Parallel memory processing by the CA1 region of the dorsal hippocampus and the basolateral amygdala

Abstract

There is abundant literature on the role of the basolateral amygdala (BLA) and the CA1 region of the hippocampus in memory formation of inhibitory avoidance (IA) and other behaviorally arousing tasks. Here, we investigate molecular correlates of IA consolidation in the two structures and their relation to NMDA receptors (NMDArs) and β-adrenergic receptors (β-ADrs). The separate posttraining administration of antagonists of NMDAr and β-ADr to BLA and CA1 is amnesic. IA training is followed by an increase of the phosphorylation of calcium and calmodulin-dependent protein kinase II (CaMKII) and ERK2 in CA1 but only an increase of the phosphorylation of ERK2 in BLA. The changes are blocked by NMDAr antagonists but not β-ADr antagonists in CA1, and they are blocked by β-ADr but not NMDAr antagonists in BLA. In addition, the changes are accompanied by increased phosphorylation of tyrosine hydroxylase in BLA but not in CA1, suggesting that β-AD modulation results from local catecholamine synthesis in the former but not in the latter structure. NMDAr blockers in CA1 do not alter the learning-induced neurochemical changes in BLA, and β-ADr blockade in BLA does not hinder those in CA1. When put together with other data from the literature, the present findings suggest that CA1 and BLA play a role in consolidation, but they operate to an extent in parallel, suggesting that each is probably involved with different aspects of the task studied.

Memory consolidation of one-trial inhibitory avoidance (IA) relies on the CA1 region of the dorsal hippocampus, the basolateral amygdala (BLA), and the entorhinal, parietal, and cingulate cortex (1–6). CA1 plays a key role in consolidation of IA and other fear-motivated learning tasks using the sequence of molecular events that underlies long-term potentiation (LTP) in that area (1, 2, 6, 7). This sequence is initiated by activation of NMDA receptors (NMDArs) followed by up-regulation of calcium/calmodulin-dependent protein kinase II (CaMKII), cAMP-dependent protein kinase (PKA), extracellular signal-regulated kinases 1 and 2 (ERK1/2) (8), several transcription factors (1), and mRNA and protein synthesis (1, 2).

There are doubts as to whether the BLA plays a role in IA consolidation (9), its modulation by emotional arousal or stress (1–4), both consolidation and modulation (1, 9, 10), or general regulation of hippocampal functions (11–13). The term “BLA” is used by most authors more or less generically (1, 3, 4, 10, 14); some have attributed its role rather to the lateral amygdala (15, 16), and other amygdala nuclei may also participate (4, 10). Evidence indicates that CaMKII and ERK signaling pathways are necessary for IA consolidation in CA1 (1, 2, 8) and for the consolidation of contextual and conditioned fear in BLA (14, 15). Functional inactivation (5, 17) or posttraining infusion of specific antagonists of NMDAr or β-adrenergic receptors (β-ADrs) into BLA or CA1 (9, 18–20) causes retrograde amnesia for several fear-motivated learning tasks and/or blocks the memory-enhancing effect of a variety of substances (1, 9). NMDArs are believed to mediate activity-dependent synaptic changes inherent to memory formation (1, 2), whereas β-ADrs are usually attributed a modulatory role secondarily affecting CaMKII and GluR1 phosphorylation and ERK1/2 and PKA signaling (1, 2, 8, 20).

The amygdala affects hippocampal LTP and memory formation by the hippocampus (10, 11, 20–24). It is generally agreed that it does so through influences mediated by entorhinal and perirhinal neurons (25) and, in many cases (26, 27), but not all (28–30), probably the dentate gyrus (26–32).

Do NMDAr and β-ADr blockers affect similar metabolic pathways in CA1 and BLA? Does NMDAr blockade after training affect the molecular events that accompany or underlie consolidation in BLA? Are CaMKII and ERK1/2 signaling pathways involved in the consolidation of IA in the BLA? Given that there are reciprocal pathways between the amygdala and the hippocampus (26) does hippocampal activity affect the BLA in relation to memory formation? In particular, do the hippocampal molecular changes underlying IA consolidation (1, 2, 6, 7, 8) affect the amygdala? Several studies have shown that ERK1/2 regulate the phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis (33–36). Given that ERK1/2 activity increases in the hippocampus (8) and, as will be seen, also in BLA after IA training, does this influence local TH activity and therefore catecholamine production? Most accounts on hippocampal or amygdala modulation by β-ADr or dopaminergic receptors (1, 2, 37–39) assume that their function is regulated mostly by arousal influences at their site of origin, rather than by variables acting at the catecholaminergic terminals in the temporal lobe (1–4).

