c-Rel, an NF-kappaB family transcription factor, is required for hippocampal long-term synaptic plasticity and memory formation

Abstract

Transcription is a critical component for consolidation of long-term memory. However, relatively few transcriptional mechanisms have been identified for the regulation of gene expression in memory formation. In the current study, we investigated the activity of one specific member of the NF-kappaB transcription factor family, c-Rel, during memory consolidation. We found that contextual fear conditioning elicited a time-dependent increase in nuclear c-Rel levels in area CA1 and DG of hippocampus. These results suggest that c-rel is active in regulating transcription during memory consolidation. To identify the functional role of c-Rel in memory formation, we characterized c-rel(-/-) mice in several behavioral tasks. c-rel(-/-) mice displayed significant deficits in freezing behavior 24 h after training for contextual fear conditioning but showed normal freezing behavior in cued fear conditioning and in short-term contextual fear conditioning. In a novel object recognition test, wild-type littermate mice exhibited a significant preference for a novel object, but c-rel(-/-) mice did not. These results indicate that c-rel(-/-) mice have impaired hippocampus-dependent memory formation. To investigate the role of c-Rel in long-term synaptic plasticity, baseline synaptic transmission and long-term potentiation (LTP) at Schaffer collateral synapses in c-rel(-/-) mice was assessed. c-rel(-/-) slices had normal baseline synaptic transmission but exhibited significantly less LTP than did wild-type littermate slices. Together, our results demonstrate that c-Rel is necessary for long-term synaptic potentiation in vitro and hippocampus-dependent memory formation in vivo.

Figure 1.

Temporal change of nuclear levels of c-Rel during long-term memory formation. (A) c-Rel protein expression in area CA1 in the hippocampus was analyzed by Western blotting. c-Rel is expressed predominantly in the cytoplasm of the CA1 region of the hippocampus and slightly in the nucleus. c-rel^−/− mice do not express c-Rel in either the cytoplasm or the nucleus. (B) Cytoplasmic marker MKP-3 and nuclear marker lamin A were used to show the purity of cytoplasmic and nuclear extracts. (C) Mice were trained using a contextual fear conditioning paradigm and sacrificed at different times after training (15 min, 30 min, 1 h, and 3 h). Nuclear fraction was prepared from area CA1 and the DG of the hippocampus, and amount of c-Rel protein was assessed using Western blotting. Nuclear marker protein lamin A was used as a loading control. Level of c-Rel in the nucleus was significantly increased at 30 min after training in area CA1 (F_[4.19] = 5.505; P < 0.01; n = 5); **P < 0.01. (D) Level of c-Rel in the nucleus was significantly increased at1 h after fear conditioning in DG (F_[4.15] = 5.900; P < 0.01; n = 4); **P < 0.01.

Figure 2.

Stereological analysis of total numbers of pyramidal neurons in hippocampal area CA1 and quantification of hippocampal volume. (A) Cresyl violet-stained horizontal section of hippocampus from c-rel^−/− and wild-type littermate mice were compared for the possible presence of gross morphological changes in c-rel^−/− mice. We found no gross morphological changes or differences in the hippocampus between c-rel^−/− and wild-type littermate mice. Total hippocampal volume (B) and CA1 pyramidal neuron number (C) were determined from horizontal cresyl violet sections. The hippocampal volume and CA1 neuron numbers were not different between c-rel^−/− and wild-type littermate mice. n = 3 wild-type and 3 c-rel^−/− (male only). Scale bars, 100 μm.

Figure 3.

