ERK phosphorylation is required for retention of trace fear memory

Neurobiology of Learning and Memory 85 (2006) 44–57 www.elsevier.com/locate/ynlme

ERK phosphorylation is required for retention of trace fear memory Julissa S. Villarreal *, Edwin J. Barea-Rodriguez Department of Biology, University of Texas at San Antonio, TX 78249, USA Received 31 May 2005; revised 5 August 2005; accepted 8 August 2005 Available online 22 September 2005

Abstract The extracellular signal-regulated kinase (ERK) has been previously associated with long-term memory formation. Earlier studies have demonstrated a role for phospho-ERK in delay fear conditioning and it has been shown to disrupt trace fear memory when inhibited after training. cAMP response element binding protein (CREB) is a key transcription factor that has been implicated in long-term memory formation across different species. It has also been shown to be modulated by ERK. In our study, we used the drug SL327 to prevent ERK phosphorylation. Two groups of Fischer 344 male rats (2–4 months) were injected intraperitoneally with 100% DMSO (2 ml/kg) or SL327 (100 mg/kg/2 ml dissolved in DMSO) 45 min before 10 trials of trace fear conditioning. Each trial consisted of a tone paired with a footshock with a 30-s interval separating the stimuli. Twenty-four hours later, rats were tested for fear to the tone. Our results showed that SL327-treated rats displayed memory deficits 24 h after training. Western blot analyses of total hippocampal protein revealed a significant increase in phosphorylated ERK immediately after training. There were also decreases in phosphorylated ERK at 45 and 90 min post-injection of SL327-treated rats as compared to DMSO-treated rats, but levels of phosphorylated CREB remained the same. These findings indicate that ERK phosphorylation is increased immediately after trace fear conditioning and inhibiting this increase is correlated with memory deficits in trace fear conditioning 24 h later. These findings support a role for ERK phosphorylation in the formation of trace fear memories.
2005 Elsevier Inc. All rights reserved. Keywords: Trace fear conditioning; Extracellular-signal regulated kinase; cAMP response element binding protein; Learning and memory

1. Introduction The mitogen-activated protein kinases (MAPK) are a family of kinases that take part in signal transduction pathways from the membrane to the nucleus. They participate in a variety of cellular programs which include cell division, cell differentiation, cell movement, and cell death. The family members include the extracellular signal-regulated kinases 1 and 2 (ERK 1 and ERK 2), cJun N-terminal kinase/stress-activated protein kinases (JNK/SAPK), p38 MAPK, ERK 3, and ERK 5 (English et al., 1999; Schaeffer & Weber, 1999; Seger & Krebs, 1995). Although classically studied in cellular growth processes, ERK signaling pathways are now being implicat*

Corresponding author. Fax: +1 210 458 7498. E-mail address: [email protected] (J.S. Villarreal).

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2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2005.08.005

ed in regulation of neuronal function. The ERK cascade is composed of three kinases that are consecutively phosphorylated. The first kinase is Raf-1, which in turn phosphorylates the second kinase, MEK (mitogen-activated, ERK-activating kinase) by serine/threonine phosphorylation. MEK then activates ERK through dual phosphorylation of both a threonine and tyrosine residue (English et al., 1999; Seger & Krebs, 1995). Interestingly, immunohistochemical studies show that ERK is prominently found in the neocortex, hippocampus, striatum, amygdala, and cerebellum, which are all areas involved in learning and memory processing (Fiore et al., 1993; Flood et al., 1998; Ortiz et al., 1995). More specifically, ERK is found in the cell bodies and dendrites of the dentate gyrus, CA3, and CA1 hippocampal areas (Fiore et al., 1993; Flood et al., 1998). Because ERK is found in several brain regions associated with learning and memory, numerous studies are

