Alcohol-specific Transcriptional Dynamics Of Memory Reconsolidation And Relapse Part 1

Relapse, a critical issue in alcohol addiction, can be attenuated by disruption of alcohol-associated memories. Memories are thought to temporarily destabilize upon retrieval during the reconsolidation process. 

The relationship between alcohol addiction and memory is a hotly debated topic. Many people believe that alcohol abuse can damage brain function and lead to memory loss, but the reality is not that simple.

Alcohol is a neurotoxin, and excessive drinking can damage brain cells, leading to cognitive decline and possible conditions such as amnesia. However, moderate drinking can be helpful for memory. Studies show that drinking one to two glasses of red wine daily can boost cognitive and memory skills.

The ingredients in red wine can enhance the health of blood vessels, improve blood flow, and increase oxygen supply, which is very important for brain health. In addition, alcohol can stimulate brain cells to produce new neurons, speed up the process of nerve regeneration, and enhance brain connections and learning capabilities, thereby improving memory. But this only works in moderation. Excessive drinking can cause damage to the brain.

Therefore, if you are an alcoholic and want to improve your memory, it is best to stop drinking or reduce the amount you drink. At the same time, you can also ensure a healthy diet by eating more foods that improve memory, such as soybeans, fish, lemons, green vegetables, etc., and perform correct mental training, such as learning new skills and reading books, to promote brain activity and memory ability. This will not only improve the health of alcoholics but also help make the brain healthier and improve the quality of life. 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 minced meat comes from its various active ingredients, including acid, polysaccharides, flavonoids, etc. These ingredients can promote brain health in a variety of ways.

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Here, we provide evidence for unique transcriptional dynamics underpinning alcohol memory reconsolidation. Using a mouse place-conditioning procedure, we show that alcohol memory retrieval increases the mRNA expression of immediate-early genes in the dorsal hippocampus and medial prefrontal cortex and that alcohol-seeking is abolished by post-retrieval non-specific inhibition of gene transcription, or by downregulating ARC expression using antisense-oligodeoxynucleotides. 

However, since the retrieval of memories for a natural reward (sucrose) also increased the same immediate-early gene expression, we explored alcohol-specific transcriptional changes using RNA sequencing. 

We revealed a unique transcriptional fingerprint activated by alcohol memories, as the expression of this set of plasticity-related genes was not altered by sucrose-memory retrieval.
Our results suggest that alcohol memories may activate two parallel transcription programs: one is involved in memory reconsolidation in general, and another is specifically activated during alcohol-memory processing.

INTRODUCTION

Alcohol use disorder (AUD) is a detrimental neuropsychiatric disorder with severe medical, social, and economic burdens [1], yet available pharmacotherapy is limited [2]. Nearly 70% of patients relapse within the first year of abstinence [3], marking relapse as a major clinical challenge. 

Relapse is often triggered by a craving for alcohol, evoked by environments and cues previously associated with alcohol [4]. Therefore, the disruption of memories that evoke alcohol-related behaviors is expected to reduce or even prevent cue-induced relapse [5, 6].
It is increasingly accepted that well-consolidated memories can be reactivated upon retrieval. 

Retrieved memories undergo temporary destabilization and subsequent re-stabilization, termed reconsolidation [7–11]. Thus, memory reactivation initiates a temporary "reconsolidation window", lasting a few hours, during which memory is labile for certain manipulations [7, 8, 11, 12]. 

Indeed, interference with the reconsolidation of drug memories was shown to attenuate their subsequent expression and cue-induced relapse, thus providing a potential strategy for relapse prevention [13, 14].

Although the exact mechanisms underpinning the processing of reactivated drug memories have yet to be characterized, reconsolidation of drug and alcohol memories was generally shown to be interrupted by the inhibition of NMDA [15–17] or beta-adrenergic receptors[17, 18]; or by preventing protein synthesis [5, 10, 15]. 

According to recent fear and drug memory studies, memory reconsolidation requires not only protein synthesis but also gene transcription [19]. Moreover, the transcription of certain immediate early genes (IEGs), including Arc, encoding an activity-regulated cytoskeleton-associated protein and the transcription factor-encoding Egr1 (Zif268), was implicated in the reconsolidation of various types of memory [19–22], implying that similar dynamics might control the reconsolidation of alcohol memories. 

Similarly, we previously showed that inhibition of the mechanistic target of rapamycin complex 1 (mTORC1), which controls the synthesis of a subset of dendritic proteins [23], disrupted the reconsolidation of alcohol memories [5], and additional studies have shown that it also disrupted the reconsolidation of memories associated with fear [24] or with post-ingestive nutrients [25].

However, there is also evidence that some of the mechanisms underlying alcohol seeking may differ from those controlling natural reward seeking [5, 26–28]. Furthermore, there is evidence that memories for different rewards (including different drugs of abuse) are differentially processed [29–32]. 

Therefore, alcohol memory reconsolidation may be characterized by a unique transcriptional profile. 

As such, we sought to determine the transcriptional dynamics that underlie alcohol memory reconsolidation within the dorsal hippocampus (DH) and medial prefrontal cortex (mPFC) [5, 33, 34], brain regions implicated in alcohol use disorder [35, 36] and in the formation, retention, and expression of drug memories [5, 37, 38].