Basal behavioral characterization of c-rel^−/− mice. Several behavioral tasks were performed in both c-rel^−/− and wild-type littermate mice to measure their overall levels of locomotor activity, anxiety, nociception, and motor learning. (A) c-rel^−/− mice have similar levels of locomotor activity relative to wild-type littermates as assessed by total distance traveled during a 15 min session in the open field task. n = 25 wild-type (14 male, 11 female) and 27 c-rel^−/− (18 male, nine female). (B) c-rel^−/− mice have similar levels of basal anxiety relative to wild-type littermate as assessed by the ratio of the center:total distance traveled in the open field task. n = 25 wild-type (14 male, 11 female) and 27 c-rel^−/− (18 male, nine female). (C) No significant difference was seen in thermal sensitivity between c-rel^−/− and wild-type littermates, indicating that nociception is comparable between the two groups of mice. n = 25 wild-type (14 male, 11 female) and 27 c-rel^−/− (18 male, nine female). (D) Motor learning of c-rel^−/− mice was measured using an accelerating rotarod test. In this test, c-rel^−/− mice did not show motor learning deficits compared with wild-type littermates. n = 18 wild-type (11 male, seven female) and 20 c-rel^−/− (13 male, seven female).

Figure 4.

c-rel^−/− mice have impaired hippocampus-dependent fear memory. c-rel^−/− and wild-type littermate mice were trained in two different fear conditioning paradigms and then tested for freezing behavior. Hippocampus-dependent long-term memory formation was assessed via a contextual fear conditioning paradigm, and amygdala-dependent long-term memory formation was assessed via a cued fear conditioning paradigm. Mice were trained with modest training protocol (one exposure to cue and shock), and freezing behavior was measured 24 h after training. (A) In the contextual test, c-rel^−/− mice had a significant deficit in freezing behavior assessed 24 h after training (*P < 0.05). n = 9 wild-type (seven male, two female) and 9 c-rel^−/− (seven male, two female). (B) In the cued test, no significant difference was seen in freezing behavior between c-rel^−/− and wild-type littermates when mice were tested in the absence or the presence of an auditory cue 24 h after training. n = 9 wild-type (seven male, two female) and 9 c-rel^−/− (seven male, two female). (C) To assess short-term contextual memory in c-rel^−/− mice, in a parallel experiment, freezing behavior was measured 1 h after training via a context re-exposure test. c-rel^−/− mice did not show any deficits in short-term fear memory in this test. n = 6 wild-type (four male, two female) and 7 c-rel^−/− (five male, two female). (D) c-rel^−/− mice showed no deficits in long-term fear memory with a more robust training paradigm consisting of three training trials instead of one. n = 12 wild-type (seven male, five female) and 13 c-rel^−/− (eight male, five female).

Figure 5.

Impaired novel object recognition memory in c-rel^−/− mice. The object recognition task is based on the natural tendency of mice to investigate a novel object rather than a familiar object, and object recognition memory also depends on hippocampus. During the training day, two identical objects were placed in the open field box. Each mouse was then placed in the middle of box and allowed to freely investigate the objects for 10 min. On the test day (24 h after training), one of the familiar objects was replaced with a novel object, and the amount of time the mouse explored each object was recorded. In this test, wild-type littermate showed statistical significant preference to the novel object (*P < 0.05). However, c-rel^−/− mice did not distinguish the novel object from the familiar object. These results indicate that c-rel^−/− mice have a deficit in hippocampus-dependent object recognition memory. n = 13 wild-type (nine male, four female) and 14 c-rel^−/− (10 male, four female).

Figure 6.

Impaired short-term and long-term synaptic plasticity in c-rel^−/− mice. (A) LTP at Schaffer collateral synapses in hippocampal slices of c-rel^−/− mice was compared with wild-type littermate controls, after two trains of 100-Hz stimulation for 1 sec separated by 20 sec at 30°C. c-rel^−/− slices exhibited significantly less potentiation after LTP induction relative to wild-type littermate slices. F_[1,1504] = 433.00, P < 0.0001; n = 10 wild-type and 9 c-rel^−/− (male only). (B) To analyze basal synaptic transmission of c-rel^−/− mice, input/output functions for stimulus intensity versus fEPSP was measured. The input/output function in c-rel^−/− mice was not different from wild-type littermate. (C) Basal synaptic transmission was also assessed by examining the correlation between the slopes of fEPSP and fiber volleys, known as the input/output relationship. c-rel^−/− mice and wild-type littermate had comparable input/output relationships.