Reactivating Hippocampal-mediated Memories During Reconsolidation To Disrupt Fear Part 2

Next, we assessed the long-term effects of our manipulation. We replicated the above findings with a similar experimental design.

Arguably, there is a strong connection between long-term effects and memory. On the one hand, long-term effects can have profound effects on memory, and on the other hand, memory can affect our long-term effects.

First of all, the long-term impact on memory is mainly reflected in two aspects: one is the impact on people's physiological functions, and the other is the impact on people's psychological state.

Long-term effects can affect our physiology, such as in areas such as diet, sleep, and exercise. If we live in an unhealthy environment for a long time, such as unbalanced eating, poor sleep quality, long-term lack of exercise, etc., these long-term adverse effects will leave a certain memory in our body, thereby affecting our brain activity and memory.

At the same time, long-term effects will also affect our mental state, such as excessive tension, anxiety, and stress, which will hurt our memory.

The long-term impact of memory is also very important. A good memory can give us greater clarity, independence, and confidence, giving us greater control over our own lives and long-term consequences. It can help us better adjust our state, thereby reducing stress, anxiety, and other adverse factors, improving our ability to resist frustration, and enhancing our ability to adapt.

Therefore, we should pay attention to the impact of long-term effects on us in our daily lives, pay attention to health and good mental state, and cultivate healthy living habits and good adaptability, to give full play to our memory and better cope with long-term effects, Achieve substantial growth of its own. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors. These substances are very important for memory and learning. In addition, Cistanche deserticola can also improve blood flow and promote oxygen delivery, which can ensure that the brain receives sufficient nutrients and energy, thereby improving brain vitality and endurance.

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However, instead of giving mice a reinstatement test after immediate shock, we left mice undisturbed in their home cage for 2 weeks after extinction and then gave them a test for the spontaneous recovery of fear (Fig. 1n). 

Twenty-four hours after fear-conditioning (Fig.1o), during recall, both positive and neutral-ChR2 groups froze less in the last half of the session compared to eYFP controls (Fig. 1p). We saw no group differences during extinction (Fig. 1q and Supplementary Fig. 1c). 

Consistent with effects seen during recall, the spontaneous recovery test revealed that both positive and neutral-ChR2 mice froze less compared to eYFP controls and compared to the negative-Chr2 group demonstrating that our manipulation produced enduring effects on fear memory retrieval processes evident two weeks after extinction (Fig. 1r, s). 

Importantly, we showed the decreases in freezing observed after tagging and stimulating a positive memory, cannot be attributed solely to the viral injection or light stimulation alone. Specifically, we found that reactivation of a tagged positive memory (female exposure) (Supplementary Fig. 3a), after mice were FC to show increased postshock freezing (Supplementary Fig. 3b, c), resulted in less freezing in only ChR2 mice that received laser stimulation. 

This was in comparison to ChR2 mice that did not receive stimulation, eYFP mice that did receive stimulation, and the no virus/no laser group. This was true during recall (Supplementary Fig. 3d), on the first day of extinction, and during the first 3 min of extinction (Supplementary Fig. 3e, f). As expected, we saw no group differences during immediate shock (Supplementary Fig. 3g) but saw a significant decrease in freezing in the ChR2-Laser On group compared to all other groups at reinstatement (Supplementary Fig. 3h, I). 

Moreover, this effect was only present when mice received artificial reactivation of a tagged memory in the FC context (Supplementary Fig. 4a). Next, we tagged a positive memory (female exposure) and then FC mice that demonstrated postshock freezing (Supplementary Fig. 4b). 

When this memory was reactivated in a novel context and mice were returned to the conditioning context 24 h later, freezing did not decrease in the ChR2 group during recall (Supplementary Fig. 4c). 

Freezing remained similar to eYFP controls throughout extinction and reinstatement (Supplementary Fig. 4d, e). These findings provide evidence that our manipulation occurs via reconsolidation, as it is specific to when mice experience natural recall of the fear memory, which in this case, is prompted by exposure to the conditioning context.