Here, we provide some answers to the questions posed above and possibly raise others.

Results

Amnesic Effect of NMDAr and β-ADr Antagonists.

To analyze whether consolidation of avoidance memory requires NMDAr and β-ADr in BLA and CA1, male Wistar rats were trained in IA and immediately after that received bilateral intra-BLA (Fig. 1A) or intradorsal CA1 (Fig. 1B) infusions of vehicle (0.1% DMSO in saline), the NMDAr antagonists d-2-amino-5-phosphonovaleric acid (AP5) (5 μg per side) and 5R,10S-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) (1 μg per side) or the β-ADr antagonists S-(-)-1-(t-butylamino)-3-[(4-morpholino-1,2,5-thiadiazol-3-yl)oxy]-2-propanol maleate [timolol (TIM)] (1 μg per side) and (S)-1-Isopropylamino-3-(1-naphthyloxy)-2-propanol hydrochloride [propranolol (PRO)] (1 μg per side). Memory retention was evaluated in a nonreinforced test session carried out 24 h later (test 1). AP5, MK-801, TIM, and PRO significantly reduced test step-down latency (Fig. 1; test 1), indicating that retention of IA long-term memory involves the early participation of NMDAr and β-ADr in BLA and CA1. A second test carried out 48 h after the first one (test 2) showed that the amnesia was long-lasting and probably caused by blockade of the consolidation process.

Fig. 1.

Consolidation of IA memory requires functional NMDAr and β-ADr in BLA and the CA1 region of the dorsal hippocampus. Effect of the bilateral intra-BLA (A) or intra-CA1 (B) infusion of vehicle (VEH), AP5 (5 μg per side), MK-801 (MK; 1 μg per side), TIM (1 μg per side), or PRO (1 μg per side) on IA memory retention as evaluated 24 h (test 1) or 72 h (test 2) after training. Data are expressed as median ± interquartile range. ***, P < 0.001 and **, P < 0.01 vs. VEH in the respective session in Dunn's multiple comparison analysis after Kruskal-Wallis test; n = 10 per group.

Signaling Pathways Activated by NMDAr and β-ADr in BLA and CA1 During IA Memory Consolidation.

NMDAr-dependent LTP in the amygdala and the hippocampus is impaired by CaMKII inhibitors (40, 41) whereas β-ADr activation modulates LTP in both regions (see refs. 1 and 27 for references). In turn, the GluR1 subunit of AMPA receptors is one of the best-studied neuronal substrates of CaMKII (42, 43) and several pieces of evidence indicates that ERK1/2-mediated phosphorylation of TH at Ser-31 is critical for activation of this enzyme in different experimental systems (33, 34). Thus, to investigate whether IA consolidation is indeed associated with activation of NMDAr- and β-ADr-coupled signaling in BLA and CA1 we analyzed the effect of IA training in the phosphorylation state of CaMKII (pThr286CaMKII; pCaMKII) and ERK2 (pThr202/Tyr204ERK2; pERK) and their substrates GluR1 (pSer831GluR1; pGluR1) and TH (pSer-31TH; pTH).

IA training did not modify the levels of pCaMKII and pGluR1 in BLA (Fig. 2, BLA) but induced a rapid and reversible increase in the phosphorylation of these two proteins in dorsal CA1 (Fig. 2, CA1). In BLA, pERK and pTH peaked 30 min posttraining and returned to control values within 180 min (Fig. 2, CA1). Conversely, in the CA1 region pERK2, but not pTH, increased after IA training. No changes in total CaMKII, GluR1, ERK2, or TH levels were observed in BLA or CA1 at the posttraining times analyzed (data not shown).

Fig. 2.

IA training induces the activation of ERK2 and CaMKII in the CA1 region of the dorsal hippocampus and the activation of ERK2 in the BLA. Representative immunoblots and densitometric quantification show the time course of the IA training-induced increase in αCaMKII (pCaMKII), GluR1 (pGluR1), ERK2 (pERK2), and TH (pTH) phosphorylation levels in total homogenates prepared from the BLA or the CA1 region of the dorsal hippocampus (CA1). IA-trained rats were killed immediately (0), 30 or 180 min after training. n = Naïve animals, i.e., rats that were killed without submitting them to any specific behavioral protocol. Data are expressed as mean ± SEM. ***, P < 0.001, **, P < 0.01, and *, P < 0.05 vs. naïve in Dunnett's test after ANOVA; n = 5 per group.