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now focusing on ERK in the regulation of neuronal function and memory processing. Numerous studies have shown that the ERK cascade is important in both invertebrate and vertebrate memory systems. Long-term facilitation in Aplysia is inhibited with the addition of antibodies against phosphorylated ERK or MEK inhibitors in the presynaptic cell (Martin et al., 1997). Crow, Xue-Bian, Siddiqi, Kang, and Neary (1998) also show that in vitro one-trial conditioning and multi-trial Pavlovian conditioning in Hermissenda results in the activation of ERK. More recently, phosphorylation of extra-nuclear ERK in the crab Chasmagnathus was important for long-term memory consolidation (Feld, Dimant, Delorenzi, Coso, & Romano, 2005). Additionally, levels of phosphorylated ERK increase in the insular cortex by a taste experience that produces a long-term taste memory. Equally important, inhibiting ERK phosphorylation in the insular cortex before exposure to a novel taste in a conditioned taste aversion task causes long-term taste memory deficits (Berman, Hazvi, Rosenblum, Seger, & Dudai, 1998). Hippocampally associated tasks such as the Morris water maze, contextual fear conditioning, and inhibitory avoidance require ERK phosphorylation as well. Using a specific inhibitor (SL327) of MEK to prevent phosphorylation of ERK, studies show that mice are impaired in contextual learning as well as spatial learning (Selcher, Atkins, Trzaskos, Paylor, & Sweatt, 1999). Additionally, another inhibitor of MEK, PD098059, blocked long-term spatial memory in rats trained in the water maze (Blum, Moore, Adams, & Dash, 1999). Atkins, Selcher, Petraitis, Trzaskos, & Sweatt (1998) show that ERK is activated in the hippocampus after tone and contextual conditioning and is needed for consolidation of this learning. Furthermore, studies using PD098059 have shown that hippocampal ERK is involved in retention of an inhibitory avoidance task, specifically in a time-dependent manner (Walz et al., 1999, 2000). Transient increases in ERK phosphorylation can also be seen in the amygdala after delay fear conditioning, and infusion of U0126 into the lateral amygdala impairs long-term memory of this fear conditioning (Schafe et al., 2000). These studies strongly support a role for ERK phosphorylation in learning and memory processes. cAMP response element binding protein (CREB) is a part of a large family of structurally related proteins that bind to cAMP response element (CRE)-containing promoter sites of different genes. The critical event in CREB activation is the phosphorylation of Serine133 in the kinase-inducible domain (KID), which includes concensus phosphorylation sites for protein kinase A, protein kinase C, calcium/calmodulin kinases, and RSK2. This phosphorylation can be regulated by increases or decreases in the levels of cAMP and calcium, which activate these kinases [as reviewed in (Silva, Kogan, Frankland, & Kida, 1998; Lamprecht, 1999)]. The involvement

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of CREB in learning and memory processes has been extensively studied across different species (Aplysia, Drosophila, rat, and mouse). Long-term facilitation in Aplysia cultured neurons was disrupted after injection of oligonucleotides with CRE sequences (Dash, Hochner, & Kandel, 1990). In Drosophila, pretraining disruption of CREB function through transgenic expression of a dominant-negative CREB protein caused long-term memory deficits in olfactory learning and memory (Yin et al., 1994). Mice with a CREBaD mutation have long-term memory impairments in contextual fear conditioning, the Morris water maze, and the social transmission of food preferences (Bourtchuladze et al., 1994; Kogan et al., 1997). Guzowski & McGaugh (1997) showed that pretraining intrahippocampal infusion of CREB antisense oligodeoxynucleotides (ODN) produce disruptions in CREB protein levels and that this disruption impaired retention in rats trained in the Morris water maze, suggesting an importance of CREB in the consolidation of memory processes initiated at the time of training. Trace fear conditioning is a task in which the subject must learn to associate a conditioned stimulus (CS; tone) with an unconditioned stimulus (US; foot shock) when they are separated over time. This information is referred to as being temporally discontiguous. Rawlins (1985) has suggested that most tasks sensitive to hippocampal damage contain some form of temporally discontiguous information, which must be used to solve the learning task correctly. Young rats with hippocampal lesions are impaired in both trace eye blink and trace fear conditioning (McEchron, Bouwmeester, Tseng, Weiss, & Disterhoft, 1998; Weiss, Bouwmeester, Power, & Disterhoft, 1999). Thus, the hippocampus is important for connecting temporally discontiguous events. Recently, Runyan, Moore, & Dash (2004) examined the role of the medial prefrontal cortex in trace fear conditioning through inhibition of the ERK cascade. Pre-training prefrontal cortex infusions of the MEK inhibitor, U0126, impaired trace memory retention. In subsequent experiments, post-training infusions of U0126 in the hippocampus also disrupted trace memory retention. However, their behavioral protocol differed from ours in that they used a shorter tone, a shorter trace period, and a lower shock intensity and duration. Also, freezing was measured only during the trace period of testing. Additionally, post-training hippocampal infusions of U0126 were used, which only allow for study of consolidation and does not address acquisition of trace fear conditioning. In our experiments, we used pre-training intraperitoneally (IP) injections of the MEK inhibitor SL327 to investigate acquisition and consolidation of trace fear conditioning. Furthermore, they investigated the levels of phosphorylated ERK after trace fear conditioning and found an increase at 1 h after training (Runyan et al., 2004).

