A cFos activation map of remote fear memory attenuation

Abstract

Rationale: The experience of strong traumata leads to the formation of enduring fear memories that may degenerate into post-traumatic stress disorder. One of the most successful treatments for this condition consists of extinction training during which the repeated exposure to trauma-inducing stimuli in a safe environment results in an attenuation of the fearful component of trauma-related memories. While numerous studies have investigated the neural substrates of recent (e.g., 1-day-old) fear memory attenuation, much less is known about the neural networks mediating the attenuation of remote (e.g., 30-day-old) fear memories. Since extinction training becomes less effective when applied long after the original encoding of the traumatic memory, this represents an important gap in memory research.

Objectives: Here, we aimed to generate a comprehensive map of brain activation upon effective remote fear memory attenuation in the mouse.

Methods: We developed an efficient extinction training paradigm for 1-month-old contextual fear memory attenuation and performed cFos immunohistochemistry and network connectivity analyses on a set of cortical, amygdalar, thalamic, and hippocampal regions.

Results: Remote fear memory attenuation induced cFos in the prelimbic cortex, the basolateral amygdala, the nucleus reuniens of the thalamus, and the ventral fields of the hippocampal CA1 and CA3. All these structures were equally recruited by remote fear memory recall, but not by the recall of a familiar neutral context.

Conclusion: These results suggest that progressive fear attenuation mediated by repetitive exposure is accompanied by sustained neuronal activation and not reverted to a pre-conditioning brain state. These findings contribute to the identification of brain areas as targets for therapeutic approaches against traumatic memories.

Keywords: Amygdala; Contextual fear conditioning; Cortex; Extinction; Hippocampus; Neuronal network; PTSD; Remote memory; Thalamus; cFos.

Fig. 1

Remote fear memory is efficiently attenuated by a spaced extinction paradigm. a Schematic representation of the experimental setting. Mice underwent contextual fear conditioning (CFC) and were re-exposed to the conditioned context 30 days later (Recall). On the following day, the animals were subjected to spaced extinction where they were re-exposed twice per day for 4 days to the conditioned context in the absence of foot shock. One and 15 days later, both groups received an additional context exposure to test their extinction memory (EM) and spontaneous recovery (SR). b Freezing levels across the spaced extinction procedure. During recall, freezing was significantly increased compared to baseline (BL, 3 min context exposure before conditioning), and during the last extinction session, extinction memory and spontaneous recovery freezing was significantly decreased compared to recall (ANOVA, F(4, 51) = 6.76, P = 0.0002, n = 8–16). c Experimental design for cFos analysis. d Freezing levels of all experimental animals further used for cFos analysis. At remote recall and during the first extinction sessions, freezing was significantly increased in animals that received the foot shock (“Home Cage,” “Recall,” and “Extinction”) compared to animals that received no shocks in the conditioning session (“Context Only”). At the end of extinction, animals that did or did not receive the foot shock in the conditioning context showed no significant differences in freezing (two-way ANOVA, F(1, 24) = 33.3, P < 0.0001, n = 10–16 per group). *P < 0.05 by Holm-Sidak post-hoc test

Fig. 2

cFos activation in cortex. a Schematic representation of the cortical structures selected for cFos density analysis. b Representative pictures of cFos immunohistochemistry in the PL. Scale bar = 200 μm. c cFos density in “Home Cage,” “Context Only,” “Recall” and “Extinction” groups in the ACC (ANOVA, F(3, 24) = 3.3, P < 0.04, n = 5–8), PL (ANOVA, F(3, 24) = 18.1, P < 0.0001, n = 5–8), IL (ANOVA, F(3, 24) = 13.7, P < 0.0001, n = 5–8), and RSP (ANOVA, F(3, 24) = 4.4, P = 0.01, n = 5–8). *P < 0.05; **P < 0.01; ***P < 0.001, by Sidak post-hoc tests

Fig. 3

cFos activation in thalamus. a Schematic representation of the thalamic structures selected for cFos density analysis. b Representative pictures of cFos immunohistochemistry in the NRe. Scale bar = 100 μm. c cFos density in “Home Cage,” “Context Only,” “Recall,” and “Extinction” groups in the CM (ANOVA, F(3, 25) = 0.8, P = 0.5, n = 5–8), aNRe (ANOVA, F(3, 15) = 0.9, P < 0.4, n = 3–7), PVT (ANOVA, F(3, 25) = 4.4, P = 0.01, n = 6–8), and NRe (ANOVA, F(3, 25) = 10.5, P = 0.00001, n = 6–8). *P < 0.05; **P < 0.01; ***P < 0.001, by Sidak post-hoc tests

Fig. 4

cFos activation in amygdala. a Schematic representation of the amygdalar structures selected for cFos density analysis. b Representative pictures of cFos immunohistochemistry in the BLA and CEA. Scale bar = 250 μm. c cFos density in “Home Cage,” “Context Only,” “Recall,” and “Extinction” groups in the CEA (ANOVA, F(3, 25) = 4.0, P = 0.01, n = 6–8) and BLA (ANOVA, F(3, 25) = 7.9, P = 0.0007, n = 6–8). *P < 0.05; **P < 0.01; ***P < 0.001, by Sidak post-hoc tests

Fig. 5

cFos activation in hippocampus. a Schematic representation of the hippocampal structures selected for cFos density analysis. b cFos density in “Home Cage,” “Context Only,” “Recall,” and “Extinction” groups in the dDG (ANOVA, F(3, 25) = 3.9, P = 0.02, n = 6–8) and dCA3 (ANOVA, F(3, 25) = 0.7, P = 0.5, n = 6–8), dCA1 (ANOVA, F(3, 25) = 1.2, P = 0.3, n = 6–8), vDG (ANOVA, F(3, 25) = 4.1, P = 0.02, n = 6–8), vCA3 (ANOVA, F(3, 25) = 9.6, P = 0.0002, n = 6–8) vCA1 (ANOVA, F(3, 25) = 5.2, P = 0.006, n = 6–8). *P < 0.05; **P < 0.01; ***P < 0.001, by Sidak post-hoc tests. c Representative pictures of cFos immunohistochemistry in the vCA1. Scale bar = 400 μm

Fig. 6

Cross-correlation and network connectivity analysis of cFos activationin in (from top to bottom) the “Home Cage”, “Context only”, “Recall” and “Extinction” groups. a Pearson correlation matrices showing inter-regional correlations for cFos activation density. Axes represent brain regions. Colors reflect Pearson correlation coefficients (scale, below) and labels within squares correspond to P values of correlations. *P < 0.05; **P < 0.01; ***P < 0.001. R values that were calculated using fewer than four pairs of cFos densities are shown as gray boxes. b Network connectivity graphs indicate only the strongest correlations (r value ≥ 0.5, P value ≤ 0.1, n value > 4). Connecting line transparency represents correlation strength (r value. Scale, below). Regions are color-grouped by major brain subdivisions and node size is proportional to the fold-change of cFos activation density between the indicated experiment and Home cage