ERK Plays a Physiologic Role in CA1, and Both ERK and TH Play a Role in BLA.

When given into BLA immediately after training, the TH inhibitor α-methyl-p-tyrosine methyl ester hydrochloride (AMPT; 0.5–50 μg per side; ref. 44) hindered IA long-term memory (Fig. 3Left). AMPT had no effect on memory retention when given in CA1 immediately posttraining (Fig. 3 Right). Intra-BLA posttraining infusion of TIM (1 μg per side) or the ERK1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene ethanolate (U0126) (1 μg per side), but not AP5 (5 μg per side), blocked the IA-induced increase in ERK2 and TH phosphorylation in BLA (Fig. 4A). U0126 has a profound amnesic effect in this task when given into these and other structures (45), as does another MEK1/2 inhibitor, PD098059 (46). On the other hand, intra-CA1 infusion of AP5 and U0126, but not TIM, hindered the IA training-induced phosphorylation of ERK2 in CA1 (Fig. 4B). Control animals that received a foot shock identical to that used for IA training but were not submitted to the IA procedure also showed increased pERK2 and pTH levels in BLA but not in CA1. The time course of these changes was similar to that observed after IA training (Fig. 4 C and D). Moreover, the phosphorylation of ERK2 and TH that occurred in the BLA of shocked animals was also blocked by TIM (Fig. 4E). Thus, the activation of the ERK pathway that happens in the BLA after IA training might be related to the pain and/or emotional arousal induced by exposure to the foot shock inherent to the IA paradigm rather than to the learning of the task itself. Alternatively, it is possible that animals in the shocked control group formed some kind of fear-motivated association, however, different from that produced by IA training, and that the increase in pERK2 and pTH observed in BLA after the foot shock was related to that learning. In fact, presentation of the foot shock alone in the training apparatus resembles a contextual fear conditioning training protocol (1, 8, 41).

Fig. 3.

Inhibition of TH in the BLA but not in the CA1 region of the hippocampus blocks consolidation of IA memory. Effect of the bilateral intra-BLA (Left) or intra-CA1 (Right) infusion of vehicle (VEH) or AMPT on IA memory retention as evaluated 24 h posttraining. Data are expressed as median ± interquartile range. ***, P < 0.001 vs. VEH in Dunn's multiple comparison analysis after Kruskal-Wallis test; n = 10 per group.

Fig. 4.

Activation of the ERK signaling pathway induced by IA training requires functional β-ADr in BLA and NMDAr in the CA1 region of the dorsal hippocampus. (A) Representative immunoblots show the effect of the intra-BLA infusion of TIM, U0126 (U0), and AP5 on the increase in ERK2 (pERK2) and TH (pTH) phosphorylation induced in that region by IA training. Total homogenates prepared from the BLA of IA-trained rats that received TIM (1 μg per side) or U0126 (1 μg per side) immediately after training and were killed 30 min thereafter, exhibited significantly lower levels of pERK2 (t = 5.51, P < 0.05 for TIM; t = 9.76, P < 0.001 for U0; n = 4 per group) and pTH (t = 3.25, P < 0.05 for TIM; t = 7.23, P < 0.01 for U0; n = 4 per group) than those obtained from trained animals that received intra-BLA vehicle (T groups). The intra-BLA infusion of AP5 (5 μg per side) immediately posttraining had no effect on the training-induced increase in pERK2 (t = 0.89, P > 0.1; n = 4) and pTH levels (t = 1.49, P > 0.1; n = 4) in that region. N = Naïve animals, i.e., rats that were killed without submitting them to any specific behavioral protocol. (B) Representative immunoblots show the effect of the intra-CA1 infusion of TIM, U0126, and AP5 on the increase in pERK2 induced in that region by IA training. Total homogenates prepared from the dorsal CA1 region of trained rats that received U0126 (1 μg per side) or AP5 (5 μg per side) immediately after training and were killed 30 min thereafter exhibited significantly lower levels of pERK2 (t = 10.47, P < 0.001 for U0; t = 3.58, P < 0.05 for AP5; n = 4 per group) than those obtained from trained animals that received intra-CA1 vehicle (T groups). The intra-CA1 infusion of TIM (1 μg per side) immediately posttraining had no effect on the training-induced increase in pERK2 in that region (t = 0.71, P > 0.1; n = 4). (C and D) Densitometric quantification (C) and representative immunoblots (D) show the time course of the increase in pERK2 and pTH levels induced in total homogenates prepared from BLA by a 0.5-mA, 2-s electric foot shock. Shocked animals were killed immediately (0), 30 or 180 min after the electric footshock. Data are expressed as mean ± SEM. ***, P < 0.001 vs. Naïve in Dunnett's test after ANOVA; n = 5 per group. (E) Representative immunoblots show the effect of the intra-BLA administration of TIM on the increase in pERK2 and pTH induced in that region by a 0.5-mA, 2-s electric foot shock. Total homogenates prepared from the BLA of shocked rats that received TIM (1 μg per side) immediately after the shock and were killed 30 min thereafter exhibited significantly lower levels of pERK2 (t = 4.2, P < 0.05; n = 4 per group) and pTH (t = 3.45, P < 0.05; n = 4 per group) than those obtained from shocked animals that received intra-BLA vehicle (S groups).

