| Objective:Based on the neuronal information coding and synaptic plasticity,to investigate the neurobiological mechanism of raw-fried Ziziphi Spinosae Semen drug pair against state anxiety induced by conditioned fear in rats.Methods: ?.Behavioral evaluation of conditioned fear induced state anxiety in ratsMale SD rats were randomly divided into the blank control group and the model group after 7 days of adaptation in the experimental environment.Then,the blank control group and the model group were randomly divided into 1-day,7-day and 10-day groups respectively.The rats in each group were placed in the stimulation box of the conditioned fear analysis system for replication of state anxiety.After adaptation for 180s,sound stimulation(75 d B,1000 Hz,5 s)was given first in the first stimulation cycle,and electrical stimulation(0.5m A,1s)was given in the last 1s of sound stimulation.The interval between each two adjacent stimuli was 60 s,and it was repeated for 15 times.The animals were taken out after 180 s of adaptation.The rats in the blank control group were placed in the box and only given sound stimulation without any electrical stimulation.Other conditions were the same as above.The rats in each group were trained continuously for 4 days according to the above steps.Behavioral evaluation was conducted for each group of animals by EPM(5 min)on the 1st,7thand 10thday after state anxiety replication respectively.?.Study on the changes of neuronal information coding in related brain regions with state anxiety induced by conditioned fear in ratsMale SD rats are selected,and after 7 days of adaption in the experimental environment,the 16-channel vivo recording electrodes were implanted respectively in Pr L region of 1/3 of the animals,v HPC region of 1/3 of the animals and LA region of 1/3 of the animals under the condition of anesthesia and stereotaxometer.Three days after recovery,the experimental animals,which have implanted electrodes in their three different brain regions,were randomly divided into the blank control group and the model group for each brain region.Then,the blank control group and the model group were randomly divided into 1stday,7thday and 10thday groups respectively.Each group of experimental animals were placed in the conditioned fear analysis system to replicate state anxiety,with the same parameters as above.When the animals were awake and free to move,the Plexon in vivo multi-channel neural signal recording and analysis system was used to collect signals(Gain:1000)in each group on the 1st,7thand 10thday after state anxiety replication,respectively:first,the basic discharge signals of neurons in vivo in the daily feeding environment(5 min)were collected,and then the discharge signals of neurons in vivo in EPM were collected at the same time of EPM(5 min).Offline Sorter and Neuro Explorer software were used to process the collected electrical signals of neurons and calculated the spike firing rate of each type of neurons(?Neuron=spike firing rate in the EPM-baseline firing rate).Histological determination of the position of electrode implantation was carried out after all the electrical signals were collected.?.Study on the correlation between the behavioral changes and neuronal information coding in related brain regions with state anxiety induced by conditioned fear in ratsBased on the results of“I.Behavioral evaluation of conditioned fear induced state anxiety in rats”and“II.Study on the changes of neuronal information coding in related brain regions with state anxiety induced by conditioned fear in rats”.Using SPSS 26.0 statistical software and bivariate correlation analysis,the correlation between open arm/total movement time(%)and open arm/total entry times(%)in EPM and each type of?Neuron in Pr L,v HPC and LA region of rats with state anxiety was examined.?.Study on the changes of synaptic functional plasticity in related brain regions with state anxiety induced by conditioned fear in ratsMale SD rats were randomly divided into the blank control group and the model group after 7 days of adaptation in the experimental environment.The experimental animals in the blank control group and model group were divided into 1stday(Pr L region,v HPC region,LA region)group,7thday(Pr L region,v HPC region,LA region)group and 10thday(Pr L region,v HPC region,LA region)group according to the detection time and brain region.Each group of experimental animals were placed in the conditioned fear analysis system to replicate state anxiety,with the same parameters as above.Using BL-420F biological function experiment system under the condition of anesthesia and stereotaxometer,LTP induction and detection were performed(sampling rate:1k Hz,mode:coarse voltage single