Reactivating Hippocampal-mediated Memories During Reconsolidation To Disrupt Fear Part 1

Memories are stored in the brain as cellular ensembles are activated during learning and reactivated during retrieval. Using the Tet-tag system in mice, we label dorsal dentate gyrus neurons activated by positive, neutral, or negative experiences with channelrhodopsin-2. 

Cell groups are the basic units of various organs in the human body and are also highly collaborative and complex biological systems. Cell groups play a very important role in the formation of memory.

Memory is divided into short-term memory and long-term memory. Different types of memories require different cell groups to store and process. Short-term memory is mainly stored in nerve cells on the surface of the cerebral cortex. In contrast, long-term memory requires continuous strengthening and strengthening of connections between neurons and is ultimately stored in cell groups deep in the cerebral cortex.

The quantity and quality of cell populations play a crucial role in improving memory. In life, we can activate and strengthen brain cell groups in many ways, such as reading, learning languages, exercising, and living daily life.

In addition, some healthy lifestyle habits, such as eating right, getting enough sleep, and avoiding excessive drinking and smoking, can also help maintain the health of cell groups, thereby promoting memory improvement.

In short, there is an inseparable relationship between cell populations and memory. We should actively cultivate and protect our cell populations to keep our brains healthy and flexible, improving our memory and cognitive abilities. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory because Cistanche deserticola is a traditional Chinese medicinal material that has many unique effects, one of which is to improve memory. The efficacy of Cistanche deserticola comes from the multiple active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health through a variety of pathways.

Click know 10 ways to improve memory

Following fear conditioning, these cells are artificially reactivated during fear memory recall. Optical stimulation of a competing positive memory is sufficient to update the memory during reconsolidation, thereby reducing conditioned fear acutely and enduringly. 

Moreover, mice demonstrate operant responding for reactivation of a positive memory, confirming its rewarding properties. These results show that interference from a rewarding experience can counteract negative affective states. 

While memory updating, induced by memory reactivation, involves a relatively small set of neurons, we also find that activating a large population of randomly labeled dorsal dentate gyrus neurons is effective in promoting reconsolidation. 

Importantly, memory updating is specific to the fear memory. These findings implicate the dorsal dentate gyrus as a potential therapeutic node for modulating memories to suppress fear.

Maladaptive conditioned fear, caused by dysregulated fear circuits, plays a significant role in the etiology of anxiety disorders such as specific phobias and post-traumatic stress disorder (PTSD). 

PTSD can develop in individuals who have experienced a traumatic event and it is often characterized by persistent memories of the trauma1. Consequently, contextual fear-conditioning (CFC), which is highly conserved across species2, has been used as a representative model in animals to study certain aspects of PTSD, such as fear generalization, exaggerated fear responses, and enhanced stress reactivity3–5 .
The most widely used CFC paradigms involve pairing an emotionally neutral conditioned stimulus (CS) such as a training context, with an aversive unconditioned stimulus (US) like a foot shock that typically elicits activity bursts that lead to conditioned freezing responses in rodents. A learned association emerges, and the CS acquires aversive properties that facilitate retrieval of the conditioned fear memory in the absence of the US. 

In rodent models, this results in a conditioned fear response upon re-exposure to the context demonstrating this learned relationship6. In humans, pathological conditioned fear can occur for decades even in the absence of the exact context in which the traumatic event took place. 

Even though anxiety disorders are extremely prevalent in the general population, and many individuals experience pathological anxiety as a form of an exaggerated fear state, there are few ways to attenuate maladaptive conditioned fear. 

Reconsolidation, however, has potential as a therapeutic mechanism for diminishing Pavlovian fear7. Reconsolidation theory posits that memories become destabilized during recall as they enter a transient state of malleability where they can be modulated when it takes them to restabilize7,8. 