Blockade of NMDAr Signaling in Dorsal CA1 Does Not Influence Learning-Induced Molecular Changes in BLA.

Because intrahippocampal administration of β-ADr and NMDAr antagonists interferes with the consolidation of contextual fear conditioning memory (47) we analyzed whether posttraining blockade of β-ADr and NMDAr in dorsal CA1 affects the biochemical changes induced by IA training in BLA. To do that, IA-trained animals received bilateral intra-CA1 infusions of an amnesic dose of AP5 or TIM immediately after training and were killed 30 min thereafter. As can be seen in Fig. 5A, the intra-CA1 infusion of TIM or AP5 did not affect the phosphorylation of ERK2 and TH that happens in BLA after IA training. However, intra-CA1 AP5, but not TIM, completely abolished the IA training-induced increases in pCaMKII, pGluR1 (Fig. 5B), and pERK (see Fig. 4B) that occur in dorsal CA1.

Fig. 5.

Blockade of β-ADr and NMDAr in dorsal CA1 does not affect the biochemical changes induced by IA training in BLA, and blockade of β-ADr and NMDAr in BLA does not affect the biochemical changes induced by IA training in dorsal CA1. (A) Representative immunoblots show the lack of effect of the intra-CA1 infusion of TIM and AP5 on the increase in ERK2 (pERK2; t = 0.95, P > 0.1 for TIM; t = 0.27, P > 0.1 for AP5; n = 4 per group) and TH (pTH; t = 0.16, P > 0.1 for TIM; t = 1.19, P > 0.1 for AP5; n = 4 per group) phosphorylation induced in BLA by IA training. (B) Representative immunoblots show the effect of the intra-CA1 administration of TIM and AP5 on the increase in CaMKII (pCaMKII; t = 0.24, P > 0.1 for TIM; t = 5.68, P < 0.01 for AP5; n = 4 per group) and GluR1 (pGluR1; t = 0.12, P > 0.1 for TIM; t = 3.54, P < 0.05 for AP5; n = 4 per group) phosphorylation induced in that region by IA training. (C) Representative immunoblots show the lack of effect of the intra-BLA infusion of TIM and AP5 on the increase in pCaMKII (t = 0.84, P > 0.1 for TIM; t = 0.40, P > 0.1 for AP5; n = 4 per group), pGluR1 (t = 0.03, P > 0.1 for TIM; t = 0.34, P > 0.1 for AP5; n = 4 per group), and pERK2 (t = 0.32, P > 0.1 for TIM; t = 0.66, P > 0.1 for AP5; n = 4 per group) phosphorylation induced in the dorsal CA1 region by IA training.

Blockade of β-ADr and NMDAr Signaling in BLA Does Not Influence Learning-Induced Molecular Changes in Dorsal CA1.

To evaluate whether blockade of β-ADr and NMDAr in BLA affect the biochemical changes induced by IA training in dorsal CA1, animals received bilateral intra-BLA infusions of TIM or AP5 immediately after IA training and were killed 30 min thereafter. Neither TIM nor AP5 given into BLA affected the IA-induced phosphorylation of CaMKII, GluR1, or ERK2 in dorsal CA1 (Fig. 5C).

