Charlotte R. Flavell

Neocortical Tet3-mediated accumulation of 5-hydroxymethylcytosine promotes rapid behavioral adaptation

Significance We have discovered a critical role for ten-eleven translocation 3-mediated hydroxylation of 5-methycytosine in the adult prefrontal cortex in mediating rapid behavioral adaptation. 5-hydroxymethylcytosine (5-hmC) is highly dynamic in response to fear extinction training, and rather than simply reflecting a functional intermediary of active DNA demethylation, the learning-induced intergenic accumulation of 5-hmC creates an epigenetic state that promotes experience-dependent gene expression and behavioral adaptation. 5-hydroxymethylcytosine (5-hmC) is a novel DNA modification that is highly enriched in the adult brain and dynamically regulated by neural activity. 5-hmC accumulates across the lifespan; however, the functional relevance of this change in 5-hmC and whether it is necessary for behavioral adaptation have not been fully elucidated. Moreover, although the ten-eleven translocation (Tet) family of enzymes is known to be essential for converting methylated DNA to 5-hmC, the role of individual Tet proteins in the adult cortex remains unclear. Using 5-hmC capture together with high-throughput DNA sequencing on individual mice, we show that fear extinction, an important form of reversal learning, leads to a dramatic genome-wide redistribution of 5-hmC within the infralimbic prefrontal cortex. Moreover, extinction learning-induced Tet3-mediated accumulation of 5-hmC is associated with the establishment of epigenetic states that promote gene expression and rapid behavioral adaptation.

Behavioural memory reconsolidation of food and fear memories

The reactivation of a memory through retrieval can render it subject to disruption or modification through the process of memory reconsolidation. In both humans and rodents, briefly reactivating a fear memory results in effective erasure by subsequent extinction training. Here we show that a similar strategy is equally effective in the disruption of appetitive pavlovian cue–food memories. However, systemic administration of the NMDA receptor partial agonist D-cycloserine under the same behavioural conditions did not potentiate appetitive memory extinction, suggesting that reactivation does not enhance subsequent extinction learning. To confirm that reactivation followed by extinction reflects a behavioural analog of memory reconsolidation, we show that prevention of contextual fear memory reactivation by the LVGCC blocker nimodipine interferes with the amnestic outcome. Therefore, the reconsolidation process can be manipulated behaviourally to disrupt both aversive and appetitive memories.

Activation of BDNF signaling prevents the return of fear in female mice.

There are significant sex differences in vulnerability to develop fear-related anxiety disorders. Females exhibit twice the rate of post-traumatic stress disorder (PTSD) as males and sex differences have been observed in fear extinction learning in both humans and rodents, with a failure to inhibit fear emerging as a precipitating factor in the development of PTSD. Here we report that female mice are resistant to fear extinction, and exhibit increased DNA methylation of Bdnf exon IV and a concomitant decrease in mRNA expression within the medial prefrontal cortex. Activation of BDNF signaling by the trkB agonist 7,8-dihydroxyflavone blocks the return of fear in female mice after extinction training, and thus represents a novel approach to treating fear-related anxiety disorders that are characterized by a resistance to extinction and increased propensity for renewal.

Long Noncoding RNA-Directed Epigenetic Regulation of Gene Expression Is Associated With Anxiety-like Behavior in Mice

BACKGROUND RNA-directed regulation of epigenetic processes has recently emerged as an important feature of mammalian differentiation and development. Perturbation of this regulatory system in the brain may contribute to the development of neuropsychiatric disorders. METHODS RNA sequencing was used to identify changes in the experience-dependent expression of long noncoding RNAs (lncRNAs) within the medial prefrontal cortex of adult mice. Transcripts were validated by real-time quantitative polymerase chain reaction and a candidate lncRNA, Gomafu, was selected for further investigation. The functional role of this schizophrenia-related lncRNA was explored in vivo by antisense oligonucleotide-mediated gene knockdown in the medial prefrontal cortex, followed by behavioral training and assessment of fear-related anxiety. Long noncoding RNA-directed epigenetic regulation of gene expression was investigated by chromatin and RNA immunoprecipitation assays. RESULTS RNA sequencing analysis revealed changes in the expression of a significant number of genes related to neural plasticity and stress, as well as the dynamic regulation of lncRNAs. In particular, we detected a significant downregulation of Gomafu lncRNA. Our results revealed that Gomafu plays a role in mediating anxiety-like behavior and suggest that this may occur through an interaction with a key member of the polycomb repressive complex 1, BMI1, which regulates the expression of the schizophrenia-related gene beta crystallin (Crybb1). We also demonstrated a novel role for Crybb1 in mediating fear-induced anxiety-like behavior. CONCLUSIONS Experience-dependent expression of lncRNAs plays an important role in the epigenetic regulation of adaptive behavior, and the perturbation of Gomafu may be related to anxiety and the development of neuropsychiatric disorders.

Mechanisms governing the reactivation-dependent destabilization of memories and their role in extinction.

The extinction of learned associations has traditionally been considered to involve new learning, which competes with the original memory for control over behavior. However, a recent resurgence of interest in reactivation-dependent amnesia has revealed that the retrieval of fear-related memory (with what is essentially a brief extinction session) can result in its destabilization. This review discusses some of the cellular and molecular mechanisms that are involved in the destabilization of a memory following its reactivation and/or extinction, and investigates the evidence that extinction may involve both new learning as well as a partial destabilization-induced erasure of the original memory trace.

