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.