Engrams: From Behavior to Brain-Wide Networks.

Animals utilize a repertoire of behavioral responses during everyday experiences. During a potentially dangerous encounter, defensive actions such as "fight, flight, or freeze" are selected for survival. The successful use of behavior is determined by a series of real-time computations combining an animal's internal (i.e., body) and external (i.e., environment) state. Brain-wide neural pathways are engaged throughout this process to detect stimuli, integrate information, and command behavioral output. The hippocampus, in particular, plays a role in the encoding and storing of the episodic information surrounding these encounters as putative "engram" or experience-modified cellular ensembles. Recalling a negative experience then reactivates a dedicated engram ensemble and elicits a behavioral response. How hippocampus-based engrams modulate brain-wide states and an animal's internal/external milieu to influence behavior is an exciting area of investigation for contemporary neuroscience. In this chapter, we provide an overview of recent technological advancements that allow researchers to tag, manipulate, and visualize putative engram ensembles, with an overarching goal of casually connecting their brain-wide underpinnings to behavior. We then discuss how hippocampal fear engrams alter behavior in a manner that is contingent on an environment's physical features as well as how they influence brain-wide patterns of cellular activity. Overall, we propose here that studies on memory engrams offer an exciting avenue for contemporary neuroscience to casually link the activity of cells to cognition and behavior while also offering testable theoretical and experimental frameworks for how the brain organizes experience.

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.

Basolateral Amygdala Astrocytes Are Engaged by the Acquisition and Expression of a Contextual Fear Memory.

Astrocytes are key cellular regulators within the brain. The basolateral amygdala (BLA) is implicated in fear memory processing, yet most research has entirely focused on neuronal mechanisms, despite a significant body of work implicating astrocytes in learning and memory. In the present study, we used in vivo fiber photometry in C57BL/6J male mice to record from amygdalar astrocytes across fear learning, recall, and three separate periods of extinction. We found that BLA astrocytes robustly responded to foot shock during acquisition, their activity remained remarkably elevated across days in comparison to unshocked control animals, and their increased activity persisted throughout extinction. Further, we found that astrocytes responded to the initiation and termination of freezing bouts during contextual fear conditioning and recall, and this behavior-locked pattern of activity did not persist throughout the extinction sessions. Importantly, astrocytes do not display these changes while exploring a novel context, suggesting that these observations are specific to the original fear-associated environment. Chemogenetic inhibition of fear ensembles in the BLA did not affect freezing behavior or astrocytic calcium dynamics. Overall, our work presents a real-time role for amygdalar astrocytes in fear processing and provides new insight into the emerging role of these cells in cognition and behavior.SIGNIFICANCE STATEMENT We show that basolateral amygdala astrocytes are robustly responsive to negative experiences, like shock, and display changed calcium activity patterns through fear learning and memory. Additionally, astrocytic calcium responses become time locked to the initiation and termination of freezing behavior during fear learning and recall. We find that astrocytes display calcium dynamics unique to a fear-conditioned context, and chemogenetic inhibition of BLA fear ensembles does not have an impact on freezing behavior or calcium dynamics. These findings show that astrocytes play a key real-time role in fear learning and memory.

Trpm4 ion channels in pre-Bötzinger complex interneurons are essential for breathing motor pattern but not rhythm.

Inspiratory breathing movements depend on pre-Bötzinger complex (preBötC) interneurons that express calcium (Ca2+)-activated nonselective cationic current (ICAN) to generate robust neural bursts. Hypothesized to be rhythmogenic, reducing ICAN is predicted to slow down or stop breathing; its contributions to motor pattern would be reflected in the magnitude of movements (output). We tested the role(s) of ICAN using reverse genetic techniques to diminish its putative ion channels Trpm4 or Trpc3 in preBötC neurons in vivo. Adult mice transduced with Trpm4-targeted short hairpin RNA (shRNA) progressively decreased the tidal volume of breaths yet surprisingly increased breathing frequency, often followed by gasping and fatal respiratory failure. Mice transduced with Trpc3-targeted shRNA survived with no changes in breathing. Patch-clamp and field recordings from the preBötC in mouse slices also showed an increase in the frequency and a decrease in the magnitude of preBötC neural bursts in the presence of Trpm4 antagonist 9-phenanthrol, whereas the Trpc3 antagonist pyrazole-3 (pyr-3) showed inconsistent effects on magnitude and no effect on frequency. These data suggest that Trpm4 mediates ICAN, whose influence on frequency contradicts a direct role in rhythm generation. We conclude that Trpm4-mediated ICAN is indispensable for motor output but not the rhythmogenic core mechanism of the breathing central pattern generator.

Dbx1 Pre-Bötzinger Complex Interneurons Comprise the Core Inspiratory Oscillator for Breathing in Unanesthetized Adult Mice.

The brainstem pre-Bötzinger complex (preBötC) generates inspiratory breathing rhythms, but which neurons comprise its rhythmogenic core? Dbx1-derived neurons may play the preeminent role in rhythm generation, an idea well founded at perinatal stages of development but incompletely evaluated in adulthood. We expressed archaerhodopsin or channelrhodopsin in Dbx1 preBötC neurons in intact adult mice to interrogate their function. Prolonged photoinhibition slowed down or stopped breathing, whereas prolonged photostimulation sped up breathing. Brief inspiratory-phase photoinhibition evoked the next breath earlier than expected, whereas brief expiratory-phase photoinhibition delayed the subsequent breath. Conversely, brief inspiratory-phase photostimulation increased inspiratory duration and delayed the subsequent breath, whereas brief expiratory-phase photostimulation evoked the next breath earlier than expected. Because they govern the frequency and precise timing of breaths in awake adult mice with sensorimotor feedback intact, Dbx1 preBötC neurons constitute an essential core component of the inspiratory oscillator, knowledge directly relevant to human health and physiology.