Inhibition and enhancement of contextual fear memory destabilization

The reactivation of a memory can result in its destabilization, necessitating a process of memory reconsolidation to maintain its persistence. Here we show that the destabilization of a contextual fear memory is potentiated by the cannabinoid CB1 receptor agonist Arachidonyl-2-chloroethylamide (ACEA). Co-infusion of ACEA and the IkappaB kinase (IKK) inhibitor sulfasalazine (Sulf) into the dorsal hippocampus impaired contextual fear memory reconsolidation. This observation was achieved under behavioral conditions that, by themselves, did not result in a reconsolidation impairment by Sulf alone. Moreover, we show that the destabilization of a contextual fear memory is dependent upon neuronal activity in the dorsal hippocampus, but not memory expression per se. The effect on contextual fear memory destabilization of intra-hippocampal ACEA was replicated by systemic injections, allowing an amnestic effect of MK-801. These results indicate that memory expression and destabilization, while being independent from one another, are both dependent upon memory reactivation. Moreover, memory destabilization can be enhanced pharmacologically, which may be of therapeutic potential.

The olivo-cerebellar system and its relationship to survival circuits

How does the cerebellum, the brain’s largest sensorimotor structure, contribute to complex behaviors essential to survival? While we know much about the role of limbic and closely associated brainstem structures in relation to a variety of emotional, sensory, or motivational stimuli, we know very little about how these circuits interact with the cerebellum to generate appropriate patterns of behavioral response. Here we focus on evidence suggesting that the olivo-cerebellar system may link to survival networks via interactions with the midbrain periaqueductal gray, a structure with a well known role in expression of survival responses. As a result of this interaction we argue that, in addition to important roles in motor control, the inferior olive, and related olivo-cortico-nuclear circuits, should be considered part of a larger network of brain structures involved in coordinating survival behavior through the selective relaying of “teaching signals” arising from higher centers associated with emotional behaviors.

Post-training unilateral amygdala lesions selectively impair contextual fear memories

The basolateral amygdala (BLA) and the dorsal hippocampus (dHPC) are both structures with key roles in contextual fear conditioning. During fear conditioning, it is postulated that contextual representations of the environment are formed in the hippocampus, which are then associated with foot shock in the amygdala. However, it is not known to what extent a functional connection between these two structures is required. This study investigated the effect on contextual and cued fear conditioning of disconnecting the BLA and dHPC, using asymmetrically placed, excitotoxic unilateral lesions. Post-training lesions selectively impaired contextual, but not cued, fear, while pretraining lesions resulted in a similar but nonsignificant pattern of results. This effect was unexpectedly observed in both the contralateral disconnection group and the anticipated ipsilateral control, which prompted further examination of individual unilateral lesions of BLA and dHPC. Post-training unilateral dHPC lesions had no effect on contextual fear memories while bilateral dHPC lesions and unilateral BLA lesions resulted in a near total abolition of contextual fear but not cued conditioned fear. Again, pretraining unilateral BLA lesions resulted in a strong but nonsignificant trend to the impairment of contextual fear. Furthermore, an analysis of context test-induced Fos protein expression in the BLA contralateral to the lesion site revealed no differences between post-training SHAM and unilateral BLA lesioned animals. Therefore, post-training unilateral lesions of the BLA are sufficient to severely impair contextual, but not cued, fear memories.

Reconsolidation-disruption diminishes spontaneous and stress-induced cocaine seeking

BACKGROUND: There has been a recent surge of interest in exploiting the process of reconsolidation to weaken maladaptive memories, in the hope this will drive the next wave of innovation in psychotherapy. Reconsolidation normally functions to stabilise and maintain memories in the long-term, and is critical in enabling memory updating. However, this process can be disrupted pharmacologically to weaken memories, or harnessed to allow destructive interference of a memory trace. Work has already begun to exploit this mechanism to disrupt pavlovian fear memories in the treatment of maladaptive anxiety and threat processing, and additionally being able to target instrumental memories may provide further clinical benefit. METHODS: Expanding our rat intravenous (i.v.) self-administration paradigm, we tested whether disruption of instrumental memory reconsolidation with the non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 could reduce relapse of cocaine seeking in response to stress, drug-priming or presentation of a drug-associated cue. RESULTS: Spontaneous responding for i.v. cocaine was reduced by reconsolidation-disruption. Furthermore, responding was not rescued by pharmacologically-induced stress. However, responding was restored following systemic administration of the drug, or presentation of a drug-associated cue. CONCLUSIONS: These data are consistent with hypothesis that there exist multiple routes to relapse, and suggest that at least some of these routes could be blocked by reconsolidation-disruption. This work provides important proof-of-principle that reconsolidation based therapies are a viable means of reducing the rates of relapse in substance use disorders.BACKGROUND There has been a recent surge of interest in exploiting the process of reconsolidation to weaken maladaptive memories, in the hope this will drive the next wave of innovation in psychotherapy. Reconsolidation normally functions to stabilise and maintain memories in the long-term, and is critical in enabling memory updating. However, this process can be disrupted pharmacologically to weaken memories, or harnessed to allow destructive interference of a memory trace. Work has already begun to exploit this mechanism to disrupt pavlovian fear memories in the treatment of maladaptive anxiety and threat processing, and additionally being able to target instrumental memories may provide further clinical benefit. METHODS Expanding our rat intravenous (i.v.) self-administration paradigm, we tested whether disruption of instrumental memory reconsolidation with the noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 could reduce relapse of cocaine seeking in response to stress, drug-priming or presentation of a drug-associated cue. RESULTS Spontaneous responding for i.v. cocaine was reduced by reconsolidation-disruption. Furthermore, responding was not rescued by pharmacologically-induced stress. However, responding was restored following systemic administration of the drug, or presentation of a drug-associated cue. CONCLUSIONS These data are consistent with hypothesis that there exist multiple ‘routes to relapse’, and suggest that at least some of these routes could be blocked by reconsolidation-disruption. This work provides important proof-of-principle that reconsolidation based therapies are a viable means of reducing the rates of relapse in substance use disorders.