Fig. 4: Chemogenetic reactivation of a hippocampal extinction engram inhibits circuit-induced relapse of extinguished fear.

A Adult male and female rats were placed on doxycycline (DOX) diet and injected with AAV-cFos-tTA and AAV-TRE-hM3Dq into the dorsal hippocampus (DH); a guide cannula was implanted into the nucleus reuniens (RE). After recovery from surgery, all animals underwent auditory fear conditioning. After conditioning, animals underwent extinction. After the last extinction session, animals were removed from the DOX diet and underwent an extinction retrieval session to capture DH extinction ensembles (“engram capture”). After engram capture, animals were returned on DOX chow diet for the remainder of the experiment. Retrieval testing consisted of two counterbalanced sessions to assess whether reactivation of the extinction engram affects circuit-induced relapse. Prior to the retrieval tests, animals received intra-RE infusions of either saline (SAL) or muscimol (MUS)—this drug assignment did not change for the two tests. In addition, animals received a systemic (i.p.) injection of either vehicle (VEH) or the designer drug clozapine-N-oxide (CNO); each animal was injected with these drugs in a counterbalanced manner across the two retrieval tests. B Left panel, Schematic representation of viral expression within the DH for all the animals (viral expression common to all rats is indicated by the most darkly shaded areas). Left panel, top row, representative immunofluorescent image of AAV-cFos-tTA and AAV-TRE-hM3Dq mCherry expression in the DH, Right panel, top row representative thionin-stained coronal section showing a midline cannula placement in the RE, Right panel, bottom row Cannula placements for all rats that were included in the analysis. Atlas figures are adapted from Swanson (2018). C Left panel displays percentage of freezing [mean ± standard error of the mean (SEM)] during fear conditioning during the baseline (BL) period and final CS. The group labels indicate the intracranial drug assignments for subsequent retrieval tests [saline (RE-SAL), n = 15; muscimol (RE-MUS), n = 13).; middle panel, displays percentage of freezing (mean ± SEM) during the extinction session for the first 5-trial block and the final extinction 5-trial block; right panel displays the percentage freezing (mean + SEM) for the engram capture session. White-filled and light blue-filled triangles represent female animals, while dark gray-filled and dark blue-filled diamonds represent male animals in the bar graph. D Percentage of freezing (mean ± SEM) for the extinction retrieval test, animals received either VEH or CNO injections, in a counterbalanced manner, to reactivate the DH extinction engram. Systemic CNO decreased freezing in animals receiving either SAL or MUS infusions into the RE thereby reducing the magnitude of circuit-induced relapse. *p < 0.05; n.s. = not significant.