| Objective:Based on experiments,to elucidate the effect and mechanism of ZhuShaAnShen Pill on fear memory in conditioned fear rats.Methods:Part 1.The effect of ZhuShaAnshen Pill on fear memory in conditioned fear rats.1.The effects of ZhuShaAnShen Pill on the acquisition of fear memory in conditioned fear ratsConditional fear model replication:The experimental animals were randomly divided into the blank control group,the model control group and the drug experimental group,with 10 rats in each group.The experimental animals in the drug experimental group were given ZhuShaAnShen Pill suspension(given by intragastric administration,0.81mg/g),the experimental animals in the blank control group and the model control group were given the same volume of distilled water,1 times/day for 7 consecutive days.After the last drug delivery,the experimental animals in the model control group and the drug experimental group were placed in the conditioned fear stimulus box to reproduce the conditioned fear model,adapt to 120 seconds.In 120th seconds,the experimental animals were given a single frequency speech signal(2000Hz,75dB).In 125th seconds,on the basis of the sound signal,the experimental animals were given inescapable plantar electrical stimulation(0.75mA).In 130th seconds,the sound signal and electrical stimulation stop at the same time,and no processing is done in 130?190 seconds.In 190th seconds,repeat the 120?190 second process for 20 consecutive times.The experimental animals in the blank control group,only the voice signal was given without electrical stimulation,and the other operations were the same.After that,let the experimental animals stay in the stimulus box for 120 seconds before putting them back to the cage.Fear memory acquisition test:On the next day of replication conditioned fear model,the experimental animals in each group were put into the stimulus box in turn,and adapted to 120 seconds.In 120th seconds,the experimental animals were given the single frequency speech signal(2000Hz,75dB).In 130th seconds,the sound signal stop,no processing is done in 130?190 seconds,recording the time of freezing time of the experimental animals within 60 seconds after stopping the sound signal.In 190th seconds,repeat the 120?190 second process for 5 consecutive times.After that,let the experimental animals stay in the stimulus box for 120 seconds before putting them back to the cage.As an observation index to explore the influence of ZhuShaAnShen Pill on fear memory acquisition in conditioned fear rats,the freezing time was recorded.2.The effect of ZhuShaAnshen Pill on the decline of fear memory in conditioned fear ratsThe experimental animals were replicated the conditioned fear model according"The effects of ZhuShaAnShen Pill on the acquisition of fear memory in conditioned fear rats".After the acquisition test,the experimental animals were put in the conditioned fear stimulus box again on the first,third,fifth and seventh days,the operation of repeated fear memory acquisition test.As an observation index to investigate the effect of ZhuShaAnShen Pill on the decline of fear memory in conditioned rats,the Freezing time was recorded.3.The effect of ZhuShaAnShen Pill on autonomic activity in conditioned fear ratsThe experimental animals were replicated the conditioned fear model according"The effects of ZhuShaAnShen Pill on the acquisition of fear memory in conditioned fear rats".On the next day,the experimental animals in each group were placed in the opening experiment box of the rat,and in order to explore the effect of ZhuShaAnShen Pill on the autonomic activity of conditioned fear rats,the movement time and distance of the animals were recorded.Part 2.The effects of ZhuShaAnShen Pill on hippocampal synaptic plasticity in conditioned fear rats.1.The effect of ZhuShaAnShen Pill on the hippocampal synaptic functional plasticity in conditioned fear ratsThe experimental animals were replicated the conditioned fear model according"The effects of ZhuShaAnShen Pill on the acquisition of fear memory in conditioned fear rats".On the next day,the experimental animals in each group were anesthetized with urantan,the stimulation electrode and the recording electrode are buried.After record the basic level of population spike,Long time potentiation(LTP)was induced by high frequency stimulation(HFS).The population spikes(PS)in hippocampal CA1 area of the experimental animals in each group were recorded,according to the change rate of PS,the effect of ZhuShaAnShen Pill on the hippocampal synaptic functional plasticity in conditioned fear rats was investigated.2.The effect of ZhuShaAnShen Pill on the hippocampal synaptic structural plasticity in conditioned fear ratsThe experimental animals were replicated the conditioned fear model according"The effects of ZhuShaAnShen Pill on the acquisition of fear memory in conditioned fear rats".On the next day,three experimental animals were randomly selected in each group.after anesthetize experimental animals by intraperitoneal injection of chloral hydrate,the hippocampus was taken out and the ultrathin sections of the transmission electron microscope were made to observe the synaptic morphology around the neuropil in Hitachi(H-7650)transmission electron microscope magnified 30000 times.Measure the length of the synaptic active zone,the thickness of postsynaptic density,and the width of the synaptic gap in hippocampal CA1 area of experimental animal by Image-Pro Plus6.0 to explore the effect of ZhuShaAnShen Pill on the hippocampal synaptic structural plasticity in conditioned fear rats.3.The protective effect of ZhuShaAnShen Pill for the hippocampal neurons in conditioned fear ratsThe experimental animals were replicated the conditioned fear model according"The effects of ZhuShaAnShen Pill on the acquisition of fear memory in conditioned fear rats".On the next day,three experimental animals were randomly selected in each group.after anesthetize experimental animals by intraperitoneal injection of chloral hydrate,the hippocampus was taken out and the ultrathin sections of the transmission electron microscope were made to observe the neuronal morphology around the neuropil in Hitachi(H-7650)transmission electron microscope magnified 8000 times,and observe the intracellular organelle morphology around the neuropil in magnified 15000 times.Results:Part 1.Part 1.The effect of ZhuShaAnShen Pill on fear memory in conditioned fear rats.1.The effects of huShaAnShen Pill on the acquisition of fear memory in conditioned fear ratsThe experimental results show that:At the time point of 1min,3min,5min and the time point of 2min and 4min in the acquisition of conditioned fear memory,the freezing time of the experimental animal in model control group was higher than that of the blank