Foreground contextual fear memory consolidation requires two independent phases of hippocampal ERK/CREB activation

Abstract

Fear conditioning is a popular model for investigating physiological and cellular mechanisms of memory formation. In this paradigm, a footshock is either systematically associated to a tone (paired conditioning) or is pseudorandomly distributed (unpaired conditioning). In the former procedure, the tone/shock association is acquired, whereas in the latter procedure, the context/shock association will prevail. Animals with chronically implanted recording electrodes show enhanced amplitude of the extracellularly recorded field EPSP in CA1 pyramidal cells for up to 24 h after unpaired, but not paired, fear conditioning. This is paralleled by a differential activation of the ERK/CREB pathway in CA1, which is monophasic in paired conditioning (0-15 min post-conditioning), but biphasic (0-1 h and 9-12 h post-conditioning) in unpaired conditioning as revealed by immunocytochemistry and Western blotting. Intrahippocampal injection of the MEK inhibitor U0126 prior to each phase prevents the activation of both ERK1/2 and CREB after unpaired conditioning. Block of any activation phase leads to memory impairment. We finally reveal that the biphasic activation of ERK/CREB activity is independently regulated, yet both phases are critically required for the consolidation of long-term memories following unpaired fear conditioning. These data provide compelling evidence that CA1 serves different forms of memory by expressing differential cellular mechanisms that are dependent on the training regime.

Figure 1.

Enhancement of synaptic efficacy in the CA1 of the dorsal hippocampus after unpaired fear conditioning. Mice were chronically implanted with stimulating electrodes in the ventral hippocampal commissural pathway and recording electrodes in the contralateral pyramidal layer of the CA1. Field potentials evoked by a single-pulse stimulation of the ventral hippocampal commissural pathway were recorded at different time points either before conditioning to determine the baseline level, or after each type of conditioning (unpaired or paired conditioning). (A) Kinetic of the effect of each type of conditioning on the evoked field potential amplitudes. At different time intervals after either conditioning protocol, evoked field potentials were recorded in a neutral context. Unpaired conditioning (n = 12) induced a significant increase in evoked field potential amplitude in the CA1 of the hippocampus at any analyzed time points after conditioning. Paired conditioning (n = 8) induced no variation as revealed by statistical analyses. (B) Examples of recording curves obtained just before or 1 h after unpaired conditioning. Dashed lines represent the evoked field potential amplitudes used to establish the quantifications represented in A. Statistics: *P < 0.05.

Figure 2.

ERK1/2 and CREB phosphorylation induced by unpaired compared to paired conditioning. (A) Immunocytochemical analyses of pERK1/2 (left) and pCREB (right) at different time intervals following context conditioning. pERK1/2 labeling was prominent in both the dendritic and somatic compartments with peak levels attained at 15 min and 9 h post-training. At baseline (home cage animals; ctrl), all somata were pCREB positive, but the intensity increased at 15 min and 9 h post-training. (B) Quantitative analyses of immunocytochemical labeling of pERK1/2 (left) and pCREB (right) revealed different activation patterns in paired (top row) and unpaired conditioning (bottom row). Paired conditioning induced an early transient activation phase of both pERK1/2 and pCREB in the CA1 of the dorsal hippocampus (n = 6 for each experimental group, n = 13 for the control group) with no further activation at later time points. Unpaired conditioning, in contrast, produced two peaks of pERK1/2 and pCREB at 0–1 h and around 9 h post-training (6 < n < 10 for each experimental group). Mean +SEM. (C) Representative Western blots showing pERK1/2 and ERK1/2 (left) as well as CREB and pCREB levels (right) from hippocampal extracts from mice sacrificed at different time intervals after unpaired fear conditioning. The biphasic pattern of phosphorylation as seen in immunocytochemistry was confirmed (for ERK1/2, mainly on the ERK1 [p44] isoform). Note that total ERK1/2 and CREB protein levels were not affected at any delay. Statistics: *P  <  0.05; **P  <  0.01; ***P  <  0.001.

Figure 3.

Transient inhibition of the MAPK/ERK1/2 pathway induced long-term memory deficits depending on the type of conditioning. Means ±SEM. (A) The MEK inhibitor U0126 or vehicle (DMSO 2% in aCSF) was infused bilaterally in the CA1 area of the dorsal hippocampus, 30 min before (U0126e), 2 h (U0126i), or 7.5 h (U0126l) after conditioning. Memory retention measured as freezing was evaluated 24 h later by re-exposing animals to the tone in a neutral context (tone test), and 2 h later to the context. Sham and vehicle-infused animals, which were spread out according to the different time points of injection, were pooled into one control group (paired: n = 26; unpaired: n = 27) since no differences were detected between groups (P ≥ 0.12). (B) U0126 infusion 30 min before (U0126e, n = 8) or 7.5 h after (U0126l, n = 10), but not 2 h after (U0126i, n = 8), conditioning blocked freezing responses to the context normally observed. Freezing to the tone was absent and not altered by drug. (C) Mice trained in paired conditioning exhibited a high freezing response to the tone when tested 24 h later. U0126 infused 30 min before the conditioning (U0126e, n = 8) impaired the retention to the tone, whereas the injection at later time points had no effect (U0126i, n = 8; U0126l, n = 10). Moreover, although these animals displayed a high level of freezing when re-exposed to the context of conditioning, U0126 had no effect on this response. Statistics: *P < 0.05; ***P < 0.001.

Figure 4.

Short-term memory processes were not affected by U0126 administered 30 min before paired (left, n = 4) or unpaired (right, n = 4) conditioning. Short-term memory was tested 1 h after training. Means ±SEM.

Figure 5.

CREB phosphorylation depends on the phosphorylation of ERK1/2, but the second peak of ERK1/2 is independent of the first phase. (A) Microphotography showing the inhibitory effect of U0126 (left panel) intrahippocampal infusion on pERK1/2 labeling compared to vehicle (right panel) in the CA1 of the dorsal hippocampus 15 min after unpaired conditioning. (B) U0126 infusion into the dorsal hippocampus 30 min before unpaired conditioning reduced pERK1/2 immunoreactivity (bottom left) compared to control (top left) 15 min post-training. Similarly, immunoreactivity for pCREB was also reduced in U0126 animals (bottom right) compared to controls (top right). Quantified data (Mean +SEM) are depicted in C confirming the reduction in pERK1/2 (left) and pCREB (right) after U0126 treatment. (D) Immunoreactivity for pERK1/2 (left column) and pCREB (right column) after U0126 infusion 30 min before (U0126, n = 4; vehicle, n = 3), 2 h after (U0126, n = 4; vehicle, n = 3), and 7.5 h after (U0126, n = 4; DMSO, n = 3) conditioning. Animals were sacrificed at 9 h post-training. Both pERK1/2 and pCREB labeling was reduced when U0126 was administered 7.5 h post-training. Data are quantified (Mean + SEM) in E for pERK1/2 (left) and pCREB (right).