stimulation)in each group on the 1st,7thand 10thday after state anxiety replication:in each group of Pr L region,recording electrodes were implanted in Pr L region,and stimulation electrodes were implanted in v HPC region;in each group of v HPC region,recording electrodes were implanted in v HPC region and stimulation electrodes were implanted in LA region;in each group of LA aregion,recording electrode was implanted in LA region and stimulation electrode was implanted in Pr L region.The PS amplitude change rate(%)of each group was recorded,and the data without successful LTP induction were not included in the statistical results.Histological determination of the position of electrode implantation was carried out after all the electrical signals were collected.?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on the behavior with state anxiety induced by conditioned fear in ratsMale SD rats were randomly divided into blank control group,model group,positive drug group and raw-fried Ziziphi Spinosae Semen compatibility group(hereinafter referred to as ZSS group)after 7 days of adaptation in the experimental environment.Each group of experimental animals were placed in the conditioned fear analysis system to replicate state anxiety,with the same parameters as above.On the second day after the replication of state anxiety,rats in the ZSS group were given raw-fried Ziziphi Spinosae Semen compatibility lyophilized powder solution(20 g/kg)by intragastric administration,positive drug group were given diazepam suspension(3.6 mg/kg),blank control group and model group were given the same volume of distilled water,once a day for consecutive 10 days.After 30minutes of administration on the last day,behavioral evaluation was performed by EPM(5min)for each group of animals.?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on neuronal information coding in related brain regions with state anxiety induced by conditioned fear in ratsMale SD rats are selected,and after 7 days of adaption in the experimental environment,the 16-channel vivo recording electrodes were implanted respectively in Pr L region of 1/3 of the animals,v HPC region of 1/3 of the animals and LA region of 1/3 of the animals under the condition of anesthesia and stereotaxometer.Three days after recovery,the experimental animals,which have implanted electrodes in their three different brain regions,were randomly divided nto blank control group,model group,positive drug group and ZSS group for each brain region.Each group of experimental animals were placed in the conditioned fear analysis system to replicate state anxiety,with the same parameters as above.On the second day after the replication of state anxiety,rats in the ZSS group were given raw-fried Ziziphi Spinosae Semen compatibility lyophilized powder solution(20 g/kg)by intragastric administration,positive drug group were given diazepam suspension(3.6 mg/kg),blank control group and model group were given the same volume of distilled water,once a day for consecutive 10 days.When the animals were awake and free to move,the Plexon in vivo multi-channel neural signal recording and analysis system was used to collect signals in each group after 30 minutes of administration on the last day according to the above method,respectively.and the?Neuron of each type of neuron was recorded. ?.Study on the correlation between the intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on the behavior and the neuronal information coding in related brain regions with state anxiety induced by conditioned fear in ratsBased on the results of“V.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on the behavior with state anxiety induced by conditioned fear in rats”and“VI.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on neuronal information coding in related brain regions with state anxiety induced by conditioned fear in rats”.Using SPSS 26.0 statistical software and bivariate correlation analysis,the correlation between open arm/total movement time(%)and open arm/total arm entry times(%)in EPM and each type of?Neuron in Pr L,v HPC and LA region of state anxiety induced by conditioned fear in rats under the intervention of raw-fried Ziziphi Spinosae Semen drug pair was examined.?