Memories are thought to be stored in neuronal ensembles throughout the brain, or engrams. These ensembles are defined as the neuronal populations active during learning, that undergo lasting cellular changes due to the learning, and are necessary for memory retrieval. Genetic strategies that utilize immediate early genes (IEGs), which are expressed upon cellular stimulation, have been developed to identify engrams. These tools allow for the labeling of the cells active during memory encoding, which can then be compared with those active during retrieval, advancing our knowledge of how single memories are stored in the brain. Despite these advances, little is known about how multiple memories are encoded and stored in the brain. This limitation is due to the current methods, which restrict our ability to visualize multiple ensembles in the brain. Here, I developed a multiple labeling system, based on the IEG Arc, that allows us to investigate how single and multiple memories are stored in the brain. In Chapter 2, we first show the validity of using an existing Arc-based labeling system to investigate how fear memory ensembles are modulated by propranolol, a β-adrenergic receptor antagonist. We found that propranolol modulates fear retrieval and decreases the reactivation of fear ensembles in the dorsal dentate gyrus (DG). In Chapter 3, I show the development of a novel, multiple Arc (mArc) labeling system that allows for the tagging of multiple Arc ensembles in the brain. We validated this system by investigating how context, time, and valence influence ensemble reactivation in the DG. We show that similar contextual experiences and experiences occurring close in time are stored in overlapping ensembles. The mArc system provides a powerful approach for investigating how multiple memories are organized in the brain and will be useful for multiple areas of investigation.

Switching between a state of fear and safety is a critical aspect of adaptive behavior. Aversive and non-aversive associations must be formed quickly and reliably but remain malleable as these associations change dynamically. When these associations become biased towards aversive associations by traumatic and stressful circumstances, as in PTSD, fear generalization and impaired fear extinction arise. These changes are associated with reduced activity in the medial prefrontal cortex (mPFC) and enhanced activity in the basolateral amygdala (BLA). It has been hypothesized that the mPFC mediates top-down control of the BLA to signal safety. It has previously been demonstrated that synchronous activity within the mPFC-BLA circuit is strongly engaged during fear conditioning, but it is unknown how activity in this circuit changes to mediate aversive discrimination. We investigated how the mPFC and BLA cooperate to mediate successful discrimination between aversive and non-aversive stimuli both for learned and innately-valent associations. Extracellular elecrophysiological recordings were obtained simultaneously form the mPFC and BLA in mice during innate anxiety, fear discrimination, and fear extinction. Local field potentials were recorded in both structures along with single unit recordings from the BLA. We discovered that fear was associated with enhanced theta-frequency synchrony and theta-gamma coupling within the mPFC-BLA circuit. On the other hand, safety was associated with predominant mPFC-to-BLA directionality of synchronous information flow and enhanced fast gamma frequency activity in both structures. Interestingly, gamma oscillations in the BLA were strongly coupled to theta frequency activity arising in the mPFC. This data is consistent with entrainment of inhibitory circuits in the BLA by mPFC input to mediate safety.

Organisms must continuously navigate complex environments, balancing the drive to seek rewards with the need to avoid potential threats. This tradeoff between approach and avoidance behaviors, known as approach-avoidance conflict, is a critical determinant of survival. The medial prefrontal cortex (mPFC) plays a key role in regulating these behaviors, with the ventromedial (vmPFC) and dorsomedial components thought to suppress and promote, respectively, behavioral responses to threats. Within the vmPFC, neural populations expressing the opioid peptide dynorphin (Dyn) and its receptor, the kappa opioid receptor (KOR), have been implicated in stress responses. However, the specific role of the vmPFC Dyn system in encoding threat-related information and shaping behavioral responses remains largely unexplored. To address this, we employed a multi-faceted approach, utilizing fiber photometry, calcium imaging, shRNA-mediated knockdown, and DREADD-mediated inhibition to investigate the vmPFC Dyn system in various threat-related paradigms. These included the platform-mediated avoidance (PMA) task, which assesses approach-avoidance conflict; the repeated looming disk test, a pain-free model of innate fear suppression; and standard fear conditioning, a well-established paradigm for studying learned fear responses. Our findings reveal that while the vmPFC Dyn system is not differentially regulated under non-threatening baseline conditions, it is actively recruited upon threat exposure. Fiber photometry recordings during the PMA task showed that vmPFC Dyn neurons bidirectionally signal features related to approach and avoidance behaviors in the presence of threat. Furthermore, shRNA-mediated downregulation of Dyn in the vmPFC led to enhanced avoidance in the repeated looming disk test, indicating that Dyn is necessary for suppressing avoidance in this context. Calcium imaging of the pan-neuronal vmPFC population in conjunction with Dyn knockdown revealed that loss of Dyn impairs cortical activity, as evidenced by reduced synchrony and decreased performance of a logistic regression decoder. These findings suggest that Dyn plays a critical role in shaping the activity of vmPFC neurons during threat processing. Taken together, our results highlight a specific role for the vmPFC Dyn system in toggling threat-driven behavioral responses, particularly in the context of approach-avoidance conflict. By demonstrating how Dyn shapes both behavior and neural activity in the vmPFC during threat exposure, this study provides novel insights into an understudied area of opioidergic circuitry. Moreover, our findings contribute to a deeper understanding of how distinct cell types within the vmPFC encode threat-related features to promote or suppress avoidance behaviors, shedding light on the neural mechanisms underlying adaptive responses to environmental challenges.