Loss of Ensemble Segregation in Dentate Gyrus, but not in Somatosensory Cortex, during Contextual Fear Memory Generalization

Abstract

The details of contextual or episodic memories are lost and generalized with the passage of time. Proper generalization may underlie the formation and assimilation of semantic memories and enable animals to adapt to ever-changing environments, whereas overgeneralization of fear memory evokes maladaptive fear responses to harmless stimuli, which is a symptom of anxiety disorders such as post-traumatic stress disorder (PTSD). To understand the neural basis of fear memory generalization, we investigated the patterns of neuronal ensemble reactivation during memory retrieval when contextual fear memory expression is generalized using transgenic mice that allowed us to visualize specific neuronal ensembles activated during memory encoding and retrieval. We found preferential reactivations of neuronal ensembles in the primary somatosensory cortex (SS), when mice were returned to the conditioned context to retrieve their memory 1 day after conditioning. In the hippocampal dentate gyrus (DG), exclusively separated ensemble reactivation was observed when mice were exposed to a novel context. These results suggest that the DG as well as the SS were likely to distinguish the two different contexts at the ensemble activity level when memory is not generalized at the behavioral level. However, 9 days after conditioning when animals exhibited generalized fear, the unique reactivation pattern in the DG, but not in the SS, was lost. Our results suggest that the alternations in the ensemble representation within the DG, or in upstream structures that link the sensory cortex to the hippocampus, may underlie generalized contextual fear memory expression.

Keywords: PTSD; context; hippocampus; memory engram; mice.

Figure 1

Contextual fear memory is generalized over time. (A) Images of chambers used in the fear conditioning experiments. (B) A schematic of the experimental design. Mice were trained for fear conditioning in context A. They were subjected to a memory retrieval test by exposure to either context A or context B 1 day (1d) and 9 days (9d) later. Different groups of animals were used for each test (1d: context A, n = 12; context B, n = 12. 9d: context A, n = 12; context B, n = 13). (C) Percentage of time spent frozen during the retrieval tests in conditioned context A (white bars) and in novel context B (checkered bars) at different time points. Two-way ANOVA followed by Tukey’s multiple comparisons test, **p < 0.01, ***p < 0.001. (D) Levels of the discrimination index at different time points after conditioning. Discrimination of context A and context B was significantly decreased over 9 days after fear conditioning. Unpaired t-test, ***p < 0.001.

Figure 2

The transgenic system used to label neuronal ensembles activated during the acquisition and retrieval of memory. (A) Schematic representation of the transgenic system. When neuronal activity induces activation of the c-fos promoter in the absence of doxycycline (Dox), transactivator (tTA) drives the tetO promoter and induces the expression of tau-lacZ and the mutant tTA^H100Y (tTA*). Because tTA* can activate the tetO promoter even in the presence of Dox, neurons once activated during the off-Dox time window will continue to express tau-lacZ. (B) Schematic time-course representation of the genetic tau-lacZ (green line) and the endogenous ZIF (red dotted lines) expressions induced by two separated events. Neurons activated in response to fear conditioning during off-Dox expressed tau-lacZ persistently and endogenous ZIF transiently. Neurons activated by the retrieval test expressed endogenous ZIF, but the tetO-dependent tau-lacZ is not newly synthesized due to the presence of Dox. (C) Representative fluorescent confocal images showing cells labeled with tau-lacZ (green), ZIF (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue) in the dentate gyrus (DG) of the transgenic mice. White arrows: neurons labeled only with tau-lacZ. White arrowheads: neurons labeled only with ZIF. Yellow arrowheads: neurons double-labeled with tau-lacZ and ZIF. Scale bar, 50 μm. (D) Representative fluorescent confocal images showing the expression of tau-lacZ (green) in various regions. Mice were either fear-conditioned (FC) or remained in the home cage (HC) after Dox removal. Scale bar, 100 μm. (E) The proportion of tau-lacZ positive cells in the dorsal DG (HC, n = 11; FC, n = 24), dorsal hippocampal CA1 (HC, n = 9; FC, n = 24) and CA3 (HC, n = 6; FC, n = 21), somatosensory cortex (SS; HC, n = 11; FC, n = 24), and prelimbic cortex (PrL; HC, n = 6; FC, n = 15). Black bars: HC, white bars: fear conditioned. Unpaired t-test: *p < 0.05, **p < 0.01, ***p < 0.001. (F) The proportion of ZIF positive cells in the dorsal DG (HC, n = 13; RET, n = 12), dorsal hippocampal CA1 (HC, n = 11; RET, n = 12) and CA3 (HC, n = 6; RET, n = 10), (SS; HC, n = 13; RET, n = 12), and (PrL; HC, n = 5; RET, n = 7). Black bars: HC, white bars: retrieved. Unpaired t-test: *p < 0.05, **p < 0.01.

