Highly stable long-term memory in a mouse model of post-traumatic stress disorder
Authors: Zamorina T. A. , Toropova K. A. , Ivashkina O. I. , Anokhin K. V. 
Published: May 2024

The retention of information represents a defining trait of cognitive systems. The primary function of an organism’s memory is to retain engrams of unique experiences for extended periods, without interrupting memory trace during new learning processes. One of the cardinal unsolved issues in neuroscience lies in unveiling the mechanisms responsible for long-term memory. Despite this, a trustworthy experimental animal model for single-trial long-term memory remains undeveloped. To address this challenge, we aimed to create an animal model to study lifelong memory formation with a single trial. We used a mouse model of post-traumatic stress disorder (PTSD) for this purpose [1]. In this model, mice exposed to a powerful electrical foot shock develop highly persistent traumatic memories, which results in long-term behavioral changes [1]. Our hypothesis was that this model could uncover the mechanisms related to lifelong memory.
The primary rationale for adopting the PTSD model as a model of lifelong memory pertains to the heightened durability of traumatic memories in comparison to conventional aversive memories [2]. Protein synthesis inhibitors, which are needed for long-term memory consolidation, can be used as amnesic agents to evaluate memory stability [3]. In a PTSD model using predator scent, the administration of a protein synthesis blocker prior to a traumatic experience disrupts the development of PTSD in mice [4]. However, it remains unclear how traumatic memory impairment affects PTSD development in the footshock model and whether the impairment persists long-term.
Based on the evidence that the formation of PTSD necessitates the consolidation of associative memory, which is reliant on protein synthesis and coincides with changes in the stress response system, Siegmund and Watzhek [1] present a two-part proposal concerning the onset of PTSD. The two-part hypothesis regarding PTSD posits that PTSD formation involves sensory conditioning and sensitization processes that mutually reinforce one another. In line with this theorem, we considered the effects of studying context and exposure timing on the development of post-traumatic stress disorder, as it could potentially interfere with sensory conditioning formation [5].
The aim of this study is to create an experimental approach for developing enduring long-term memory through a single trial event on adult mice. We methodically analyzed behavioral expressions and the endurance of normal and traumatic fear memory, as well as their sensitivity to protein synthesis inhibition. Additionally, we investigated the effects of separating the timing of the associative and aversive elements of traumatic memory on PTSD development in a mouse model.
The experiment involved male C57Bl/6 mice, aged between 3–4 months and 15–18 months (for an investigation into aged mice), which were placed in an electrified chamber. After 170 seconds, the mice experienced either one footshock (1.5 mA, 2 s) to elicit fear memory or three footshocks (1.5 mA, 10 s) for PTSD induction. After receiving the footshock, the mouse was held in the chamber for 60 seconds before being returned to its home cage. A memory test was conducted seven days later by placing the mice back in the same context. To assess the existence of standard PTSD symptoms, like sensitization and generalization, the creatures were exposed to an unfamiliar context and an unexpected auditory stimulus, respectively. In the experiment, animals were placed in unfamiliar or familiar safe contexts that differed from their previous ones. The duration of freezing was measured to assess fear and evaluate memory retention, sensitization, and generalization. Anxiety levels were evaluated using the elevated plus maze test.
In the process of constructing a highly stable long-term memory model, we evaluated the behavioral performance of PTSD-induced (PTSD), fear-conditioned (FC), and active control (AC) groups of animals at 7 days, 1 month, and 3 months after exposure. As a result of PTSD induction, mice displayed increased fear levels for up to 3 months in the training context, along with heightened fear sensitization and generalization at 7 days following exposure, relative to the FC and AC groups. The PTSD group exhibited heightened freezing behavior within a month and a decreased number of entries to the closed arms during the initial three months following exposure when contrasted with the FC and AC groups. We additionally noted that the FC group displayed raised fear levels in contrast to the control animals up to 3 months post-exposure, albeit lower in magnitude than that of the PTSD group. The FC group, similar to the PTSD group, showed increased sensitization and generalization compared to control animals at 1 week post-exposure, but to a lesser extent than we observed in the PTSD group. The findings suggest that traumatic memory remains present for a minimum of three months, whereas fear memory in the fear conditioning paradigm diminishes in intensity during this time. Hence, PTSD induction effectively functions as an animal model of profoundly stable long-term memory, in opposition to the fear conditioning paradigm.
As the high stable long-term memory model is designed for testing in senior animals, the impact of aging on traumatic memory formation and storage becomes an important inquiry at six months and one year following exposure. We analyzed the formation of both aversive and traumatic memory in mice aged 15–18 months, one week after footshock exposure. In the study involving older mice, the PTSD group exhibited increased levels of fear memory and sensitization when compared with both the FC and AC groups, in addition to displaying higher levels of generalization and anxiety when compared with the AC group. Furthermore, mice that underwent rear-conditioning showed heightened levels of fear during learning, fear sensitization, and generalization, although not anxiety. These findings demonstrate the formation of traumatic and aversive memory in aged mice, indicating that conducting memory tests six months and a year after exposure is an appropriate approach.
To evaluate the durability of traumatic and aversive memory, we administered a protein synthesis inhibitor called cycloheximide (Chm) to interfere with memory formation. Mice were given a cycloheximide solution (95 mg/kg) via intraperitoneal injection 30 minutes before footshock (Chm-PTSD and Chm-FC groups) while control animals received a saline injection (Sal-PTSD, Sal-FC). After training, the Chm-FC group displayed a decrease in freezing rate when compared to the saline-injected group at the 7- and 30-day marks. The Chm-PTSD group exhibited fear levels comparable to those of the Sal-FC group in the training context, and did not display sensitization or generalization of fear. These findings suggest that inhibiting protein synthesis during memory formation resulted in complete amnesia for standard aversive memory and merely weakened traumatic memory. As a result, only the associative component of traumatic memory remained intact, while nonspecific symptoms of PTSD were absent. The strong resilience of memory in the PTSD model to severe disruptions, such as protein synthesis blockade, also confirms its stability, which facilitates exploration of lifelong memory mechanisms in the PTSD model.
To examine the timing effect of the associative and aversive component of PTSD development, we evaluated traumatic memory formation when contextual memory formation was absent, and contextual exploration occurred three days before shock cessation. A week after exposure to immediate and intense footshock, mice in the experimental group exhibited lower levels of fear, reduced fear sensitization, and less anxiety compared to those in the PTSD-induced group. If an intense foot shock was preceded by a contextual study three days earlier, the animals exhibited lower levels of fear in an unfamiliar, safe context, and reduced anxiety compared to the mice induced with PTSD. In both cases, the fear level after the shock cessation was identical to that of the mice who experienced an immediate, moderate shock. Based on our findings, we concluded that aversive memory forms in the absence or prior formation of contextual memory in relation to traumatic exposure. However, PTSD induction does not occur under such circumstances. This indicates that contextual memory needs to be formed simultaneously with the traumatic event for the development of traumatic memory in the mouse model of PTSD.
In summary, our findings indicate that aversive memory tends to fade over time, while traumatic memory remains stable for a minimum of 3 months following its induction. Therefore, PTSD proves to be a fitting candidate laboratory model for high, stable, and long-term memory. Aged mice aged between 15–18 months display the formation of traumatic memory and experience PTSD symptoms in a manner similar to adult animals aged between 3–4 months in the PTSD model. For the induction of PTSD in mice, contextual memory formation about the traumatic environment and the traumatic event occurrence may coincide, which is crucial.