Valence matters: Artificial reactivation of a neutral home cage experience during reconsolidation is not sufficient to disrupt a fear memory

The above results illustrate that hippocampal interference resulting from the reactivation of positive or neutral engrams is more effective at reducing conditioned fear than engrams associated with a negative experience. 

To further gauge the importance of valence, first, we tested if the novel clean cage experience was indeed "neutral", rather than positive or negative given that novel stimuli can engage a complex set of approach and avoidance dopaminergic pathways related to salience, reward, and neophobia48–52. Therefore, for the neutral component of the next experiment, we used a home cage38 experience instead, where mice were left undisturbed. 

Secondly, we asked whether a separate positive experience that did not involve female exposure was sufficient to reduce fear. Consequently, we used acute cocaine exposure53 as our next positive experience. Finally, we asked if the inability to reduce freezing via stimulation of a negative engram during fear memory recall was the result of those memories overlapping. 

To test this, we tagged dDG cells active when mice were FC in context C as our next negative experience as this interfering engram would theoretically be composed of some of the same cells as the fear memory acquired in context A due to generalization. To address these questions, we again opened a tagging window off DOX and labeled a positive, neutral, or negative memory in the dDG (Fig. 2a). 

Mice assigned to negative groups were initially FC in context C demonstrating significant post-shock freezing (Fig. 2b). The following day they were FC as before in context A. Mice FC the previous day showed higher freezing than the other groups pre- and postshock (Fig. 2c). During recall, negative ChR2 mice continued to exhibit more freezing compared to other ChR2 groups (Fig. 2d), which was observed throughout the session. 

However, there were no real-time decreases in freezing in any of the ChR2-groups, compared to eYFP counterparts in any part of the recall session (Fig. 2d). No group differences were observed during extinction (Fig. 2e and Supplementary Fig 1d) nor immediate shock (Fig. 2f). 

Optical stimulation of the home cage memory was not sufficient to compete with the fear memory given that during reinstatement, we observed that only positive-ChR2 mice showed less freezing compared to eYFP controls and the negative groups (Fig. 2g). Negative-ChR2 mice demonstrated equal freezing to controls (Fig. 2g, h). 

These results corroborate our previous findings showing that optical stimulation of a competing positive memory, but not a neutral or negative memory, is sufficient to disrupt reconsolidation of fear.

The reduction in freezing observed is not due to increases in locomotion

In a separate cohort of mice, dDG cells involved in encoding an acute cocaine exposure were tagged off DOX (Fig. 2i). The next day, to assess whether activation of a cocaine engram would induce hyper locomotor activity, we tested mice in the open field where we reactivated the cocaine engram in the last 5 min of the 10 min test. 

We found no group differences in the total number of line crossings (Fig. 2j), distance traveled (Fig. 2k), or speed (Fig. 2l) suggesting decreases in freezing observed in the previous experiment were not due to increased locomotion. 

Additionally, time spent in the center region revealed no group differences (Fig. 2m) suggesting that artificial activation of a cocaine-related memory is neither anxiogenic nor anxiolytic.

Mice will perform an operant response for the artificial reactivation of a positive memory

While the hippocampus is implicated in processing positive experiences, it is thought to do so in concert with several regions involved in neuromodulation, including the ventral tegmental area (VTA). 

The VTA is a critical component of the brain's reward system, and negative affective states (e.g., anxiety) are mediated by VTA dysregulation. It is well established that intracranial self-stimulation (ICSS) of the VTA is a powerfully rewarding operant behavior, where rodents maintain delivery of electrical impulses resulting in dopamine release54,55. 

This procedure has been previously adapted56–61 to incorporate in vivo optogenetic stimulation of dopaminergic neurons in the VTA. To tag and reactivate dDG cells active during this positive experience, we selectively expressed ChR2 in dopaminergic VTA cells using transgenic mice which express Cre under control of the dopamine transporter (DAT). 

We injected our viral vectors AAV5-Ef1a-DIO-(hChR2-E123A)- eYFP and implanted an optic fiber unilaterally into the VTA. We also injected c-Fos-tTA-TRE-(ChR2)-eYFP and implanted optic fibers bilaterally aimed at the dDG (Fig. 3a). Mice were initially habituated to the operant chamber and given access to a wheel with no consequences, which served as a baseline measure. 