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on neuronal morphology in related brain regions with state anxiety induced by conditioned fear in ratsMale SD rats were randomly divided into blank control group,model group,positive drug group and ZSS group after 7 days of adaptation in the experimental environment.Each group of experimental animals were placed in the conditioned fear analysis system to replicate state anxiety,with the same parameters as above.On the second day after the replication of state anxiety,rats in the ZSS group were given raw-fried Ziziphi Spinosae Semen compatibility lyophilized powder solution(20 g/kg)by intragastric administration,positive drug group were given diazepam suspension(3.6 mg/kg),blank control group and model group were given the same volume of distilled water,once a day for consecutive 10days.After 30 minutes of administration on the last day,the brains of the animals in each group are taken out.The number and ultrastructure of neurons in Pr L,v HPC and LA region of each group of rats were observed by using Nissl staining and transmission electron microscopy.?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on synaptic functional plasticity in related brain regions with state anxiety induced by conditioned fear in ratsMale SD rats were randomly divided into blank control group,model group,positive drug group and ZSS group after 7 days of adaptation in the experimental environment.The experimental animals of each group were divided into Pr L region,v HPC region,LA region group according to the detection brain region.Each group of experimental animals were placed in the conditioned fear analysis system to replicate state anxiety,with the same parameters as above.On the second day after the replication of state anxiety,rats in the ZSS group were given raw-fried Ziziphi Spinosae Semen compatibility lyophilized powder solution(20 g/kg)by intragastric administration,positive drug group were given diazepam suspension(3.6 mg/kg),blank control group and model group were given the same volume of distilled water,once a day for consecutive 10 days.Using BL-420F biological function experiment system under the condition of anesthesia and stereotaxometer,LTP induction and detection were performed in each group after 30 minutes of administration on the last day according to the above method,respectively.and the PS amplitude change rate(%)in each group was recorded.?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on synaptic structural plasticity in related brain regions with state anxiety induced by conditioned fear in ratsMale SD rats were randomly divided into blank control group,model group,positive drug group and ZSS group after 7 days of adaptation in the experimental environment.Each group of experimental animals were placed in the conditioned fear analysis system to replicate state anxiety,with the same parameters as above.On the second day after the replication of state anxiety,rats in the ZSS group were given raw-fried Ziziphi Spinosae Semen compatibility lyophilized powder solution(20 g/kg)by intragastric administration,positive drug group were given diazepam suspension(3.6 mg/kg),blank control group and model group were given the same volume of distilled water,once a day for consecutive 10days.After 30 minutes of administration on the last day,the brains of the animals in each group are taken out.The synaptic ultrastructure in Pr L,v HPC and LA region of rats were observed by using transmission electron microscopy.Results:?.Behavioral evaluation of conditioned fear induced state anxiety in ratsThe results showed that,(1)1st,7th,and 10thdays after replication of state anxiety,compared with the blank control groups,the open arm movement time(s),open arm/total movement time(%),open arm entry times and open arm/total entry times(%)of model groups were significantly decreased(P<0.05,P<0.01),while the total arm entry times and exploration times had no significant changes(P>0.05).(2)1st,7th,and 10thdays after replication of state anxiety,(1)the behavioral indexes of the blank control groups showed no obvious change with the increase of time,there was no significant difference between each two(P>0.05);(2)the behavioral indexes of the model groups showed a decreasing trend with the increase of time,but there was no significant difference between each two(P>0.05).?.Study on the changes of neuronal information coding in related brain regions with state anxiety induced by conditioned fear in ratsThe results showed that,(1)1st,7th,and 10thdays after replication of state anxiety,compared with the blank control groups,?Neuron Pr L-1,?Neuron Pr L-2,?Neuron LA-1and?Neuron LA-2 of model groups were significantly increased(P<0.05,P<0.01),?Neuron Pr L-3,?Neuron v HPC-1,?Neuron v HPC-2 and?Neuron v HPC-3 were significantly decreased(P<0.05,P<0.01).