Hippocampal Engrams Generate Variable Behavioral Responses and Brain-Wide Network States.

Freezing is a defensive behavior commonly examined during hippocampal-mediated fear engram reactivation. How these cellular populations engage the brain and modulate freezing across varying environmental demands is unclear. To address this, we optogenetically reactivated a fear engram in the dentate gyrus subregion of the hippocampus across three distinct contexts in male mice. We found that there were differential amounts of light-induced freezing depending on the size of the context in which reactivation occurred: mice demonstrated robust light-induced freezing in the most spatially restricted of the three contexts but not in the largest. We then utilized graph theoretical analyses to identify brain-wide alterations in cFos expression during engram reactivation across the smallest and largest contexts. Our manipulations induced positive interregional cFos correlations that were not observed in control conditions. Additionally, regions spanning putative "fear" and "defense" systems were recruited as hub regions in engram reactivation networks. Lastly, we compared the network generated from engram reactivation in the small context with a natural fear memory retrieval network. Here, we found shared characteristics such as modular composition and hub regions. By identifying and manipulating the circuits supporting memory function, as well as their corresponding brain-wide activity patterns, it is thereby possible to resolve systems-level biological mechanisms mediating memory's capacity to modulate behavioral states.

In vivo genome editing using novel AAV-PHP variants rescues motor function deficits and extends survival in a SOD1-ALS mouse model.

CRISPR-based gene editing technology represents a promising approach to deliver therapies for inherited disorders, including amyotrophic lateral sclerosis (ALS). Toxic gain-of-function superoxide dismutase 1 (SOD1) mutations are responsible for ~20% of familial ALS cases. Thus, current clinical strategies to treat SOD1-ALS are designed to lower SOD1 levels. Here, we utilized AAV-PHP.B variants to deliver CRISPR-Cas9 guide RNAs designed to disrupt the human SOD1 (huSOD1) transgene in SOD1G93A mice. A one-time intracerebroventricular injection of AAV.PHP.B-huSOD1-sgRNA into neonatal H11Cas9 SOD1G93A mice caused robust and sustained mutant huSOD1 protein reduction in the cortex and spinal cord, and restored motor function. Neonatal treatment also reduced spinal motor neuron loss, denervation at neuromuscular junction (NMJ) and muscle atrophy, diminished axonal damage and preserved compound muscle action potential throughout the lifespan of treated mice. SOD1G93A treated mice achieved significant disease-free survival, extending lifespan by more than 110 days. Importantly, a one-time intrathecal or intravenous injection of AAV.PHP.eB-huSOD1-sgRNA in adult H11Cas9 SOD1G93A mice, immediately before symptom onset, also extended lifespan by at least 170 days. We observed substantial protection against disease progression, demonstrating the utility of our CRISPR editing preclinical approach for target evaluation. Our approach uncovered key parameters (e.g., AAV capsid, Cas9 expression) that resulted in improved efficacy compared to similar approaches and can also serve to accelerate drug target validation.

Hippocampal cells segregate positive and negative engrams.

The hippocampus is involved in processing a variety of mnemonic computations specifically the spatiotemporal components and emotional dimensions of contextual memory. Recent studies have demonstrated cellular heterogeneity along the hippocampal axis. The ventral hippocampus has been shown to be important in the processing of emotion and valence. Here, we combine transgenic and all-virus based activity-dependent tagging strategies to visualize multiple valence-specific engrams in the vHPC and demonstrate two partially segregated cell populations and projections that respond to appetitive and aversive experiences. Next, using RNA sequencing and DNA methylation sequencing approaches, we find that vHPC appetitive and aversive engram cells display different transcriptional programs and DNA methylation landscapes compared to a neutral engram population. Additionally, optogenetic manipulation of tagged cell bodies in vHPC is not sufficient to drive appetitive or aversive behavior in real-time place preference, stimulation of tagged vHPC terminals projecting to the amygdala and nucleus accumbens (NAc), but not the prefrontal cortex (PFC), showed the capacity drive preference and avoidance. These terminals also were able to change their capacity to drive behavior. We conclude that the vHPC contains genetically, cellularly, and behaviorally segregated populations of cells processing appetitive and aversive memory engrams.

Digging behavior discrimination test to probe burrowing and exploratory digging in male and female mice.

