Weak cued fear memory strengthening by re-activating the engram
Authors: Toropova K. A. , Ivashkina O. I. , Yurin A. M. , Anokhin K. V. 
Published: May 2024

One of neurobiology’s primary objectives is to discover the mechanisms by which past experiences influence current behavior and learning. While it is acknowledged that previous cognitive experiences can facilitate the creation of new memories in both humans and animals, investigations into the behavioral and neuronal aspects of the phenomenon of new memory being dependent on past cognitive experiences remain to be explored. The aim is to evaluate the theory that the facilitating effects of prior experiences on the creation of new memories are solely evident if these occurrences involve shared groups of neurons in the brain. To this end, a novel method was devised to lightly instruct mice to instinctively freeze when presented with a cue by exposing them to a conditioned signal for a brief (5 seconds) period, followed immediately by a gentle electrodermal stimulation. We discovered that weak memory can be strengthened through repeated weak learning in cumulative learning, despite the lack of formation of long-term memory caused by weak learning itself. Strengthening of memory only occurs when the animal is trained on the same conditioned signal during both weak training times. On the other hand, if the conditioned signals used during the first and second weak training are insignificantly different, no memory formation will occur for either. However, memory reinforcement in cumulative learning depended on context, fully manifesting only when multiple training sessions occurred in the same environment. We examined if long-term memory formation of the conditioned signal during cumulative learning relied on the animals’ past experiences, which were subject to alteration through repeated training. We discovered that successful long-term memory formation for the conditioned signal occurred when two weak learnings were separated by more than 30 minutes (up to 30 days), but not when the interval between learnings was 30 seconds or 5 minutes. Thus, weak memory reinforcement in cumulative learning occurs only when the second training is separated from the first by a time interval sufficient to form a latent memory of the first. The data obtained suggest that weak training of the conditioned reflex freeze results in long-term plastic rearrangements in the brains of mice. These changes can be strengthened through repeated weak training to the same conditioned signal, resulting in the behavioral manifestation of memory. We then investigated the activity of different brain regions during cumulative learning. We demonstrated that repeated weak training elicits selective activation of mouse brain regions that are crucial for long-term memory formation, including associative cortical regions, amygdala, and hippocampus. Conversely, such activation was absent after a single weak training, where the brain activity was equivalent to the mice that received the conditioned stimulus without reinforcement and remained untrained. This study suggests that a weak single training can form a memory trace in the brain, which can be strengthened through repetition. However, identifying this trace at the level of entire brain structures appears to be unattainable. Therefore, we directly evaluated the main hypothesis of this study by examining the overlap of neuronal populations in transgenic Cre-lineage mice. We labeled neurons involved in the first weak learning with fluorescent protein expression and cells active in the second learning with immunohistochemical staining for the native Arc protein. It was demonstrated that during cumulative learning, over 30% of neurons in the prelimbic cortex, auditory cortex, and amygdala were reactivated, while only 10% of cells were reactivated when two weak learnings were performed on different conditioned signals that did not lead to the formation of memories. Consequently, the present study examines the phenomenon of newly formed memory dependence on individual experience history at both a behavioral and neuronal level. The study demonstrated that the enhancing effect of previous experiences on memory creation relies on the repeated activation of identical neurons.