Despite the long history of experimental reconsolidation-related interventions using a variety of pharmacological agents, behavioral treatments, and stimulation protocols to disrupt or enhance memory9–11, these studies have yielded mixed results.
Only recently has the potential for developing improved reconsolidation-based treatments and novel interventions been recognized12,13. Nevertheless, most effective therapies for PTSD are trauma-focused, meaning the treatment focuses on the memory of the traumatic event14. 

Memory is thought to be stored in the sparse activity patterns of neuronal populations within a distributed network15–17, or as Wilder Penfield described memory as "the writing left behind the brain by conscious experience" 18. 

We often refer to these ensembles, active during memory encoding, as memory traces or engrams19,20, and these engrams are reactivated during retrieval17,21. Findings from several studies have shown that specific memories, including fear memories, can be disrupted by inhibition of associated engrams15,22–26. Specifically, the dorsal dentate gyrus (dDG) of the hippocampus is important for encoding contextual fear memories27–30 and has been implicated in the pathophysiology of several anxiety disorders28,31. 

Of particular relevance to PTSD, contextual information, which includes more than spatial information, can modulate fear and safety4. Valence (e.g., negative memories) can be considered an aspect of context, which has the potential to promote exaggerated fear responses and fear generalization through associations formed in the hippocampus. 

Importantly, the DG also plays a role in disambiguating trauma-related and non-trauma-related contextual information32,33 as well as in extinction learning34 and PTSD patients exhibit impairments in both3. Moreover, we have previously shown that artificial reactivation of a positive memory stored in the dDG can acutely rescue stress-induced, depression-related behavior35. 

Here, we propose an innovative intervention based on the hypothesis that using optogenetics to artificially reactivate a previously formed, dDG-mediated memory during reconsolidation will permanently alter and disrupt the original fear memory. We used the Tet-tag system36 to label dDG neurons activated by exposure to positive, neutral, or negative experiences with channelrhodopsin-2 (ChR2). 

Mice were subsequently fear-conditioned and given a fear memory recall test wherein these tagged neurons were optically reactivated. We hypothesized that this intervention during the reconsolidation window would update the fear memory with attributes from the competing engram, thereby reducing the behavioral expression of conditioned fear. 

Moreover, as we have previously shown that stress-induced behaviors can be rescued by optically reactivating dDG cells previously active during a positive experience35 and others have shown that positive emotions counteract a subset of aftereffects of negative emotions37, we proposed that this effect would be more pronounced when the competing engram was associated with a positive experience compared to a neutral or negative experience. 

Here we show that reconsolidation-based hippocampal interference induced by optical reactivation of a competing, positive memory is sufficient to update a fear memory at the ensemble level resulting in an attenuation of maladaptive conditioned fear.

Results

Artificial reactivation of hippocampal-mediated memories during fear memory reconsolidation reduces fear acutely and enduringly

We used a viral, activity-dependent, and inducible neuronal tagging strategy in wild-type c57BL/6 mice (Fig. 1a). Male mice were injected with a virus (either ChR2 or eYFP) and implanted with bilateral optic fibers before being taken off DOX to open a tagging window17,38. 

They were split into three groups, and each assigned a differentially valenced behavioral experience (Fig. 1a). All mice were placed into a novel clean cage and either left undisturbed (neutral)21, placed with a female (positive)35,39, or placed into a restraint tube with air holes (negative)35 and then placed into the cage. Mice were returned to their home cages 1 h later back on DOX to close the tagging window. 

The following day, mice were fear conditioned (FC) in context A and 24 h later given a 20 min recall test in the same context. During this test, in which we assessed conditioned fear (i.e., freezing) as a proxy for retrieval of the associative fear memory, we simultaneously stimulated the tagged dDG ensembles during the first (F10) or last half (L10) of the session rather than the entire session, as we did not want to risk heat damage to the brain40 and wanted to compare light-on and light-off periods in a within-subject manner. 

We initially hypothesized that reactivating a positive memory during the last half of the recall session would promote reconsolidation since the fear memory would already be online. This would potentially alter the fear memory ensemble, updating it with positive attributes from the experience resulting in decreased freezing at subsequent time points. 