Discussion

Extending previous findings (1, 2, 9, 18), AP5, MK-801, PROP, and TIM given immediately posttraining into either BLA or CA1 are amnesic, so both NMDAr and β-ADr in these two brain regions appear necessary for memory consolidation.

The molecular changes induced by IA training are different in CA1 and BLA. In the former, there is an increased activity of ERK2 and CaMKII and an increased phosphorylation of the substrate of the latter, GluR1. No change in TH phosphorylation is observed in CA1. In BLA there are no changes of CaMKII or GluR1 but there is also an increase in ERK2 and TH phosphorylation levels, the latter a substrate of ERK in this structure. In CA1, the increases in CaMKII and ERK2 phosphorylation are prevented by posttraining infusion of AP5 in that region. Here, we show that these changes are not prevented by β-ADr antagonists given into CA1. In BLA, the opposite is true concerning the posttraining increase in ERK2 phosphorylation: it is blocked by a β-ADr antagonist but not AP5.

The lack of influence of the inhibition of CA1 molecular cascades on BLA signaling and vice versa does not agree with the numerous postulations of a reciprocal influence of one structure on the other (2, 10, 20–25). However, this does not indicate that the reciprocal influences do not exist. It merely suggests that up to some extent, perhaps a large extent, memory processing by the two structures is parallel. There have been numerous postulations of parallel processing of recently learned information by BLA and CA1 (see refs. 1, 3, 4, and 10).

In the case of posttraining CaMKII and ERK2 activation in CA1 and ERK2 activation in BLA, the results fit with findings that specific inhibitors of these two kinases are amnesic when given into CA1, but only inhibitors of ERK1/2 are amnesic when given into BLA in the IA task (1, 2, 28, 45). Also, the TH data (posttraining increase in BLA, no change in CA1) fit with the finding that AMPT is amnesic when given into BLA but not CA1. AP5 given into CA1 and TIM given in BLA caused full retrograde amnesia and blocked the molecular changes induced in the structure in which they were infused, but they did not affect the molecular changes induced by training in the other structure. The molecular changes caused by IA training in BLA and CA1, therefore, must be viewed as parallel and not sequential (1, 2).

Thus, probably NMDAr activation of CA1 signaling cascades (1, 2, 8) and β-ADr activation of BLA signaling begin more or less simultaneously but then each cascade follows its own course for several hours (1). Clearly the amygdala influences the activities of the hippocampus relevant to learning (3, 4, 9, 10, 21–25), not only acting on the hippocampus itself, but also on the caudate nucleus and other brain regions (refs. 48–50 and see refs. 3 and 4). Other brain regions, particularly the anterior cingulate and other prefrontal cortical areas (50, 51), are known to influence hippocampal activity in memory processing. The amygdala influences frontal and other areas besides the hippocampus (10, 52, 53).

In BLA, but not CA1, footshock alone enhances ERK2 and TH phosphorylation just like IA training does. This finding suggests that BLA may be more involved in the analysis of information pertinent to the unconditioned stimulus than the hippocampus, which agrees with previous behavioral observations suggesting this idea (see above).

The present findings, like many before them, fail to draw a line between consolidation and modulation of IA memory. They may be taken to suggest instead that the hippocampus and the amygdala take part in both processes. It has been argued that consolidation lasts several hours to be susceptible to modulatory influences (3, 10), which is tantamount to considering consolidation and modulation as faces of the same coin (3, 4).

The amnesic effect of AP5 and MK-801 given into BLA and the change in ERK2 phosphorylation seen in that structure after IA training are typical of structures to which a role in consolidation is attributed, like CA1 (1). Therefore, despite the fact that the ERK changes in BLA are linked to an essentially modulatory phenomenon (the increase of pTH), there are reasons to believe that BLA does play a role in consolidation in this task, as it has been suggested to have in related tasks (14–16,18). The reasons are, of course, inferential, but they are no less solid than those that have been used to substantiate the role of the hippocampus in memory making (1–7). Given that inhibition of NMDAr signaling in CA1 hamper CA1 but not BLA learning-induced biochemical changes (and vice versa), it seems likely that the two brain structures process different aspects of the IA task, maybe in parallel. It was proposed long ago that BLA may be in charge of the more emotionally arousing aspects of consolidation and CA1 may be in charge of the more cognitive (spatial, contextual) aspects of consolidation (1–4, 9, 10, 52). The former causes long-lasting brain image changes visible in PET scan (54) or fMRI (55) studies, which may depend on mood (56).

Materials and Methods

IA Training.

