice as the research object,the PTSD model was established by using one-way short-term electric shock mode,and the effects of D3R knockout on the formation and regression of fear memory induced by electric shock were studied in order to clarify the behavioral phenotype of D3R involved in the regulation of PTSD fear memory.Then,the distribution of D3R in the ventral tegmental area of the mesencephalic limbic cortical system and the basolateral amygdala of the emotion regulation center,and the types of neurons expressed in D3R were studied by RNA scope technique.Furthermore,in PTSD animal,D3R highly selective antagonist YQA14 was injected into ventral tegmental area and basolateral expressed in D3R were studied by RNA scope technique.Furthermore,in PTSD animal,D3R highly selective antagonist YQA14 was injected into ventral tegmental area and basolateral amygdala of mice before and after training,respectively,to deter mine the specific effect of D3R on fear memory and anxiety in PTSD.Finally,in the PTSD animal model,the excitability of dopaminergic neurons in the ventral tegmental area of the PTSD model and the changes of dopamine content in the reward nucleus accumbens and the basolateral amygdala of the emotional center were studied by optical fiber recording system,and compared with those after knockout or blockage of D3R,to clarify the neurobiological mechanism of D3R receptor involved in the regulation of fear memory in PTSD.On this basis,chemical genetic techniques were used to specifically up-regulate or down-regulate the excitability of dopaminergic neurons in the ventral tegmental area of the midbrain,and to observe their effects on the formation and regression of fear memory in the PTSD model,so as to clarify and verify the direct relationship and important contribution between the midbrain marginal cortex dopaminergic system and the occurrence and development of PTSD.[Result]1.In the PTSD animal model,the behavioral phenotype of D3R knockout mice during the regression of fear memory.Using the one-way short-term electric shock model,the results showed that in the situational fear test on the 2nd,7th,10th and 14th day after modeling,the fear freeze time of D3R knockout mice was significantly lower than that of wild-type mice.It is suggested that D3R knockout attenuates the acute fear response caused by electric shock.When the fear freeze behavior of the model group WT mice and the model group D3R-KO mice disappeared,the low intensity current(0.5mA)electric shock stimulation was carried out to test the fear arousal.Compared with the WT mouse model group,the D3R-KO mouse model group showed significantly lower fear freeze behavior,suggesting that D3R knockout can reduce the arousal of fear memoryThrough the above research,we make it clear that in the PTSD model,knockout D3R not only affects the formation of fear memory and weakens the arousal of fear behavior.2.The neuronal expression type of D3R in the midbrain limbic systemIn this study,high sensitivity and high specificity RNA scope technique was used to study the expression and distribution of mRNA level of D3R in physiological state.The results showed that D3R was distributed in both VTA and BLA brain regions,and was expressed in dopamine(DA)neurons and y-aminobutyric acid(GABA)neurons in VTA brain regions.The expression proportion of D3R in DA neurons was 50%of the total number of neurons in the observed range,while the positive cells of GABA neurons accounted for 15.29%of the total number of neurons in the same observation range.D3R was expressed in both glutamate(Glu)neurons and GABA neurons in the BLA brain region,in which the positive rate of Glu neurons was 55.69%,while the positive rate of GABA neurons was 14.37%in the same observation range.These results suggest that D3R is mainly expressed in DA neurons in VTA brain region and Glu neurons in BLA brain region.It provides a good structural basis for the next step of brain region-specific intervention.3.Effects of blocking D3R by different nuclei in different periods of PTSD model on fear memory and anxiety(1)Blocking the effect of D3R on basic physiological functionIn this experiment,we first studied the dose at which YQA14 did not affect the spontaneous activity and basic pain threshold of animals.In spontaneous activity experiment and Hargreaves test,YQA14(0.1 μg/0.2 μl/side and 1 μg/0.2 μl/side),a highly selective D3R blocker,was microinjected bilaterally in VTA or BLA brain regions,respectively.The results showed that YQA14(0.1 μg/0.2 μl/side and 1 μg/0.2 μl/side)had no effect on spontaneous activity and pain threshold.Therefore,YQA14(1 μg/0.2 μl/side)was selected as the dose of nuclear group for the intervention study of PTSD.(2)The effect of specific nuclei intervening D3R in different periods on fear memory of PTSD1)Pre-electric shock intervention:During the training period of electric shock stimulation for two consecutive days,YQA14(1 μg/0.2 μl/side)was injected bilaterally into VTA or BLA brain area every day,and electric shock stimulation was performed after 10 minutes.The situational fear test was carried out on the 3rd and 8th day.The results showed that the fear freeze behavior of mice in VTA or BLA brain area intervention group was significantly lower than that in solvent group.2)Latent intervention after electric shock:No drug treatment was given during electric shock training.24 hours after modeling,a contextual fear test was conducted.According to the test results,the stress-modeling mice were divided into solvent group and administration treatment group,so that each group kept a balanced frozen behavior before