| BackgroundLearning and memory are superior functions of human brain. Exploring the mechanism of learning and memory can be helpful to investigate functions of brain which also makes significance in treatment of impairment of learning and memory related diseases.The amygdala is one of the most crucial structures in limbic system, which closely associates with learning and memory, mood and emotion. Furthermore, it also participates in the pathologic process of a variety of nervous system disorders, for instance, schizophrenia, depression, epilepsy and posttraumatic stress disorder. Amygdala is a critical brain region where fear conditioning memory is acquired and stored. Fear conditioning memory is the result of fear conditioning learning in which an animal are subjected to a neutral conditional stimulus that is paired with an aversive unconditional stimulus.RAG1, recombination activating gene-1, was first discovered in the immune system and played an important role in specific memory activities of immune cells. The encoding products of RAG1can be coordinated with a variety of other proteins, which leads to the V(D)J rearrangement, resulting in generating high diversity of the immunoglobulin and T cell receptor. It has been reported that RAG1knockout animals can result in immune memory function deficiency, causing severe combined immunodeficiency disease. Previous researches have found transcription of RAG1in the central nervous system, most apparently in the hippocampus, cerebral limbic system and other areas, both of which are related for learning and memory. However, whether RAG1are involved in learning and memory or not has not been proved clear yet.The central nervous and immune system shared the capability of encoding memory, and also it has been proved that the brain exiting a genetic recombination which is similar to that in immune system. In order to clarify whether the deletion of RAG1gene in brain will affect the learning and memory function or not, studies have been taken to examine the RAG1knockout mice and the result suggested that the deletion of RAG1caused an alteration in their nervous behavior. Nevertheless, doing research on the function of RAG1in the central nervous system by knockout the target gene remains controversial, for the deficiency of RAG1from embryo strage can cause immunodeficiency, and even have interference on the development process, resulting in affecting the results.Accordingly, we approach RNA interference technology to avoid the disadvantages in knockout method by silencing RAG1gene expression in amygdala, aiming to identify the changes in learning and memory ability in rats after RAG1gene silencing.PART1Effect of fear conditioning on the expression of RAG1in rats amygdala.ObjectiveTo investigate the impact of fear conditioning on the expression of RAG1in amygdala.Methods and resultsSD rats were randomly divided into two groups, control group and fear conditioning group. The rat fear conditioning models were built, and freezing time was scored and used as a behavioral index of fear. Real-time PCR was used to detect the expression of RAG1in rat amygdala30min and1h after fear conditioning. Results showed that the freezing time recorded from rats in the experimental group was longer than that in the control group after the rats were re-exposed to the context without electrical footshock. The difference was statistically significant. Compared with the control group, the expression of RAG1in the amygdala after fear conditioning was significantly increased.ConclusionThe expression of RAG1mRNA was increased in amygdale after fear conditioning.PART2Construction and identification of RNAi lentiviral vector targeting rat RAG1geneObjectiveTo construct effective lentiviral vectors and observe the inhibitory effect on RAG1expression.Methods and resultsTo silence RAG1expression, shRNA sequence targeting of RAG1mRNA target was designed, and a scrambled shRNA target sequence was designed as a negative control, followed by annraling, cloning and sequencing. Then the two shRNAs were cloned to the expression plasmid, pMagic4.1. The two recombinant plasmids were respectively cotransfected into293T cells with pRsv-Rev, pMDLg/pRRE and pMD2G to produce a recombinant lentivirus.The titer of virus was measured by Real-time PCR before infecting on cultured rats’ neurons in vitro. Then the expression level of RAG1was detected by western blot and Real-time PCR. The value of LV-shRNA virus titer was2.08×108TU/ml, the control virus (LV-Negative) titer was1.79×109TU/ml. Compared with nomal group, the expression levels of RAG1mRNA and protein were significantly decreased in neurons after infecting with the LV-shRNA. ConclusionAccording to the gene sequence of RAG1, the specific shRNA sequence was designed by siRNA target finder software. In this study, the lentiviral vector inhibiting the expression of RAG1was successfully constructed.PART3Effect of RNA-mediated RAG1silencing in amygdala on the ability of fear memory in ratsObjectiveThis paper was designed to study the behaviour of learning and memory after the knockdown of RAG1, and the morphological changes of amygdala neurons.Methods and resultsSD rats were randomly divided into three groups:normal group, LV-shRNA group and LV-Negative group. Lentivirus was injected into amygdala in the rat brain through stereotaxic apparatus. The expression of RAG1mRNA was detected by RT-PCR one week after stereotaxic injection, and the expression of GFP were observed by immunofluorescence. RAG1mRNA level decreased after lentivirus injecting analyzed by RT-PCR, which suggested that the expression of RAG1mRNA was restrained. The rats are exposed to behavioral test one week after lentivirus injection. In Morris water maze test, there were no statistical differences among all the groups in time finding the hidden platform and the number of target platform crossings. Then a passive-avoidance test was performed to measure learning and memory ability in rats. The result suggested that in the training trail, there was no difference in the latency to enter dark compartment among all the groups. While in the test trial, the latency of LV-shRNA group significantly decreased compared with the other two groups. Fear conditioning results showed that the freezing time of LV-shRNA group reduced in comparison to the control group and LV-Negative group. The rats were sacrificed after fear conditioning, and amygdaloid nucleus were taken out for embedding and section. Number of synapses of the LV-shRNA in amygdaloid nucleus was less than the control group through electron microscope.ConclusionThe expression of RAG1mRNA significantly decreased in amygdale after the injection of lentivirus, and the ability of fear memory and number of synapses in amygdala reduced subsequently compared with normal group. |
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