Male Wistar rats (3 months old, 260–280 g) were trained in a step-down IA paradigm, a learning task in which stepping down from a platform present in a given context is associated with a foot shock, resulting in an increase in step-down latency (1, 8). The IA training apparatus consisted of a 50 × 25 × 25-cm Plexiglas box with a 5-cm-high, 8-cm-wide, and 25-cm-long platform on the left end of a series of bronze bars that constitute the floor of the box. During training, animals were gently placed on the platform facing the left rear corner of the training box. When they stepped down and placed their four paws on the grid, they received a 2-s, 0.5-mA scrambled foot shock and were immediately withdrawn from the training box. IA memory retention was evaluated in test sessions carried out 24 and 72 h after training. At test, trained animals were put back on the training box platform until they eventually stepped down to the grid. The latency to step down was taken as an indicator of memory retention. A 180-s ceiling was imposed on step-down latency during test sessions. To perform some experiments animals were bilaterally implanted with 27-gauge indwelling guide cannulae stereotaxically aimed to the pyramidal cell layer of the CA1 region of the dorsal hippocampus or to the BLA (8, 17, 20, 41, 45). Animals were allowed to recover from surgery for 4 days before submitting them to any other procedure. At the time of drug delivery, 30-gauge infusion cannulae were tightly fitted into the guides. Bilateral infusions (0.5 μl per side) were carried out over 60 s by using an infusion pump; the cannulae were left in place for 60 additional s to minimize backflow. In all experiments the National Institutes of Health's “Principles of Laboratory Animal Care” were strictly followed. When the experimental design allowed it, placement of the cannulae was verified postmortem. To do that, 24 h after the end of the behavioral procedures, 0.5 μl of 4% methylene blue in saline were infused as indicated above. Animals were killed by decapitation 15 min later, and the brains were stored in formalin for histological localization of the infusion sites. Infusions were spread with a radius of <1.3 mm³ and found to be correct (i.e., within 1.0 mm³ of the intended site) in 94% of the animals. Only data from animals with cannulae located in the intended sites were included in the final analysis.

Drugs and Antibodies.

AP5, MK-801, TIM, PRO, AMPT, and U0126 were purchased from Sigma. All drugs were first dissolved in saline or DMSO, aliquoted, and stored protected from light at −20° until use. Right before use aliquot was thawed and further diluted to working concentration with saline or 0.1% DMSO in saline.

Immunoblot Assays.

Tissue was homogenized in ice-chilled buffer (20 mM Tris·HCL (pH 7.4), 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 μg/ml aprotinin, 15 μg/ml leupeptin, 10 μg/ml bacitracin, 10 μg/ml pepstatin, 15 μg/ml trypsin inhibitor, 50 mM NaF, and 1 mM sodium orthovanadate]. Protein concentration was determined by using the BCA protein assay (Pierce), and equal amounts of protein were fractionated by SDS/PAGE before being electrotransferred to PVDF membranes (Immobilon-P; Millipore). After verification of protein loading by Ponceau S staining, the blots were blocked in Tween-Tris buffer saline [TTBS; 100 mm Tris·HCl (pH 7.5), containing 0.9% NaCl and 0.1% Tween 20) and incubated overnight with the primary antibody to be tested. The membranes were washed in TTBS and incubated with HRP-coupled anti-IgG antibody and washed again, and the immunoreactivity was detected by using the West-Pico enhanced chemiluminescence kit (Pierce). Densitometric analysis of the films was performed with the MCID Image Analysis System (version 5.02; Imaging Research). Blots were developed to be linear in the range used for densitometry. Antibodies were from Chemicon (anti-CaMKII, anti-GluR1), Promega (anti-pCaMKII), Calbiochem (anti-TH, anti-pTH), Santa Cruz Biotechnology (anti-pGluR1, anti-ERK1/2, anti-pERK), and Cell Signaling (anti-ERK1/2, anti-pERK).

Statistical Analyses.

Because a ceiling of 180 s was imposed to step down latencies during retention tests and this variable neither follows a normal distribution nor fulfills the assumption of homoscedasticity, behavioral data are presented as median ± interquartile range and were analyzed by Kruskal-Wallis nonparametric test followed by Dunn's post hoc comparisons. Biochemical data are presented as mean ± SEM and were analyzed by two-tailed paired Student′s t test or repeated measures ANOVA followed by Dunnett′s post hoc comparisons, as appropriate.