The following day, mice were placed back into the operant box, and two nose ports were introduced, one active and one inactive. Nose pokes into the active port produced optogenetic VTA stimulation, while nose pokes into the inactive port produced no stimulation and served as a discriminative control. Mice were given three ICSS training sessions and then taken off DOX. 

They were brought back for a fourth training session, in which dDG cells responsive to VTA self-stimulation were tagged. The following day, access to the nose ports was restricted, and wheel spins produced optical dDG stimulation to reactivate the VTA self-stimulation engram. This was done to assess whether mice would perform an operant response for a positive (VTA-ChR2, dDG-ChR2) or neutral (VTA-eYFP, dDG-ChR2) experience compared to dDG-eYFP controls (Fig. 3a). 

In mice injected with ChR2 in the VTA, nose pokes into the active port were significantly higher than the inactive port, and they increased across sessions demonstrating the mice's ability to discriminate between ports and self-deliver optical stimulation for reward (Fig. 3b–e). Comparing wheel baseline measures to training and test, mice injected with ChR2 in the VTA and dDG, completed more wheel rotations, which were kept in motion for longer durations and distances (Fig. 3f–h) and produced more stimulations (Fig. 3i) compared to all other groups. 

This finding demonstrates that mice will perform an operant response to maintain the artificial reactivation of a positive memory, specifically the memory of VTA self-stimulation. Mice did not exhibit this behavior for a memory of operant box exposure in the absence of VTA stimulation. Following this test, mice underwent the same experimental protocol as before where they were FC in context A where they demonstrated post-shock freezing (Fig. 3j and Supplementary Fig. 1e) and then given a fear memory recall test. 

Reactivating the VTA self-stimulation engram (VCDC) during recall reduced freezing throughout the session (Fig. 3k). Levels remained low throughout extinction (Fig. 3l and Supplementary Fig. 1e), immediate shock (Fig. 3m), and reinstatement (Fig. 3n). 

Interestingly, between the two dDG-eYFP groups, the group that had received VTA stimulation earlier (VCDE) demonstrated a beneficial effect of this experience exhibiting intermediate levels of freezing compared to the VCDC group and the other control groups on EXT1 (Fig. 3l), and from immediate shock to reinstatement (Fig. 3o). Together, these results show that interference from a rewarding experience can counteract negative affective states.

Activation of randomly labeled dDG neurons is also sufficient to promote the reconsolidation of fear

Next, we asked, if artificial reactivation of a positive memory, which involves stimulation of a small set of neurons (<10%)38, can update a fear memory during reconsolidation, could we circumvent the positive-valence prerequisite to achieve a similar effect if we activate a larger population of neurons not necessarily tied to memory? 

Unlike previous experiments, where we used a cFos-inducible tagging strategy to label cells involved in different experiences, here, we used a virus with a constitutive promoter (CaMKIIa) to randomly tag dDG neurons with ChR2 (Fig. 4a). 

Mice were injected with either undiluted or diluted virus to label a large percentage or fraction of dDG cells, respectively. Mice demonstrated post-shock freezing after being FC (Fig. 4b and Supplementary Fig. 1f). 

During recall the next day, the labeled neurons were optically stimulated. During the first half of the session, we saw real-time decreases in freezing in both undiluted and diluted-ChR2 groups but by the second half, only the undiluted group showed less freezing (Fig. 4c). 

The undiluted-ChR2 group continued to exhibit less freezing throughout extinction (EXT1 & EXT2) (Fig. 4d) and both undiluted and diluted ChR2 groups froze less than the eYFP groups during the first 3 min of EXT1 (Supplementary Fig. 1f). 

As expected, there were no group differences during immediate shock (Fig. 4e). At reinstatement, we observed reduced freezing in both undiluted and diluted-ChR2 groups compared to eYFP controls (Fig. 4f) and compared to immediate shock (Fig. 4g). 

Our effects were greater in the undiluted group, suggesting reconsolidation-based processes can be potentially engaged by activating ensembles that are not connected to an engram of a particular valence if enough cells are activated. This memory modulation strategy may be akin to stimulation protocols currently approved for use in humans62,63.

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