control group,and there was significant difference between the two groups(P<0.01,P<0.05..Comparison with model control group,the freezing time of the experimental animal in the drug experimental group was lower than that of the model control group,but there was no significant difference between the two groups.2.The effect of ZhuShaAnShen Pill on the decline of fear memory in conditioned fear ratsThe experimental results show that:In the fade phase of conditioned fear memory,the freezing time of experimental animals in each group were gradually reduced.Compared with the blank control group,the freezing time of experimental animal in the model control group in each time period were significantly higher,there is a very significant difference between the two(P<0.01).Compared with the model control group,the freezing time of experimental animal in the drug experimental group were lower than model control group.The fifth day,the freezing time of experimental animal in the drug experimental group was lower than the model control group,there is significant differences in the comparison of the two(P<0.05).The seventh days,the freezing time of experimental animal in the drug experimental group was significantly lower than the model control group,there is a very significant difference between the two(P<0.01).3.The effect of ZhuShaAnShen Pill on autonomic activity in conditioned fear ratsThe experimental results show that:Compared with the blank control group,the movement time of experimental animal in the model control group,the movement distance were less than the blank control group,movement distance,but there is no significant difference.Compared with the model control group,the movement time of experimental animal in the medicine group drug experimental group,movement distance were higher than the model control group,but also no significant difference.Part 2.The effects of ZhuShaAnShen Pill on hippocampal synaptic plasticity in conditioned fear rats.1.The effect of ZhuShaAnShen Pill on the hippocampal synaptic functional plasticity in conditioned fear ratsThe experimental results show that:After HFS,the PS of experimental animal in each group increased more than 20%,this results showed that all groups of LTP were successful.Compared with the blank control group,the amplitude of PS in the model control group increased significantly at 40min,70min,80min,90min,100min,compared with significant difference(P<0.05).Compared with the model control group,the amplitude of PS in the drug experimental group significantly decreased at 120min,there is significant differences in the comparison of the two(P<0.05).2.The effect of ZhuShaAnShen Pill on the hippocampal synaptic structural plasticity in conditioned fear ratsThe experimental results show that:observe the number and the morphology of synapse in hippocampus CA1 area of experimental animal in each group.Compared with the blank control group,a large number of vacuoles appear in the hippocampal neuropil of experimental animal in the model control group,the number of synapses decreased significantly,the marginal boundaries of the presynaptic membrane and the postsynaptic membrane are not clear,the synaptic gap is blurred and the synaptic vesicles in the presynaptic membrane decrease and even disappear,the mitochondria were swollen in the presynaptic membrane,the internal structure dissolved,the crista decreased and even disappeared.Compared with the model group,no vacuoles were found in the nerve felt in the drug group,the number of synapses was more,the structural boundaries of each part of the synapse were clear,and the synaptic vesicles in the presynaptic membrane were also more round,clear and densely distributed,the presynaptic and posterior membranes were close to the blank group,the mitochondria structure in the presynaptic membrane was slightly swollen,but the ridges were more visible.Compare the length of synapse active zone,the thickness of synapse PSD and the width of synaptic gap in the hippocampal CA1 area of experimental animal,the length of synaptic active zone of experimental animal in the model control group(212.65 + 48.63nm)was significantly less than the biank control group(247.14 +27.57nm),compared with a very significant difference(P<0.01),the synaptic PSD thickness(37.98 + 7.84nm)was significantly less than the blank control group(51.20+ 9.48nm),compared with a very significant difference(P<0.01)and the width of synaptic cleft(31.73 + 5.20nm)were significantly higher than blank control group(22.06 + 2.80nm),compared with a very significant difference(P<0.01).The length of synapses active zone of experimental animal in the drug experimental group(238.73+ 19.20nm)was longer than the model control group,compared with significant difference(P<0.05),the synaptic PSD thickness(51.54 + 6.87nm)was significantly higher than the model control group,both with significant difference(P<0.01),the width of synaptic cleft(21.90 + 2.71nm)was significantly less than the model control There were very significant differences between the two groups(P<0.01).3.The protective effect of ZhuShaAnShen Pill for the hippocampal neurons in conditioned fear ratsThe experimental results show that:comparison of the morphology of neurons in the hippocampal CA1 area of experimental animal in each group,the structure of hippocampal neurons,cell membrane,Nuclear membrane of experimental animal in the blank group were clear,the organelles in the cytoplasm were scattered,and the structure was intact.The cell structure is complete,but the cell membrane is unclear,the nuclear membrane is not clear,and the nucleus crinkle in the model control group,vacuoles appeared in the cytoplasm,and some rough endoplasmic reticulum showed obvious expansion,most mitochondria appeared swelling and crest reduction or disappearance,even a small number of mitochondria dissolved,and vacuoles appeared inside and some lysosomes in the cytoplasm of some neurons.The structure of hippocampal neurons of experimental animal in the drug experimental group is complete and clear,Large nucleus,cell membrane and nuclear membrane was integrity,more organelles in cytoplasmic and are better morphology,vacuoles and organelle deformability in cytoplasm decreased compared with model control group,the expansion of rough endoplasmic reticulum was not obvious,most mitochondria were well formed,the crista is more,clearly visible,the small part of mitochondria is slightly swollen and the crista decreases.Conclusion:1.ZhuShaAnShen pill can promote the decline of the fear memory.2.The effect of ZhuShaAnshen Pill on the extinction of fear memory is related to the protection of hippocampal neurons,and the enhancement of hippocampal synaptic structure and functional plasticity. |
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