intervention.After that,YQA14(1 μg/0.2 μl/side)or 2%DMSO(0.2 μl/side)were injected bilaterally into VTA and BLA brain regions every day for 4 consecutive days,and then fear memory test and anxiety state test were performed on the 8th day.The results showed that the continuous administration of VTA to the brain area significantly weakened the fear and immobility behavior induced by contextual cues,but had no significant effect on the anxiety state induced by the elevated cross maze.On the contrary.continuous administration of BLA to the brain region had no obvious effect on situational fear,but significantly attenuated the fear freeze behavior induced by contextual cues,while continuous administration of BLA brain region had no significant effect on contextual fear.Combining the experimental results of the first part on D3R knockout mice and the specific blocking of D3R of brain regions can significantly change the fear memory and anxiety of PTSD in this part,it is suggestied that D3R affects the occurrence and development of PTSD by participating in the fear memory regulation of PTSD,and there are differences in the regulation of brain regions.4.D3R participates in the neurobiological mechanism of regulating PTSD.In this part,we will focus on the excitability of DA neurons in VTA,the core brain area,and the real-time change rule of DA content in NAc and BLA,the reward center of the projection target area,during the modeling of PTSD,and compare the effects of knocking out or blocking D3R on the above-mentioned specific changes,so as to clarify the neural mechanism that D3R participates in regulating PTSD.(1).In PTSD model,the excitability of DA neurons in VTA brain area and the change characteristics of DA neurotransmitters in NAc and BLA in projection target areas during electric shock.1)specific changes of excitability of VTA-DA neurons during electric shockFirstly,the neuron activation indicator protein(GCaMP6m)virus carrying Tyrosine Hydroxylase(TH)promoter was injected into VTA brain area,and the recording fiber was embedded in the same position.After two weeks of virus expression,it was confirmed by immunofluorescence that GCaMP6m was specifically expressed on TH-positive DA neurons,which could reflect the excitability of DA neurons in real time.After the virus expression was completed,the PTSD electric foot shock model was made.The mice were first acclimated to the cage for 3 minutes,and then the discontinuous electric shock with a duration of 4 minutes was started.The electric shock was given every 10 seconds,each time for 10 seconds,and the total electric shock was 12 times.The current was 0.8mA.The results showed that the calcium signal in DA neurons in VTA brain area of the model group increased instantly once and after electric shock,suggesting that DA neurons were significantly activated at the instant of electric shock and after electric shock.The above results show that DA neurons of VTA are probably directly involved in the coding of negative stress process,and the activation during electric shock promotes the learning of negative stimuli,while the activation after electric shock is the feedback regulation of negative stress and the coding of safety signals.2)Time-course change characteristics of DA neurotransmitters in NAc and BLA during electric shock.Firstly,the fluorescent probe virus of DA neurotransmitter was injected into NAc or BLA brain area,and then the recording fiber was buried in the same position.After two weeks of virus expression,it was determined that the fluorescent probe was highly expressed in NAc or BLA brain area,which can be used for real-time monitoring of DA neuro transmitters.After the virus expression was completed,the model of PTSD electric foot shock was made.The results showed that the fluorescence signal of DA probe in BLA brain area of the model group increased rapidly to plateau stage during electric shock,and then decreased gradually after electric shock until the end of the 10-second interval,on the contrary,the fluorescence signal of DA probe in NAc brain area of mice in the model group decreased rapidly during electric shock,recovered rapidly after electric shock and recovered to the basic level or slightly higher than the basic level in the intermittent period,suggesting that the DA level in BLA brain area increased significantly during electric shock and the DA level in NAc brain area decreased significantly during electric shock.The above results show that the DA nerve projection of VTA to BLA during electric shock may be involved in the learning process of negative stress,and the significant inhibition of DA nerve projection of VTA-NAc encodes the significant inhibition of reward function by stress stimulation,while the return to normal or increase after electric shock is the feedback regulation of stress stimulation,so as to promote the recovery of negative emotions.(2)In PTSD model,the change characteristics of excitability of VTA-DA neurons during electric shock after knocking out D3RCalcium indicator was specifically expressed in DA neurons in VTA brain area,and the activity change of DA neurons was detected by optical fiber recording system.The results showed that the calcium signal in VTA brain area of wild-type mice and D3R knockout mice in the model group increased instantaneously at the instant of electric shock and after electric shock,but there was no difference in the excitability of DA induced by electrical stimulation and the end of electric shock between the two genotypes.However,from the second second to the tenth second after electric shock,the calcium signal of wild-type mice in the model group dropped to the baseline level,while the calcium signal of D3R knockout mice quickly dropped below the baseline level,suggesting that the DA neurons of VTA in D3R-KO mouse model group were significantly suppressed at this time.