RESULTS

Alcohol memory reconsolidation depends on de novo gene transcription in the DH

While it has been established that the reconsolidation of alcohol memories requires de novo protein synthesis [5, 15], it remains unclear whether it is also dependent on de novo gene transcription. 

Therefore, we assessed the role of gene transcription during alcohol memory reconsolidation within the DH, a brain region implicated in alcohol use disorder [35] and involved in drug memory formation, retention, and expression [37, 38], in addition to memory reconsolidation [39, 40]. 

To form alcohol-associated memories, we employed the alcohol-conditioned place preference (CPP) paradigm. 

This paradigm has been used to examine the reinforcing properties of alcohol, as well as to explore the processing and maintenance of memories that evoke relapse to alcohol-seeking in rodents [41, 42], particularly in the DH [43].

To assess the role of hippocampal gene transcription in alcohol memory reconsolidation, we formed alcohol-associated memories in the alcohol-CPP procedure, by conditioning one compartment of the CPP-apparatus to alcohol (Fig. 1A, experimental design). 

A day after confirming the strong preference for the alcohol-paired compartment in a CPP test, the mice were re-exposed to the alcohol-paired compartment for 3 min to retrieve alcohol-associated memories, as we previously demonstrated [44, 45]. 

Immediately after memory retrieval, actinomycin D (4 µg/µl; 0.5 µl per side) or vehicle was infused into the DH [19]. In a retention test conducted 24 h later, we found that mice that received postretrieval actinomycin D did not show alcohol-CPP, whereas the preference for the alcohol-associated compartment remained high in the vehicle-treated mice (Fig. 1B; see Figure S1 for individual data). 

Thus, inhibition of gene transcription in the DH following memory retrieval led to the loss of alcohol-CPP, suggesting that the alcohol memory reconsolidation requires de novo gene transcription in the DH.

Fig. 1 Inhibition of transcription in the dorsal hippocampus after alcohol memory retrieval disrupts the expression of alcohol-conditioned place preference (CPP). A Schematic illustration of the experimental design and timeline. 

Actinomycin D (4 µg/µl) was bilaterally infused into the dorsal hippocampus of mice immediately following the retrieval of alcohol memories. B Place preference scores, expressed as means ± S.E.M. of the percent of time spent in the alcohol-paired compartment. 

Mice that showed strong alcoholCPP (t(17) = 8.31, p < 0.0001) lost alcohol-place preference when memory retrieval was followed by intra-DH infusion of actinomycin D and not the vehicle (mixed-model ANOVA: Test X Treatment (F(1,16) = 9.97, p < 0.01), post hoc: CPP test 2 (p < 0.05)). *p < 0.05, **p < 0.05; n = 9 per group).

To assess the role of hippocampal gene transcription in alcohol memory reconsolidation, we formed alcohol-associated memories in the alcohol-CPP procedure, by conditioning one compartment of the CPP-apparatus to alcohol (Fig. 1A, experimental design). 

A day after confirming the strong preference for the alcohol-paired compartment in a CPP test, the mice were re-exposed to the alcohol-paired compartment for 3 min to retrieve alcohol-associated memories, as we previously demonstrated [44, 45].
Immediately after memory retrieval, actinomycin D (4 µg/µl; 0.5 µl per side) or vehicle was infused into the DH [19]. In a retention test conducted 24 h later, we found that mice that received postretrieval actinomycin D did not show alcohol-CPP, whereas the preference for the alcohol-associated compartment remained high in the vehicle-treated mice (Fig. 1B; see Figure S1 for individual data). 

Thus, inhibition of gene transcription in the DH following memory retrieval led to the loss of alcohol-CPP, suggesting that the alcohol memory reconsolidation requires de novo gene transcription in the DH.

Retrieval of alcohol-related memories causes a time-dependent upregulation of Arc and Egr1 but not Bdnf mRNA expression in the DH and mPFC

We next assessed whether alcohol memory retrieval alters the expression of the genes previously implicated in memory reconsolidation, namely activity‐regulated cytoskeleton‐associated protein (Arc) [5, 22, 46, 47], transcription factor Egr1 (also known as Zif268) [19, 21, 48], and brain-derived neurotrophic factor (Bdnf) [49], in the DH and mPFC, brain regions implicated in the reconsolidation of drug memories [5, 33, 34, 39, 40, 50]. 

To assess Arc, Egr1, and Bdnf mRNA expression following alcohol memory retrieval, we first trained mice for alcohol-CPP (Fig. 2A, B). Twenty-four hours later, mice were re-exposed to the alcohol-paired compartment (Retrieval group) or were handled (No Retrieval group). 

We chose not to re-expose the control animals to the saline-paired context to prevent the retrieval of non-alcohol-related memories that are also characterized by changes in the expression of IEGs [46, 47, 50–52], or retrieval of a Pavlovian inhibitory alcohol memory, as the saline-paired compartment is associated with the absence of alcohol. Brain tissues were collected at five different time points after memory retrieval, and target mRNA levels were analyzed.

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