ERK pathway activation bidirectionally affects visual recognition memory and synaptic plasticity in the perirhinal cortex

Abstract

ERK 1,2 pathway mediates experience-dependent gene transcription in neurons and several studies have identified its pivotal role in experience-dependent synaptic plasticity and in forms of long term memory involving hippocampus, amygdala, or striatum. The perirhinal cortex (PRHC) plays an essential role in familiarity-based object recognition memory. It is still unknown whether ERK activation in PRHC is necessary for recognition memory consolidation. Most important, it is unknown whether by modulating the gain of the ERK pathway it is possible to bidirectionally affect visual recognition memory and PRHC synaptic plasticity. We have first pharmacologically blocked ERK activation in the PRHC of adult mice and found that this was sufficient to impair long term recognition memory in a familiarity-based task, the object recognition task (ORT). We have then tested performance in the ORT in Ras-GRF1 knock-out (KO) mice, which exhibit a reduced activation of ERK by neuronal activity, and in ERK1 KO mice, which have an increased activation of ERK2 and exhibit enhanced striatal plasticity and striatal mediated memory. We found that Ras-GRF1 KO mice have normal short term memory but display a long term memory deficit; memory reconsolidation is also impaired. On the contrary, ERK1 KO mice exhibit a better performance than WT mice at 72 h retention interval, suggesting a longer lasting recognition memory. In parallel with behavioral data, LTD was strongly reduced and LTP was significantly smaller in PRHC slices from Ras-GRF1 KO than in WT mice while enhanced LTP and LTD were found in PRHC slices from ERK1 KO mice.

Keywords: ERK1,2; perirhinal cortex; recognition memory; synaptic plasticity.

Figure 1

Activation of ERK in the Perirhinal Cortex is necessary for recognition memory consolidation. (A) Location of the perirhinal cortex (PRHC) in a schematic lateral view of the mouse brain. Shading indicates the location of the hippocampal formation (HC) and the perirhinal (PRHC), enthorinal (EC), and postrhinal cortices (POR); rs indicates the rhinal sulcus. (B) Photomicrograph of a coronal brain section showing the track (indicated by red arrow) left by a cannula inserted into the PRHC. The PRHC is also indicated. (C) U0126 infusion is effective in blocking ERK activation in the perirhinal cortex. Example of immunohistochemistry for phospho-ERK 30′ after the sample phase in one animal injected with U0126 in one PRHC and with vehicle in the contralateral PRHC. Phospho-ERK immunopositive cells are clearly present in the PRHC treated with vehicle (right) but are absent from the cortex treated with U0126 (left), showing that U0126 effectively diffused from the injection cannula and blocked ERK activation in the PRHC. In the inset, the mean number of phospho-ERK immunopositive cells per square millimeter counted in the U0126 treated PRHC (7 ± 2.6) and in the vehicle treated PRHC (38 ± 5.7; n = 4 animals) is reported. The difference between U0126 and vehicle treated side is significant (paired t-test, p = 0.035). Calibration bar: 50 μm. (D) Localization of cells immunopositive for phospho-ERK 30′ after exploration of the ORT arena with two new objects. Ect, ectorhinal cortex; Prhc, perirhinal cortex; Ent, entorhinal cortex; RhS, rhinal sulcus. Calibration bar 100 μm. (E) High power image of phospho-ERK immunopositive neurons in the perirhinal cortex. Calibration bar 15 μm. (F) Example of a double staining for phospho-ERK and GAD 67 in the perirhinal cortex. Left, cells immunopositive for phospho-ERK in the PRHC. Arrows point to two stained neurons; center, immunostaining for GAD67. Arrows point to two stained neurons; right, merge of the two images. Calibration bar 80 μm. (G) Perirhinal focal infusion of MAPK blocker UO126 impairs long term (12 h) object recognition memory. UO126 (5 mM in 50% DMSO, n = 16) or vehicle (saline in 50% DMSO, n = 16) were injected (0.5 μl bilaterally) immediately (3 min) after the sample phase of the ORT. Recognition memory was tested (test phase) 12 h after the sample phase. Left: Exploration of novel compared to familiar objects during the test phase. Vehicle injected mice explore the novel object significantly more than the familiar object (asterisk, p < 0.05, paired t-test) while UO126 injected mice do not (paired t-test, p > 0.05). Right: discrimination index in the test phase for vehicle and U0126 injected mice. The latter exhibit a significantly lower discrimination index with respect to vehicle injected mice (Wilcoxon Signed Rank test, p < 0.05, asterisk).

