Learning-induced ribosomal RNA is required for memory consolidation in mice-Evidence of differentially expressed rRNA variants in learning and memory

Abstract

The transition from short-term to long-term forms of synaptic plasticity requires protein synthesis and new gene expression. Most efforts to understand experience-induced changes in neuronal gene expression have focused on the transcription products of RNA polymerase II-primarily mRNAs and the proteins they encode. We recently showed that nucleolar integrity and activity-dependent ribosomal RNA (rRNA) synthesis are essential for the maintenance of hippocampal long-term potentiation (LTP). Consequently, the synaptic plasticity and memory hypothesis predicts that nucleolar integrity and activity dependent rRNA synthesis would be required for Long-term memory (LTM). We tested this prediction using the hippocampus-dependent, Active Place Avoidance (APA) spatial memory task and found that training induces de novo rRNA synthesis in mouse dorsal hippocampus. This learning-induced increase in nucleolar activity and rRNA synthesis persists at least 24 h after training. In addition, intra-hippocampal injection of the Pol I specific inhibitor, CX-5461 prior to training, revealed that de novo rRNA synthesis is required for 24 h memory, but not for learning. Using qPCR to assess activity-dependent changes in gene expression, we found that of seven known rRNA expression variants (v-rRNAs), only one, v-rRNA IV, is significantly upregulated right after training. These data indicate that learning induced v-rRNAs are crucial for LTM, and constitute the first evidence that differential rRNA gene expression plays a role in memory.

Fig 1. Ribosomal RNA (rRNA) synthesis is induced by spatial memory training.

A) Timeline of training protocol. B, C) APA behavioral data from each 30 min trial (pre-training, and training trials 1, 2, and 3). Three groups of mice were analyzed: 1) Untrained, 2) Yoked, and 3) Trained. B) Representative paths (grey tracings) of individual animals during pre-training and each 30 min training trial. Red circles indicate the animal’s location when a shock was received. Grey circles indicate the location of the animal when it would have received a shock had the shock zone been active. C) Left, The number of entrances into the shock zone during pre-training and each training trial. Only the APA trained mice (black circles) learned to actively avoid the zone. Right, Time to first entry into the shock zone was significantly higher for the Trained group beginning with the 2^nd training trial, compared to Yoked and Untrained controls. D) Relative levels of gene expression in mouse dorsal hippocampi one hour after third training trial, as determined by Real-time qPCR. Left, Precursor rRNA (ht-rRNA) expression is upregulated only in the Trained group, not in Untrained or Yoked controls. Middle, The IEG, c-fos, known to be activated by both learning and task related stress is elevated in both Yoked and APA trained mice compared to Untrained controls. Right, The IEG, c-jun, activated by learning but not associated stress, similarly to rRNA, is up-regulated in the Trained group but not the Untrained or Yoked controls. ^#,+, * = p<0.05, ^{##, ++,}**p<0.01.

Fig 2. rRNA variant IV expression is upregulated one hour after learning.

Real-Time qPCR relative expression show that of the five known hippocampal rRNA variants only variant IV is significantly up-regulated in the Trained group one hour after the third APA training trial: T₁₁ = 3.42; *p<0.01.

Fig 3. RNA Pol I inhibitor, CX-5461, disrupts consolidation of long-term memory, but not learning.

A) Timeline of the training protocol. Note that half of the animals were euthanized 1 h after the third training trial for qPCR analysis (see 3D) while the remainder were given a retention test 24 h later (see 3C). B) Representative paths (trajectories) of animals during pre-training, training and the 10 min retention test given 24 h after the 3^rd training trial (grey tracings). Red circles indicate the animal’s location when a shock was received. Grey circles indicate the position of the animal where it would have received a shock had the shock zone been active. C) Comparison of learning and memory between APA trained mice who received intracranial injection of vehicle (black circles) and CX-5461 (grey circles). Left, Number of entrances during the 30 min trials. No significant difference in the number of entrances indicates equivalent learning of CX-5461 treated animals and vehicle-treated controls. Right, Time to first entry the shock zone during each training trial and on the 24 h retention test. There were no significant differences in between-trial memory or between the two groups during training. In contrast, the vehicle injected animals continue to show place avoidance memory 24 h later; whereas CX-5461 injected animals do not. D) Real-Time qPCR relative expression shows that CX-5461 has no effect on Pol II dependent c-fos and c-jun transcription, but does inhibit Pol I dependent rRNA transcription. *p<0.05, **p<0.01.

Fig 4. Pol I inhibition before and during training prevents learning-induced upregulation of rRNA Variant IV.

Real-Time qPCR samples derived from dorsal hippocampi collected 1 h after APA training taken show that CX-5461 only significantly inhibits the rRNA variant IV after training. t₁₀ = 3.96; *p<0.01.

Fig 5. Elevated expression of rRNA is maintained at least 24 h after training.

A) Timeline of the training protocol. Note that half of the animals were given a retention test, while the other half were not. All animals were euthanized 24 h after training. B) Expression of ht-rRNA precursor 24 h after APA exposure. 45S ht-rRNA expression is elevated 24 h after training compared to Untrained animals. The retention test did not significantly affect rRNA expression. C) Immunohistochemistry of dorsal CA1 fixed 24 h after training with or without a retention test. Increased fibrillarin staining in the Trained animals indicates an active nucleolus. **p<0.01.