| Objective Pain and fear are considered protective for organism to avoid dangerous and harm. They strongly impact on feeling and behavior. Pain and fear, however, may become maladaptive for organism if expressed under inadequate conditions or lasted for too long time. Tree shrew, a small mammals belongs to Scandentia, has many similarity with human beings such as Anatomy, physiology, immunology, pathology and so on. Therefore, formation and erasing fear conditioning memory on tree shrew may provide better animal models for near future therapies in human being diseaseMethods1. Exp.?: The activities regularly of tree shrew in light/dark box. This experiment(Exp.) included three phases: training phase, arena familiarization phase and test phase. During the training phase, all animal was allowed to free move in device for 30 min every day. This phase was continued for 3 days. During arena familiarization phase, there were three days for animals adapt the environment and experimental rhythm. During this phase, each animal was allowed 10 min(calculated after the animal entered device 3 min) to free move in the light/dark box. The handing of animal during test phase was consistent with adaptive phase, simultaneously, the animal behavior indexing of travel distance, travel velocity, duration and frequency was analyzed during the test phase. This experiment independently repeated three times.2. Exp. ?: The suitable voltage degree for foot shock on tree shrew. In this Exp., the two chambers were all called light(the LEDs were all turned on). This Exp. was made up by two phases: Arena adaption phase and test phase. Arena adaption phase was the same as Exp.?. After three days of adaption to the apparatus, the test phase was carried out. During test phase, animals were executed 10 min with stimulus appearing in one chamber randomly. The stimulus protocol was designed as follows: 10 s stimulus of a series voltages from 0 V to 20 V with 4 V step with 30 s interval for total 10 min. The voltage degree would change till all animal had been tested in one voltage during this Exp. For searching the suitable voltage degree.3. Exp. ?: The fear conditioning memory formation in tree shrew. This Exp. was made up with four phases: Arena familiarization phase, pre-stimulus recording phase, fear memory formation phase and the upkeep of fear memory phase. Arena familiarization phase was the same as Exp.?. During the fear memory formation phase, a suitable stimulus was given once the animal got into the dark–box to form the fear conditioning memory. When the fear memory formation phase was finished, the stimulus would be removed. The stimulus protocol was the similar with Exp. ?. The two chambers were light when the animals entranced into the appliance, and then, one of chamber turned into dark randomly. Each animal was recorded once a day.4. Exp. ?: The fear conditioning memory erased in tree shrew. After fear conditioning memory formed 7 weeks, the Exp. of erasing memory was activated. This Exp. was made up with three phases: the original fear memory maintaining phase, the stage of establishing a new fear memory and the original fear memory erased stage. During the stage of establishing a new fear memory, a suitable stimulus was given once the animal got into the light–box to establishing a new fear memory. The stimulus protocol was the similar with Exp. ?. The two chambers were dark when the animals entranced into the appliance, and then, one of chamber turned into light randomly. Each animal was recorded once a day. Each animal was tested once a day.5. Data collection In all experiments, all animals in the apparatus were videotaped once a day between 14?00 ~ 18?00. During the 10 min test phase, the movie was collected by video camera fixed on the ceiling. The movie recording with succedent estimation of the data was performed using Etho Vision XT 9(Noldus, Wageningen, Netherlands). The data of duration time, travel velocity, travel distance in each chamber and frequency between two chambers were collected and analyzed. When the tree shrew was crossing the chambers, we defined the animal belongs to the chamber which its center was in.6. Data analysis and statistics All data was statistically analyzed using SPSS 16.0. Data was expressed by mean ± SEM(`x ± s) and processed by the commercially available software Graph Pad Prism 5.0. Experiment ? and experiment ? were analyzed through paired samples t-test. Experiment ? and experiment ? was analyzed through a repeated measures analysis of variance(ANOVA). Within-group effects were analyzed by a post-hoc Duncan's multiple range test with a Huynh/Feldt adjustment. The significance level was set at P < 0.05.Results1. The activities regularly of tree shrew in light/dark box Without any stimulus, the tree shrew(n = 4) was allowed to move in light/dark box. The results showed that compared with duration in dark box, the duration in light box was decline about four times [(117.075 ± 31.176) s vs(476.773 ± 26.965) s, P < 0.01]. The percentage of velocity had significant difference between light box and dark box. The percentage of distance moved had no significant difference. The frequency between two chambers was(21.42 ± 4.245). All results suggest that the tree shrew had a significant preference in the dark-box compare to the light-box.2. The suitable voltage degree for foot shock on tree shrew. The animals were put into the box which had two same chambers(A and B), and voltage pulses were applied to one of the two chambers. We found that under different voltage degree, the animals(n = 6) showed different activities characteristics. At 4 and 8 volts, all indicator of duration, travel distance, travel velocity and frequency are not significant different(P > 0.05) from the 0 volts. When the given voltage exceeds 12 volts or higher, the duration was significant different(P < 0.05) from the 0 volts. Percentage of travel distance and percentage of travel velocity showed no significant difference under 16 volts(P > 0.05). They, however, showed significant difference when the voltage degree was 20 V(P < 0.05). The Frequency showed significant difference when the voltage degree was 16 V or higher(P < 0.05). In order to get the significant different results and considered the animal care, the foot shock stimulus voltage was chosen at 16 V in following experiments, for the 20 V has serious injuries on tree shrew.3. The fear conditioning memory formation in tree shrew. All animals were divided into trial group(n = 4, with stimulus in dark-box) and control group(n = 4, without stimulus in dark-box). The results showed that two groups of tree shrew were like activity in the dark box, which conformed to the tree shrews like dark habits before the electrical stimulation. After stimulation, compared with control group, the duration and travel distance of trial group were increased in light-box(P< 0.001), the travel velocity was decreased(P < 0.001). Compared with control group, the duration and travel distance of trial group were decreased in dark-box(P < 0.001), the travel velocity was increased(P < 0.001). The frequency between two chambers was decreased significant(P < 0.001) comparing the two groups. All results showed that the fear conditioning memory with dark-box on tree shrew had been formed.4. The fear conditioning memory erased in tree shrew After fear conditioning memory maintain phase finished, the fear conditioning memory erased experiment was evoked. The results showed that before the stimulus supplying in light box, the original fear memory was no decay. As the stimulus supplying, compared with control group, the duration and travel distance of trial group were decreased in light-box(P > 0.05), the travel velocity was decreased(P > 0.05). However, compared with control group, the duration and travel distance of trial group were decreased in dark-box(P > 0.05), the travel velocity was increased(P > 0.05). The frequency between two chambers was decreased significant(P < 0.05) after increased comparing the two groups. All results showed that the fear conditioning memory with light-box on tree shrew had been formed, the fear conditioning memory with dark-box had been erased at the same time.ConclusionTree shrews fear conditioning memory can be successfully induced, and it can maintain more than 7 weeks which shows no sign of fading. Compare with the rodents, tree shrew about the natural attenuation of conditioned fear model is more stable. The conditions of the tree shrews fear memory can be selectively erased in certain circumstances. The study of tree shrews model will provide better animal model for human disease in the near future. |
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