Highlights d Acute activation of dorsal and ventral HPC engrams in mice drives reward and aversion d The ventral DG is preferentially reactivated in emotionally salient contexts d Chronic activation of HPC engrams decreases or increases context-specific freezing d Memory enhancement is disrupted when BLA cells processing fear are silenced

The formation and extinction of fear memories represent two forms of learning that each engage the hippocampus and amygdala. How cell populations in these areas contribute to fear relapse, however, remains unclear. Here, we demonstrate that, in male mice, cells active during fear conditioning in the dentate gyrus of hippocampus exhibit decreased activity during extinction and are re-engaged after contextual fear relapse. In vivo calcium imaging reveals that relapse drives population dynamics in the basolateral amygdala to revert to a network state similar to the state present during fear conditioning. Finally, we find that optogenetic inactivation of neuronal ensembles active during fear conditioning in either the hippocampus or amygdala is sufficient to disrupt fear expression after relapse. These results suggest that fear relapse triggers a partial re-emergence of the original fear memory representation, providing new insight into the neural substrates of fear relapse. IntroductionThe biological capacity to produce adaptive behavioral responses in actively changing environments is critical to an animal's survival. Contextual fear conditioning (CFC) is a form of learning whereby an animal learns to associate a conditioned stimulus (i.e. a context) with an unconditioned aversive stimulus (e.g. foot shocks) to produce a conditioned response to the conditioned stimulus (e.g. freezing). Conditioned responses can be mitigated through extinction learning via repeated exposure to the conditioned context in the absence of the foot shock. However, while extinction learning can be effective at attenuating fear, animals are susceptible to fear relapse under several conditions, including exposure to stressors, the passage of time, and re-exposure to the unconditioned stimulus (

Alcohol withdrawal directly impacts the brain's stress and memory systems, which may underlie individual susceptibility to persistent drug and alcohol-seeking behaviors. Numerous studies demonstrate that forced alcohol abstinence, which may lead to withdrawal, can impair fear-related memory processes in rodents such as extinction learning; however, the underlying neural circuits mediating these impairments remain elusive. Here, we tested an optogenetic strategy aimed at mitigating fear extinction retrieval impairments in male c57BL/6 mice following exposure to alcohol (i.e., ethanol) and forced abstinence. In the first experiment, extensive behavioral extinction training in a fear-conditioned context was impaired in ethanol-exposed mice compared to controls. In the second experiment, neuronal ensembles processing a contextual fear memory in the dorsal hippocampus were tagged and optogenetically reactivated repeatedly in a distinct context in ethanol-exposed and control mice. Chronic activation of these cells resulted in a context-specific, extinction-like reduction in fear responses in both control and ethanol-exposed mice. These findings suggest that while ethanol can impair the retrieval an extinction memory, optogenetic manipulation of a fear engram is sufficient to induce an extinction-like reduction in fear responses.

Memories are stored in the brain as cellular ensembles 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. 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.

14 The formation and extinction of fear memories represent two forms of learning that each 15 engage the hippocampus and amygdala. How cell populations in these areas contribute to fear 16 relapse, however, remains unclear. Here, we demonstrate that, in mice, cells active during fear 17 conditioning in the dentate gyrus of hippocampus and basolateral amygdala exhibit decreased 18 activity during extinction and are re-engaged after fear reinstatement. In vivo calcium imaging 19 reveals that reinstatement drives population dynamics in the basolateral amygdala to revert to a 20 network state similar to the state present during fear conditioning. Finally, we find that 21 optogenetic inactivation of neuronal ensembles active during fear conditioning in either the 22 hippocampus or amygdala is sufficient to disrupt fear expression after reinstatement. These 23 results suggest that fear reinstatement triggers a partial re-emergence of the original fear memory 24 representation, providing new insight into the neural substrates of fear relapse. 25 26 131ArchT-eYFP to drive expression of the light-sensitive protein archaerhodopsin (ArchT) in cells 132 active during CFC, and subsequently implanted optic fibers above the injection sites ( Figure 133 3a,b). Mice then underwent two EXT sessions, the reinstating shock, and recall the following day 134 (Figure 3c). Mice in both the DG and BLA experimental groups showed significant suppression 135 of freezing during optical inhibition. This manipulation was reversible, as freezing increased 136 again in the following light-off epoch (Figure 3d,e). eYFP controls did not show this decrease in 137

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ABSTRACTAlcohol withdrawal directly impacts the brain’s stress and memory systems, which may underlie individual susceptibility to persistent drug and alcohol-seeking behaviors. Numerous studies demonstrate that forced alcohol abstinence, which may lead to withdrawal, can impair fear-related memory processes in rodents such as extinction learning, however the underlying neural circuits mediating these impairments remain elusive. Here, we tested an optogenetic strategy aimed at mitigating fear extinction impairments in male c57BL/6 mice following exposure to alcohol (i.e., ethanol) and forced abstinence. In the first experiment, extensive behavioral extinction training in a fear-conditioned context was impaired in ethanol-exposed mice compared to controls. In the second experiment, neuronal ensembles processing a contextual fear memory in the dorsal hippocampus were tagged and optogenetically reactivated repeatedly in a distinct context in ethanol-exposed and control mice. Chronic activation of these cells resulted in a context-specific, extinction-like reduction in fear responses in both control and ethanol-exposed mice. These findings suggest that while ethanol can impair fear extinction learning, optogenetic manipulation of a fear engram is sufficient to induce an extinction-like reduction in fear responses.

10The hippocampus processes both spatial-temporal information and emotionally salient 11 experiences. To test the functional properties of discrete sets of cells in the dorsal dentate gyrus 12 (dDG), we examined whether chronic optogenetic reactivation of these ensembles was 13 sufficient to modulate social behaviors in mice. We found that chronic reactivation of dDG cells 14 in male mice was sufficient to enhance social behaviors in a female exposure task when 15 compared to pre-stimulation levels. However, chronic reactivation of these cells was not 16 sufficient to modulate group differences in a separate subset of social behaviors, and multi-17 region analysis of neural activity did not yield detectable differences in immediate-early gene 18 expression or neurogenesis, suggesting a dissociation between our chronic stimulation-induced 19 behavioral effects and underlying neural responses. Together, our results demonstrate that 20 chronic optogenetic stimulation of cells processing valent experiences enduringly and 21 unidirectionally modulates social interactions between male and female mice. 22 23 Chen et al., 2019). To test whether or not our chronic stimulation strategy generalizes to other 35 behaviors, here we examined whether chronic optogenetic reactivation of ensembles in the dDG 36 which are active during putative positive or negative experiences is sufficient to alter social 37 behaviors as well as the activity of multiple brain regions. 38 39 2. Methods 40 Subjects 41Wild-type C57BL/6N male mice (40-41 days; Charles River Laboratories) were housed with 42 littermates in groups of 2-5 mice per cage. Mice were acclimated to the animal facility for 72 43 hours upon delivery before experimental procedures began and kept on a 12:12-hour light cycle 44 (lights on at 7:00). Food and water were available ad libitum. Animals were put on a diet 45 containing 40 mg/kg doxycycline (dox) after the acclimation period and 24-48 hours before 46 receiving surgery between 6-7 weeks of age. Following surgery, mice were group-housed with 47