We aimed to specifically weaken the strength of the CS-US association by dampening the acquired aversion to the CS41 and altering the original fear memory through reconsolidation. Therefore, we chose a session length not longer than 20 min to ensure that our optical manipulation would be introduced during the short window post-memory reactivation when reconsolidation occurs42 and not during extinction learning43. 

This strategy permits us to measure real-time decreases in freezing with optical stimulation during recall. Moreover, it permits us to measure any long-lasting effects of our manipulation, and we thus extended our assessment to include two extinction sessions to test for stress-induced reinstatement after an immediate shock in context B. 

The shock was delivered in a new context in under 2 s so mice would not form a contextual representation of the environment, and, therefore not form a new associative fear memory, but would still experience stress. 

This method allowed us to model fear generalization and heightened stress reactivity as an example of maladaptive conditioning since the stressor was delivered in a different context 44–46. Based on previous studies39,41,47, mice first were FC using a 4-shock protocol (Fig. 1b) wherein they exhibited freezing in a stepwise manner, increasing with each successive shock presented (Supplementary Fig. 1a–c). 

We saw this pattern of freezing for all experiments (Supplementary Fig. 1a–h). Mice were returned to the context the next day for a fear memory recall test (Fig. 1c). With L10 stimulation, mice in positive and negative-ChR2 groups demonstrated a real-time reduction in freezing compared to mice in the neutral-ChR2 group and eYFP controls respectively. 

While there was a natural decline in freezing across the session due to the absence of shock, these mice showed a significantly steeper decline. While freezing levels generally declined across extinction days, no group differences were observed during extinction (Fig. 1d and Supplementary Fig. 1a) or immediate shock (Fig. 1e). During reinstatement (Fig. 1f), we saw less freezing in negative-ChR2 mice compared to neutral-eYFP mice, and in general, eYFP control mice froze more than experimental ChR2 mice at reinstatement compared to immediate shock (Fig. 1g). 

In contrast, postshock freezing following fear-conditioning (Fig. 1h) was reduced in the F10 condition (Fig. 1i), only for positive-ChR2 mice compared to eYFP controls, which occurred specifically in the last 10 min of the session. Here, neutral-ChR2 mice extinguished more quickly; however, no group differences were observed during extinction (Fig. 1j and Supplementary Fig. 1b). 

As expected, there were no group differences during immediate shock (Fig. 1k). During reinstatement, both positive and neutral-ChR2 groups demonstrated reduced fear compared to eYFP controls, while negative-ChR2 mice did not (Fig. 1l). 

Again, control mice froze more than experimental mice at reinstatement compared to immediate shock (Fig. 1m), and this was a more pronounced effect. Therefore, we adopted the F10 protocol for all subsequent experiments. 

However, as an additional control, we added an experiment to assess how effective artificial reactivation of a positive memory during the middle portion of the session (M10) would be compared to F10 or L10 stimulation during recall (Supplementary Fig. 2a). During the acquisition of the FC response, mice in all three conditions showed greater freezing post-shock (Supplementary Fig. 2b–g). 

During recall, the stimulation caused real-time decreases in freezing in the ChR2 group in the F10 and L10 conditions but not in the M10 condition (Supplementary Fig. 2h–j). F10 ChR2 mice continued to show decreased freezing in the latter half of the recall session in the absence of stimulation (Supplementary Fig. 2j). 

All three stimulation protocols produced decreased freezing in ChR2 mice in the first 3 min of extinction, but group differences were only seen with F10-stimulated mice (Supplementary Fig. 2k–m). 

Across the first extinction session, fear expression was decreased in F10 or M10 ChR2 groups (Supplementary Fig. 2n–p). There were no differences in immediate shock (Supplementary Fig. 2q–s). Finally, we again saw diminished freezing for both F10 and M10 ChR2 groups during reinstatement (Supplementary Fig. 2t–y).

For more information:1950477648nn@gmail.com