(2)1st,7th,and 10thdays after replication of state anxiety,(1)all types of?Neuron of blank control groups showed no obvious change with the increase of time,and there is no significant difference between the each two(P>0.05);(2)the?Neuron Pr L-1,?Neuron LA-1 and?Neuron LA-2 of model groups showed a gradually increasing trend with the increase of time,and compared with 1stday after replication,?Neuron Pr L-1,?Neuron LA-1 and?Neuron LA-2 in 10thday after replication were significantly increased(P<0.05);(3)the?Neuron Pr L-3,?Neuron v HPC-1,?Neuron v HPC-2 and?Neuron v HPC-3 of model groups showed a gradually decreasing trend with the increase of time,and compared with 1stday after replication,?Neuron v HPC-3 in 10thday after replication was significantly decreased(P<0.05).?.Study on the correlation between the behavior and the neuronal information coding in related brain regions with state anxiety induced by conditioned fear in ratsThe results showed that,there were significant correlation between EPM anxiety-related behavior and neuronal information coding in Pr L,v HPC and LA region in each group.The open arms/total exercise time(%)and open arms/total number of open arms(%)were highly or moderate negatively correlated with?Neuron Pr L-1,?Neuron Pr L-2,?Neuron LA-1 and?Neuron LA-2(P<0.01;0.8<?r?<1.0,0.5<?r?<0.8),were highly or moderately positively correlated with?Neuron Pr L-3,?Neuron v HPC-1,?Neuron v HPC-2 and?Neuron v HPC-3(P<0.01;0.8<?r?<1.0,0.5<?r?<0.8).?.Study on the changes of synaptic functional plasticity in related brain regions with state anxiety induced by conditioned fear in ratsThe results showed that,(1)(1)1stday after replication,compared with the blank control group,the PS amplitude change rate(%)in Pr L region,v HPC region and LA region of model group had no significant change(P>0.05).(2)7thday after replication,compared with blank control group,the PS amplitude change rate(%)in Pr L region and LA region in model group were significantly increased(P<0.05,P<0.01),and the PS amplitude change rate(%)in v HPC region was significantly decreased(P<0.01);(3)10thday after replication,compared with the blank control group,the PS amplitude change rate(%)in Pr L region and LA region in model group were significantly increased(P<0.05,P<0.01),and the PS amplitude change rate(%)in v HPC region was significantly decreased(P<0.01).(2)1st,7th,and 10thdays after replication of state anxiety,(1)the PS amplitude change rate(%)in Pr L region,v HPC region and LA region of blank control groups showed no obvious change with the increase of time,there was no significant difference between each two(P>0.05);(2)compared with 1stday after replication,the PS amplitude change rate(%)in Pr L region and LA region were significantly increased(P<0.05,P<0.01),and the PS amplitude change rate(%)in v HPC region was significantly decreased(P<0.01)in model group at 7thday after replication;(3)compared with 1stday after replication,the PS amplitude change rate(%)in Pr L region and LA region were significantly increased(P<0.05,P<0.01),and the PS amplitude change rate(%)in v HPC region was significantly decreased(P<0.01)in model group at 10thday after replication;(4)compared with 7thday after replication,the PS amplitude change rate(%)in Pr L region,v HPC region and LA region were not significantly different(P>0.05)in model group at 10thday after replication.?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on the behavior with state anxiety induced by conditioned fear in ratsThe results showed that,compared with the blank control group,the open arm movement time(s),open arm/total movement time(%),open arm entering times and open arm/total entry time(%)in the model group were significantly decreased(P<0.05,P<0.01),while the total arm entering times and exploration times had no significant changes(P>0.05).Compared with model group,the open arm movement time(s),open arm/total movement time(%),open arm entering times,open arm/total entry time(%)in positive drug group and ZSS group were significantly increased(P<0.05,P<0.01),while the total arm entering times and exploration times had no significant changes(P>0.05).?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on neuronal information coding in related brain regions with state anxiety induced by conditioned fear in ratsThe results showed that,compared with blank control group,?Neuron Pr L-1,?Neuron Pr L-2,?Neuron LA-1 and?Neuron LA-2 in model group rats were significantly increased(P<0.01),?Neuron Pr L-3,?Neuron v HPC-1,?Neuron v HPC-2 and?Neuron v HPC-3 were significantly decreased(P<0.01).Compared with model group,?Neuron Pr L-1,?Neuron Pr L-2,?Neuron LA-1 and?Neuron LA-2 in positive drug group and ZSS group were significantly decreased(P<0.05,P<0.01),?Neuron Pr L-3,?Neuron v HPC-1,?Neuron v HPC-2 and?Neuron v HPC-3 were significantly increased(P<0.01). ?.Study on the correlation between the intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on the behavior and the neuronal information coding in related brain regions with state anxiety induced by conditioned fear in ratsThe results showed that,there were significant correlation between EPM anxiety-related behavior and neuronal information coding in Pr L region,v HPC region and LA region in each groups.The open arms/total exercise time(%)and open arms/total number of open arms(%)were highly or moderate negatively correlated with?Neuron Pr L-1,?Neuron Pr L-2,?Neuron LA-1 and?Neuron LA-2(P<0.01;0.8<?r?