Abstract

The stabilization of long-term memories requires de novo protein synthesis. How can proteins, synthesized in the soma, act on specific synapses that participate in a given memory? We studied the dynamics of newly synthesized AMPA-type glutamate receptors (AMPARs) induced with learning using transgenic mice expressing the GluR1 subunit fused to green fluorescent protein (GFP-GluR1) under control of the c-fos promoter. We found learning-associated recruitment of newly synthesized GFP-GluR1 selectively to mushroom-type spines in adult hippocampal CA1 neurons 24 hours after fear conditioning. Our results are consistent with a “synaptic tagging” model to allow activated synapses to subsequently capture newly synthesized receptor and also demonstrate a critical functional distinction in the mushroom spines with learning.

Abstract

Do learning and retrieval of a memory activate the same neurons? Does the number of reactivated neurons correlate with memory strength? We developed a transgenic mouse that enables the long-lasting genetic tagging of c-fos–active neurons. We found neurons in the basolateral amygdala that are activated during Pavlovian fear conditioning and are reactivated during memory retrieval. The number of reactivated neurons correlated positively with the behavioral expression of the fear memory, indicating a stable neural correlate of associative memory. The ability to manipulate these neurons genetically should allow a more precise dissection of the molecular mechanisms of memory encoding within a distributed neuronal network.

The relay of extracellular signals into changes in cellular physiology involves a Byzantine array of intracellular signaling pathways, of which cytoplasmic protein kinases are a crucial component. In the nervous system, a great deal of effort has focused on understanding the conversion of patterns of synaptic activity into long-lasting changes in synaptic efficacy that are thought to underlie memory. The goal is both to understand synaptic plasticity mechanisms, such as long-term potentiation, at a molecular level and to understand the relationship of these synaptic mechanisms to behavioral memory. Although both involve the activation of multiple signaling pathways, recent studies are beginning to define discrete roles and mechanisms for individual kinases in the different temporal phases of both synaptic and behavioral plasticity.

•Optogenetic stimulation of RSC reactivated a contextual fear memory•Both artificial and natural retrieval of memory activated common amygdala neurons•Artificial recall by RSC stimulation was unimpaired by hippocampal inactivationDeclarative memories are thought to be stored within anatomically distributed neuronal networks requiring the hippocampus; however, it is unclear how neocortical areas participate in memory at the time of encoding. Here, we use a c-fos-based genetic tagging system to selectively express the channelrhodopsin variant, ChEF, and optogenetically reactivate a specific neural ensemble in retrosplenial cortex (RSC) engaged by context fear conditioning. Artificial stimulation of RSC was sufficient to produce both context-specific behavior and downstream cellular activity commensurate with natural experience. Moreover, optogenetically but not contextually elicited responses were insensitive to hippocampal inactivation, suggesting that although the hippocampus is needed to coordinate activation by sensory cues, a higher-order cortical framework can independently subserve learned behavior, even shortly after learning.

Oxidative stress has been implicated in the pathogenesis of numerous late-onset diseases as well as organismal longevity. Nevertheless, the genetic components that affect cellular sensitivity to oxidative stress have not been explored extensively at the genome-wide level in mammals. Here we report an RNA interference (RNAi) screen for genes that increase resistance to an organic oxidant, tert-butylhydroperoxide (tert-BHP), in cultured fibroblasts. The loss-of-function screen allowed us to identify several short hairpin RNAs (shRNAs) that elevated the cellular resistance to tert-BHP. One of these shRNAs strongly protected cells from tert-BHP and H2O2 by specifically reducing the expression of retinol saturase, an enzyme that converts all-trans-retinol (vitamin A) to all-trans-13,14-dihydroretinol. The protective effect was well correlated with the reduction in mRNA level and was observed in both primary fibroblasts and NIH3T3 cells. The results suggest a novel role for retinol saturase in regulating sensitivity to oxidative stress and demonstrate the usefulness of large-scale RNAi screening for elucidating new molecular pathways involved in stress resistance.