Digging behavior is often used to test motor function and repetitive behaviors in mice. Different digging paradigms have been developed for behaviors related to anxiety and compulsion in mouse lines generated to recapitulate genetic mutations leading to psychiatric and neurological disorders. However, the interpretation of these tests has been confounded by the difficulty of determining the motivation behind digging in mice. Digging is a naturalistic mouse behavior that can be focused toward different goals, that is foraging for food, burrowing for shelter, burying objects, or even for recreation as has been shown for dogs, ferrets, and human children. However, the interpretation of results from current testing protocols assumes the motivation behind the behavior often concluding that increased digging is a repetitive or compulsive behavior. We asked whether providing a choice between different types of digging activities would increase sensitivity to assess digging motivation. Here, we present a test to distinguish between burrowing and exploratory digging in mice. We found that mice prefer burrowing when the option is available. When food restriction was used to promote a switch from burrowing to exploration, males readily switched from burrowing to digging outside, while females did not. In addition, when we tested a model of intellectual disability and autism spectrum disorder that had shown inconsistent results in the marble burying test, the Cc2d1a conditional knockout mouse, we found greatly reduced burrowing only in males. Our findings indicate that digging is a nuanced motivated behavior and suggest that male and female rodents may perform it differently.

Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice.

CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research.

Chronic activation of fear engrams induces extinction-like behavior in ethanol-exposed mice.

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.

Odor modulates the temporal dynamics of fear memory consolidation.

Systems consolidation (SC) theory proposes that recent, contextually rich memories are stored in the hippocampus (HPC). As these memories become remote, they are believed to rely more heavily on cortical structures within the prefrontal cortex (PFC), where they lose much of their contextual detail and become schematized. Odor is a particularly evocative cue for intense remote memory recall and despite these memories being remote, they are highly contextual. In instances such as posttraumatic stress disorder (PTSD), intense remote memory recall can occur years after trauma, which seemingly contradicts SC. We hypothesized that odor may shift the organization of salient or fearful memories such that when paired with an odor at the time of encoding, they are delayed in the de-contextualization process that occurs across time, and retrieval may still rely on the HPC, where memories are imbued with contextually rich information, even at remote time points. We investigated this by tagging odor- and non-odor-associated fear memories in male c57BL/6 mice and assessed recall and c-Fos expression in the dorsal CA1 (dCA1) and prelimbic cortex (PL) 1 or 21 d later. In support of SC, our data showed that recent memories were more dCA1-dependent whereas remote memories were more PL-dependent. However, we also found that odor influenced this temporal dynamic, biasing the memory system from the PL to the dCA1 when odor cues were present. Behaviorally, inhibiting the dCA1 with activity-dependent DREADDs had no effect on recall at 1 d and unexpectedly caused an increase in freezing at 21 d. Together, these findings demonstrate that odor can shift the organization of fear memories at the systems level.

Artificially Enhancing and Suppressing Hippocampus-Mediated Memories.

Emerging evidence indicates that distinct hippocampal domains differentially drive cognition and emotion [1, 2]; dorsal regions encode spatial, temporal, and contextual information [3-5], whereas ventral regions regulate stress responses [6], anxiety-related behaviors [7, 8], and emotional states [8-10]. Although previous studies demonstrate that optically manipulating cells in the dorsal hippocampus can drive the behavioral expression of positive and negative memories, it is unknown whether changes in cellular activity in the ventral hippocampus can drive such behaviors [11-14]. Investigating the extent to which distinct hippocampal memories across the longitudinal axis modulate behavior could aid in the understanding of stress-related psychiatric disorders known to affect emotion, memory, and cognition [15]. Here, we asked whether tagging and stimulating cells along the dorsoventral axis of the hippocampus could acutely, chronically, and differentially promote context-specific behaviors. Acute reactivation of both dorsal and ventral hippocampus cells that were previously active during memory formation drove freezing behavior, place avoidance, and place preference. Moreover, chronic stimulation of dorsal or ventral hippocampal fear memories produced a context-specific reduction or enhancement of fear responses, respectively, thus demonstrating bi-directional and context-specific modulation of memories along the longitudinal axis of the hippocampus. Fear memory suppression was associated with a reduction in hippocampal cells active during retrieval, while fear memory enhancement was associated with an increase in basolateral amygdala activity. Together, our data demonstrate that discrete sets of cells throughout the hippocampus provide key nodes sufficient to bi-directionally reprogram both the neural and behavioral expression of memory.