Chemogenetic evidence that rapid neuronal de novo protein synthesis is required for consolidation of long-term memory

Abstract

46Translational control of memory processes is a tightly regulated process where the coordinated interaction and 47 modulation of translation factors provides a permissive environment for protein synthesis during memory 48 formation. Existing methods used to block translation lack the spatiotemporal precision to investigate cell-49 specific contributions to consolidation of long-term memories. Here, we have developed a novel chemogenetic 50 mouse resource for cell type-specific and drug-inducible protein synthesis inhibition (ciPSI) that utilizes an 51 engineered version of the catalytic kinase domain of dsRNA-activated protein (PKR). ciPSI allows rapid and 52 reversible phosphorylation of eIF2α causing a block on general translation by 50% in vivo. Using this resource, 53 we discovered that temporally structured pan-neuronal protein synthesis is required for consolidation of long-54 term auditory threat memory. Targeted protein synthesis inhibition in CamK2α expressing glutamatergic 55 neurons in lateral amygdala (LA) impaired long-term memory, which was recovered with artificial chemogenetic 56 reactivation at the cost of stimulus generalization. Conversely, genetically reducing phosphorylation of eIF2α in 57 CamK2α positive neurons in LA enhanced memory strength, but was accompanied with reduced memory 58 fidelity and behavior inflexibility. Our findings provide evidence for a finely tuned translation program during 59 consolidation of long-term threat memories. Introduction 82Memory is the capacity of an organism to encode, store, and retrieve information, and often guides the 83 action of the organism towards a better survival outcome. Aversive life-threatening events often lead to long-84 term associative memories between the environment in which those events were experienced in and the 85 threat, such that a salient cue from the event when presented again can elicit species-specific defensive 86 behaviors. Pavlovian cued threat conditioning is a useful experimental paradigm for understanding the 87 biological substrates of an associative threat memory, in which a neutral cue (e.g. light or tone) co-presented 88 with an innately aversive stimulus (e.g. air puff or footshock) elicits a defensive response such as freezing by a 89 repeat presentation by itself 1 . Cued threat conditioning is particularly amenable to studying memory 90 consolidation process because one-trial training is sufficient to form a persistent long-term memory and a 91 unimodal cue can be used for memory retrieval. Long-term aversive memories are thought to recruit a 92 distributed network of neurons across the brain, including subnuclei within amygdala, hippocampus, thalamus, 93 as well as the neocortex, depending on the brain state, cue complexity, and the sensory pathways engaged 2, 3 . 94Lateral amygdala is the central sensory gateway for the amygdaloid complex and is crucially engaged both in 95 the processing and storage of the associative aversive memories 4, 5, 6 . 97Decades of studies have reported that consolidation of long-te...

Opto4E-BP, an optogenetic tool for inducible, reversible, and cell type-specific inhibition of translation initiation

“…We found that mice that received light treatment 30 minutes after training exhibited significantly lower levels of freezing during testing 24 hours later compared to all the control groups (4E-BP no light, mCherry light/ no light) (Fig 5c-e). These results are consistent with previous findings in the field regarding the temporal windows of de novo protein synthesis required for memory consolidation and show for the first time that eIF4E-mediated protein synthesis in excitatory neurons in the LA is required for threat memory consolidation 13,[32][33][34] .…”

“…We proceeded to test the AAV9-EF1a-DIO-Opto4EBP-mCherry viral construct in vivo. We utilized this Opto4E-BP tool to determine temporal windows in excitatory neurons in the LA that require eIF4E-dependent protein synthesis for associative threat memory consolidation [32][33][34] . CamK2α.…”

“…2). Immediately after the injection, mice were placed the first time that eIF4E-mediated protein synthesis in excitatory neurons in the LA is required for threat memory consolidation 13,[32][33][34] .…”

“…3). We also activated Opto4E-BP 5 hours after training, which is likely outside of the protein synthesis-dependent window required for memory consolidation [32][33][34] . During the LTM test, mice in the Opto4E-BP light-treated group, exhibited normal levels of freezing similar to freezing in the no light control group (Supplemental Fig 4).…”

The integrated stress response: From mechanism to disease

“…By contrast, ISR activation by manipulations that promote eIF2-P abundance impairs long-term memory formation: hippocampal or basolateral amygdala injection of Sal003 (65,66), or pharmacogenetic activation of PKR in the hippocampus, impairs long-term memory in both rodents and birds (111,(115)(116)(117)(118).…”