Figure 3

Reactivation of cell ensembles during the retrieval test 1 day after learning. (A) Representative confocal images of immunoreactive cells with tau-lacZ (green) and ZIF (red) in the dorsal DG, the dorsal hippocampal CA1, and the SS. The number of double-labeled cells in the DG was merely detected when mice were placed in context B 1 day after learning (left panel). A significant proportion of double-labeled cells (yellow arrowheads) was found in the CA1 and the SS when mice were re-exposed to context A 1 day after learning (middle and right panels, respectively). Blue: DAPI. Scale bar, 50 μm. (B) Quantification of tau-lacZ positive cells to DAPI positive cells in the DG, CA1 and SS. (C) Quantification of ZIF positive cells to DAPI positive cells in the DG, CA1 and SS. (D) Transgenic animals, which were tested for retrieval 1 day after learning in Figure 1, were used for the ensemble reactivation analysis. The percentage of neurons double-labeled with tau-lacZ and ZIF was compared to the chance level with the Wilcoxon signed rank test in the DG (context A, n = 12; context B, n = 12), the CA1 (context A, n = 12; context B, n = 12), and the SS (context A, n = 12; context B, n = 12). When the double:chance ratio equals 1, the probability that tau-lacZ positive cells overlap with ZIF positive cells is at the chance level. If the ratio is significantly larger than the chance level, neurons were significantly reactivated during retrieval. White bars: context A; checkered bars: context B. Wilcoxon signed rank test; ⁺p < 0.05, ⁺⁺p < 0.01. Mann Whitney test; *p < 0.05, **p < 0.01. (E) Schematic drawing of a mouse brain coronal section adapted from Franklin and Paxinos (2008), showing the regions of interest selected for measurements.

Figure 4

Reactivation of cell ensembles during the retrieval test 9 days after learning. (A) Representative confocal images of immunoreactive cells with tau-lacZ (green) and ZIF (red) in the dorsal DG, the dorsal hippocampal CA1, and the SS. Yellow arrowheads indicate double-labeled cells. Blue: DAPI. Scale bar, 50 μm. (B) Quantification of tau-lacZ positive cells to DAPI positive cells in the DG, CA1 and SS. (C) Quantification of ZIF positive cells to DAPI positive cells in the DG, CA1 and SS. (D) The percentage of neurons double-labeled with tau-lacZ and ZIF was compared to the chance level with the Wilcoxon signed rank test in the DG (context A, n = 12; context B, n = 13), the CA1 (context A, n = 12; context B, n = 12), and the SS (context A, n = 12; context B, n = 11). White bars: context A; checkered bars: context B. Wilcoxon signed rank test; ⁺⁺p < 0.01. Mann Whitney test; **p < 0.01.

Figure 5

Reactivation analysis in a novel context where mice do not exhibit generalized fear memory. (A) Image of chamber used in the experiment. (B) Percentage of time spent frozen during the retrieval tests in conditioned context A (white bar, n = 12) and in context B (checkered bar, n = 13) and in context C (gray bar, n = 9) at 9 days after training. The same data from Figure 1C are used for the freezing percentage in context A and in context B. One-way ANOVA followed by Tukey’s multiple comparisons test. **p < 0.01, ***p < 0.001 (C) The percentage of neurons double-labeled with tau-lacZ and ZIF was compared to the chance level with the Wilcoxon signed rank test in the DG (n = 9), the CA1 (n = 9), and the SS (n = 9). The proportion of reactivated DG cells was significantly below the chance level. *p < 0.05. (D) Quantification of tau-lacZ and ZIF positive cells to DAPI positive cells in the DG, CA1 and SS.