(3)In PTSD model,after D3R blocker was given to VTA brain region,DA nerve projection dominated the change characteristics of DA neurotransmitters in NAc and BLA brain regions.The micro injection administration cannula was embedded in VTA brain region,and the fluorescent probe of DA neurotransmitters was injected into NAc and BLA brain regions,and the recording fiber was embedded in the same position.The results showed that after bilateral injection of D3R blocker YQA14(1μg/0.2μl/side)into VTA brain area.the signal of DA fluorescent probe in BLA and NAc was significantly enhanced,suggesting that the brain DA level in VTA can be significantly increased after blocking D3R.For BLA,it was found that blockade of D3R in the VTA brain region before electric shock on the 1 st and 2nd day,the optical fiber recording results showed that the fluorescence intensity of DA probe in BLA brain area in YQA14 treated group was significantly lower than that in vehicle treated group during electric shock on the 1st and 2nd day and when mice were reexposed to environmental cues on the 3rd and 8th.It is suggested that blocking D3R in VTA brain area before electric shock can attenuate the increase of DA content in BLA brain area induced by electric shock and contextual cues.For NAc,D3R in VTA brain was blocked before electric shock on the 1st and 2nd day.The results of optical fiber recording during electric shock modeling on the 1st and 2nd day and exposure to contextual cues in mice on the 3rd day showed that there was no significant change in fluorescence intensity of DA probe in NAc brain area between YQA14 treated group and vehicle-treated group.However,on the 8th day,it was found that the fluorescence intensity of DA probe in NAc brain area in YQA14 treated group was significantly higher than that in vehicle treated group,suggesting that blocking D3R in VTA brain area before electric shock could reverse the decrease of DA content in NAc brain region induced by contextual cues on the 8th day.To sum up,knockout of D3R before electric shock decreased the activation of DA neurons in VTA during electric shock,and the blocking of D3R in VTA brain region upregulated the basic state of BLA and NAc brain regions,which enhanced the DA release of VTA-NAc by reducing the release of DA of VTA-BLA during electric shock,enhanced the regulation ability of the body against stress,and participated in the formation of fear memory of PTSD.5.The regulation of dopamine system in limbic cortex of midbrain on fear memory of PTSD(1)In PTSD model,the excitability of DA neurons in VTA brain area was up-regulated before electric shock.Activated chemical genetic virus was injected into VTA brain area,DA fluorescent probe virus was microinjected into NAc brain area,and recording fiber was buried in the same position.After intraperitoneal administration of CNO(1mg/kg),the obvious enhancement of fluorescence intensity of DA probe in NAc was detected by optical fiber recording system,reaching the peak at about 15 minutes,and then gradually decreasing.suggesting that DA neurons of VTA were significantly activated.According to the above dynamic change characteristics of DA release after CNO administration,PTSD electric shock stimulation model was made 15 minutes after CNO administration.The behavioral results of PTSD model showed that on the 3rd,8th,15th and 30th day,the fear freeze reaction of CNO treated mice was significantly lower than that of vehicle model mice.The results suggest that up-regulating the basic level of DA nervous system in brain before electric shock can effectively control the fear response caused by high-intensity stress and accelerate the regression of fear memory.(2).In PTSD model,down-regulating the excitability of DA neurons in VTA brain areaInhibitory chemogenetic virus was injected into VTA brain region,DA fluorescent probe virus was microinjected into NAc brain region,and the recording fiber was buried in the same position.After CNO(lmg/kg)was given intraperitoneally,the obvious decrease fluorescence intensity of DA probe in NAc was obviously decreased by the optical fiber recording system,suggestcing that the DA neurons of VTA are significantly suppressed.According to the above kinetic characteristics of DA release after CNO administration,15 minutes after CNO administration,low intensity(0.5mA)electric shock stimulation was performed for two consecutive days.The behavioral results of PTSD model showed that the fear freeze behavior of mice treated with vehicle on the 3rd and 8th day in model group was significantly higher than that of mice without electric shock,in the model group,the mice treated with CNO showed stronger fear freeze reaction,which was significantly different from the vehicle model group.The results suggest that down-regulating the basic level of VTA-DA nervous system reduces the body’s ability to adjust against stress and enhances the fear reaction,thus becoming a risk factor for PTSD.[Conclusion]This study found that knocking out or blocking D3R can effectively reduce the formation of fear memory in PTSD by up-regulating the function of dopamine nervous system in mesolimbic dopamine nervous system.The basic state of mesolimbic dopamine nervous system is the key neurobiological basis for the formation and regression of fear memory in PTSD. |
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