<1.0,0.5<?r?<0.8),were highly or moderately positively correlated with?Neuron Pr L-3,?Neuron v HPC-1,?Neuron v HPC-2 and?Neuron v HPC-3(P<0.01;0.8<?r?<1.0,0.5<?r?<0.8).?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on neuronal morphology in related brain regions with state anxiety induced by conditioned fear in ratsThe results showed that,(1)compared with blank control group,the number of neurons in Pr L region,v HPC region and LA region in model group was significantly decreased(P<0.01).Compared with model group,the number of neurons in Pr L region,v HPC region and LA region in positive drug group and ZSS group were significantly increased(P<0.01).(2)Compared with the blank control group,the whole neuron cell and organelle structure in Pr L region,v HPC region and LA region of the model group were damaged,which showed as follows:the neurons were irregularly arranged and loosely distributed,the Nissl bodies in cytoplasm were decreased,and even some neurons appeared Nissl body rupture and cytoplasmic light staining,neurons swelled obviously,cell membrane and nuclear membrane were blurred,organelles were few,rough endoplasmic reticulum expanded and ribosome fell off,cristae of mitochondria decreased or disappeared,and some cells were necrotic.Compared with the model group,the whole cell and organelle structure in Pr L region,v HPC region and LA region of both the positive drug group and the ZSS group were improved to varying degrees,which were as follows:the neuronal arrangement was regular and compact,the number of Nissl bodies were increased,and the neuronal swelling were not obvious,the cell membrane and nuclear membrane were intact,the number of organelles were increased,the phenomenon of organelle swelling was rare,the mitochondrial cristae were rich and clear,the cell necrosis was reduced.?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on synaptic functional plasticity in related brain regions with state anxiety induced by conditioned fear in ratsThe results showed that,compared with blank control group,the PS amplitude change rate(%)in Pr L region and LA region of model group were significantly increased(P<0.05,P<0.01),and the PS amplitude change rate(%)in v HPC region was significantly decreased(P<0.01).Compared with model group,the PS amplitude change rate(%)in Pr L region and LA region in positive drug group and ZSS group were significantly decreased(P<0.05,P<0.01),and the PS amplitude change rate(%)in v HPC region was significantly increased(P<0.05,P<0.01).?.Intervention effect of raw-fried Ziziphi Spinosae Semen compatibility on synaptic structural plasticity in related brain regions with state anxiety induced by conditioned fear in ratsThe results showed that,compared with the blank control group,the synaptic structure in Pr L region,v HPC region and LA region of model group were damaged,which was characterized by synaptic contour and boundary were blurred,synaptic vesicles,microtubules and microfilaments were reduced or even disappeared,and vacuolization appeared.Compared with the model group,the synaptic structure in Pr L region,v HPC region and LA region of both the positive drug group and the ZSS group was improved to varying degrees,such as:synaptic contour and boundary were complete and clear,synaptic vesicles,microtubules and microfilaments were abundant and clear,and vacuolation changes were reduced.Conclusion:1.Conditional fear leads to the increase of anxiety-like behavior,which better replicates the model of animal state anxiety and has a certain time stability.2.The mechanism of state anxiety induced by conditioned fear in rats is related to changes of the neuronal information coding and synaptic functional plasticity in Pr L region,v HPC region and LA region.Meanwhile,the changes of synaptic functional plasticity in Pr L region,v HPC region and LA region of rats by conditioned fear are not obvious in the acute stage of conditioned fear,but will gradually change in the later stage.3.The raw-fried Ziziphi Spinosae Semen compatibility can antagonize the state anxiety induced by conditioned fear and reduce the anxiety-like behavior of rats.The mechanism of this action may be through regulating the neuronal information coding and synaptic functional plasticity in Pr L region,v HPC region and LA region,and improving the neuronal morphology and synaptic structural plasticity damage. |
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