Figure 2

Ras-GRF1 KO mice show long term visual recognition memory deficits. (A) Mean exploration times for the 1- and 12-h interval experiment in Ras-GRF1 KO mice and their WT littermates. The exploration time of the familiar object, tF, and the exploration time of the novel object, tN, in the test phase significantly differ (asterisk) for WT mice both at 1 h (n = 14) and at 12 h interval (n = 15; paired t-test, p < 0.001 at 1 h and p < 0.01 at 12 h) but for KO mice only at 1 h (n = 15) there is a significant difference (paired t-test, p < 0.001). At 12 h KO mice (n = 17) do not show any differential exploration of the new with respect to the familiar object (paired t-test, p = 0.69), suggesting a consolidation deficit. (B) Memory retention curve for ORT in Ras-GRF1 KO mice and their WT littermates. Discrimination index is plotted against time interval between familiarization and test. Asterisk denotes significant difference between WT and KO mice (two-way ANOVA, time × genotype, post hoc Holm–Sidak method).

Figure 3

Ras-GRF1 KO mice exhibit a reconsolidation deficit. (A) Protocols for repeated familiarizations (top) and for memory reconsolidation test (bottom). Top: mice are introduced into the arena in the presence of two identical objects for one (single familiarization phase) or eight consecutive sessions of 5 min (multiple familiarization phase). After a delay of 48 h the test phase is performed. Bottom: mice are introduced into the arena in presence of two identical objects for eight consecutive sessions of 5 min each. Twenty-four hours later mice memory trace is reactivated by re-exposing the mice to the same two familiar objects. After a delay of 10 min or of 24 h the test phase is performed. (B) Discrimination index of Ras-GRF1 KO mice 48 h after a single familiarization session or after repeated familiarization sessions. Asterisk denotes significant difference between Ras-GRF1 KO mice (n = 16 for the repeated familiarization, n = 20 for the single familiarization) in the two conditions (Mann–Whitney p = 0.012). Repeated exposure to the stimuli is sufficient to compensate for the deficit of visual recognition memory of Ras-GRF1 KO mice. (C) Discrimination index of Ras-GRF1 KO and WT mice 48 h after repeated familiarization sessions and 24 h after memory reactivation. In wt, re-exposure of the animals to the stimuli did not interfere with recognition of the familiar stimulus 24 h later (control n = 20, reactivated n = 16; p = 0.43 Mann–Whitney). By contrast a single exposure to the familiar stimulus 24 h after its memorization makes the memory trace labile in Ras-GRF1 KO mice: 24 h after reactivation, discrimination index was significantly decreased with respect to that found without reactivation (control n = 15, reactivated n = 17; p = 0.026 Mann–Whitney). (D) Exploration time of novel and familiar object of Ras-GRF1 KO mice 24 h after reactivation: there is no preferential exploration of the novel object (time of exploration of new object, tN versus time exploration familiar object, tF, control p < 0.001; reactivated p = 0.212 paired t-test). Asterisks denote significant differences. (E) Discrimination index 10 min after reactivation; the deficit in Ras-GRF1 KO mice was not present 10 min after reactivation.

Figure 4

Longer lasting visual recognition memory in ERK1 KO mice. (A) Left, quantification of phospho-ERK2 optical density, normalized to tubulin, in the perirhinal cortex of ERK1 KO (n = 11) and ERK1 WT (n = 10) mice. The difference is statistically significant (t-test, p = 0.039). Right, example of immunoblotting. (B) Memory retention curve for ORT in ERK1 KO mice (n = 23) and their WT littermates (n = 21). Discrimination index is plotted against time interval between familiarization and test. Asterisks denote significant difference between WT and KO mice (two-way ANOVA for repeated measures, time × genotype, genotype, time, and interaction all significant p < 0.001, post hoc Tukey’s test). Performance differs at 72 h. (C) Exploration times in the ORT for ERK1 KO mice (n = 23) and their WT littermates (n = 21). At 1 and 72 h intervals. Asterisks denote significant difference between tF and tN. At 1 h there is a significant difference for both groups but at 72 h only KO mice show a differential exploration of the new with respect to the familiar object (paired t-test, p < 0.001 for KO and p < 0.112 for WT mice).

Figure 5

Top: Average time course of field EPSP amplitude recorded from PRHC. The final level attained for LTP is significantly lower in Ras-GRF1 KO mice while it is higher in ERK1 KO mice with respect to WT mice (ANOVA, genotype × time, genotype p < 0.001, post hoc Student–Newman–Keuls Method). Bottom: Average time course of field EPSP amplitude recorded from PRHC. The final level attained for LTD in Ras-GRF1 KO mice is significantly lower than in WT mice; the latter is significantly lower than in ERK1 KO mice (ANOVA, genotype × time, genotype p < 0.001, post hoc Holm–Sidak method).