Fear conditioning-activated anterior basolateral amygdala neuronal ensembles are not required for remote fear memory
Authors: Hammack Robert , Daws Lynette , Toney Glenn 
Published: May 2023

Traumatic or threatening experiences can produce long-lasting fear memories whose formation reflects structural and chemical changes among engram neurons. Fear memory recall reflects reactivation of anatomically distributed ensembles of engram neurons. Over time, engram circuitry appears to reorganize such that storage of long-term memories migrates, partly or completely, to new neuronal ensembles. Here, we investigated the extent to which fear engram neurons in the anterior basolateral amygdala (aBLA), are critical for remote memory recall. We used adult offspring of TRAP2 and Ai14 mice, which allow for persistent Tdtomato expression amongst c-fos activated neurons (TRAPing). These mice were used to TRAP aBLA neurons activated during contextual fear conditioning (fear-TRAPed) or during exploration of the contextual environment (context-TRAPed). We then performed Fos-immunostaining on brains harvested following memory retrieval 3 weeks later to quantify re-activated aBLA neurons. We found that remote memory recall re-activated more aBLA neurons in fear- than context-TRAPed mice (n=6/group), suggesting that remote fear memory may be encoded by a larger aBLA engram population than is context memory. Next, we determined in separate cohorts of mice the extent to which fear behavior, indexed by postural immobility (freezing), during remote memory recall is differentially modulated by optogenetic activation and inhibition of fear-TRAPed vs. context-TRAPed aBLA neurons. We bilaterally injected (300 nL) into aBLA either AAV5-DIO-ChR2-EYFP or AAV5-FLEX-Jaws-GFP and placed fiber optic cannulae bilaterally into aBLA 2 weeks prior to context exposure or fear conditioning. Photostimulation of ChR2 (473 nm, 12 mW, 10 ms, 20 Hz)/Jaws (635 nM, 12 mW, continuous) 3 weeks later did not alter freezing behavior during memory recall in either fear-TRAPed or context-TRAPed mice (n=6-8/group). Lack of optogenetic modulation did not reflect failure of ChR2/Jaws to modulate neuronal activity as whole cell patch recordings revealed robust ChR2-driven action potential discharge (0.7 spikes/pulse) and Jaws-driven membrane hyperpolarization (-8.5 mV) (n=5-7/group). To assess the extent to which aBLA fear ensemble neurons were able to elicit fear behavior 3 weeks post TRAPing, a new cohort of mice expressing ChR2 in TRAPed aBLA neurons was photostimulated during exposure to a novel (neutral) context. Photostimulation was sufficient to drive freezing behavior in fear-TRAPed but not context-TRAPed mice (n=6-7/group). Thus, despite evidence of ensemble reactivation and the ability of aBLA fear-TRAPed neurons to drive novel context freezing behavior at a remote timepoint, neither photo-stimulation nor -inhibition altered freezing behavior during remote memory retrieval. Findings indicate that aBLA fear ensemble neurons are not a required component of the context-specific remote fear memory engram.

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This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.