Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

Figure 4

RAG1 antisense amygdalar treatment impaired consolidation of context fear conditioning. Top panel: diagram depicting the experimental design of these experiments for pretraining or posttraining amygdalar antisense or random oligonucleotide microinfusion experiments. In the pretraining microinfusion experiments, mice received RAG1 antisense or random bilateral oligonucleotide microinfusions directed at the amygdala 1 h before conditioning followed by either LTM testing or molecular evaluation. LTM was tested 24 h after conditioning. For molecular evaluation of antisense treatment effectiveness, another group of mice was sacrificed 30 min after conditioning and amygdalar RNA was used for real-time PCR. In the posttraining microinfusion experiments, mice were conditioned, returned to their home cages, and received microinfusions of antisense or random oligonucleotides 5 h after training and returned to their home cages until next day. Nineteen (19) hours later (24 h after conditioning), mice were reexposed to the conditioning chamber without any shocks in order to test LTM. (a) Mice receiving either RAG1 antisense or random oligonucleotide treatment displayed no significant differences during memory acquisition measured as the progressive enhancement of freezing behavior (Two-Way ANOVA, Treatment Factor: , ; Training Factor , ; Interaction: , ). Bonferroni posttesting analysis did not identify significant differences between the groups during the habituation or the 1st, 2nd, or 3rd trials of training (, each comparison), indicating that both groups were similarly capable of learning the task. (b) LTM was tested 24 h after conditioning. The bar graph shows that, unlike the results obtained for acquisition, mice treated with RAG1 antisense gapmer oligonucleotides displayed significantly less percent freezing to the conditioning context than random oligonucleotide controls during the LTM test (Student’s -test; ; ). (c) The molecular effectiveness of our knockdown by gapmer antisense oligonucleotide of RAG1 in the amygdala was determined by quantitative real-time PCR. Mice were infused 1 h before context fear conditioning with bilateral RAG1 antisense or random oligonucleotides and decapitated 30 min after conditioning. RAG1 mRNA normalized against gapdh mRNA showed that treatment with RAG1 antisense gapmer oligonucleotides effectively knocked down the levels of RAG1 amygdalar mRNA compared to the random controls (Student’s -test; ; ). No significant differences in the levels of gapdh were observed between treatments (data not shown). (d) We used 5 h posttraining amygdalar microinfusions of RAG1 antisense oligonucleotides or random controls with a different set of animals without any pretraining infusion and LTM was tested 24 h after training. Unlike in the pretraining microinfusion experiments, both the antisense and random posttraining-infused mice displayed similar levels of conditioned freezing during the LTM test (Student’s -test; ; ).