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In our study, we tested the hypothesis that the MEK inhibitor, SL327, prevents ERK phosphorylation and causes trace fear memory deficits in young male F344 rats. To rule out detrimental side effects of the drug, rats that received 100 mg/kg SL327 were retrained without drug treatment and retested 24 h later. We investigated the levels of ERK phosphorylation in rats that received no training (naı¨ve), that received trace fear conditioning (trace trained), or that received pseudorandom conditioning (pseudo trained) to determine whether ERK phosphorylation is regulated by trace fear conditioning. To determine the extent of inhibited ERK phosphorylation by SL327, we investigated the levels of hippocampal phosphorylated ERK and CREB proteins in rats that had received SL327 or DMSO injections at 45 and 90 min after injection. We chose the hippocampus because previous studies have shown it to be important for trace fear conditioning (McEchron et al., 1998).

2. Materials and methods 2.1. Subjects Fischer 344 male virgin rats 2–4 months of age were housed separately and provided with food and water ad libitum. We purchased our rats through the National Institute on Aging (NIA), which maintains colonies of barrier-raised rodents under contractual arrangement with Harlan Sprague–Dawley (Harlan). They were kept on a 12-h light/dark cycle throughout the experiments. All rats were handled the day before the start of training for approximately 5–10 min and right before training and testing to prevent stress-related freezing responses due to handling. Animal use procedures were approved in advance by the Institutional Animal Care and Use Committee at the University of Texas at San Antonio. 2.2. Injections For behavioral experiments, two groups of rats (n = 8 DMSO-treated; n = 8 SL327-treated) were injected with 100% DMSO (2 ml/kg) or SL327 [a structural analogue of U0126 (Favata et al., 1998)] (100 mg/kg, 2 ml/kg, dissolved in 100% DMSO) intraperitoneally (IP) 45 min before trace fear conditioning [adapted from (Atkins et al., 1998; Selcher et al., 1999)]. SL327 was provided by Bristol-Myers Squibb, Princeton, NJ. Their weights were in the range between 250 and 300 g, and the average dosage was approximately .55 ml. For Western blot analyses, two additional groups of rats received IP injections of either 100% DMSO (2 ml/kg) or SL327 (100 mg/kg, 2 ml/kg, dissolved in 100% DMSO) and hippocampi were removed either 45 or 90 min after injection. Based on our behavioral studies, we chose the 45-min time point because we wanted

to investigate the levels of ERK and CREB phosphorylation at the start of training. We chose the 90-min time point because we wanted to investigate the levels of ERK and CREB phosphorylation at the end of training. Furthermore, to control for effects of training on ERK and CREB levels, additional control groups of rats received IP injections of either 100% DMSO (2 ml/kg) or SL327 (100 mg/kg/2 ml, dissolved in 100% DMSO) 45 min prior to trace fear conditioning. Hippocampi were then removed immediately following training. 2.3. Behavioral experiments 2.3.1. Behavioral apparatus The apparatus consisted of a modular chamber (30 · 24 · 21 cm, MED Associates, VT) constructed out of aluminum (two sidewalls) and Plexiglas (rear wall, ceiling, and hinged front door) and situated within a sound-attenuating cubicle (22 · 15 · 14 in.) in an isolated room. The floor consisted of 18 stainless steel rods (4 mm diameter) spaced 1.5 cm apart that were wired to a shock generator and scrambler for the delivery of a shock. A stimulus light, ventilation fan, and a speaker for delivering acoustic stimuli were mounted to a grating on the walls of the chamber. 2.3.2. Trace fear conditioning A total of 19 rats (n = 8 DMSO-treated and n = 8 SL327-treated and n = 3 trace fear conditioning without testing) were used. During trace fear conditioning, the light was on for the duration of the conditioning and the doors of the isolation cubicle were closed. There was also a coconut scent throughout the chamber. The coconut scent is unique to the conditioning chamber and helps to make the conditioning chamber different from the testing chamber. Training began 45 min after injections. Each rat was placed inside the conditioning chamber and was allowed a 90-s acclimation period. Each rat received 10 trace fear conditioning trials with an intertrial interval (ITI) of 210 s. On each trial, the tone CS (15 s, 80 dB, 3000 Hz, 5 ms rise/fall time) was followed by a 30-s silent trace interval and then a foot shock US (1 s, 1 mA) through the grid floor. After the final trial, the rat was returned to its home cage. The chambers were cleaned between the conditioning of each rat. Our protocol was adapted from McEchron et al. (1998). 2.3.3. Pseudorandom fear conditioning For pseudorandom conditioning, the chamber was the same as the one used in trace fear conditioning. For the pseudorandom protocol, the CS and US were the same as in the trace and delay fear conditioning except these rats were trained with 10 trials of the CS alone and 10 trials of the US alone, both presented pseudorandomly, with no stimulus being presented more than two consecutive times. The pseudorandom conditioning

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session lasted the same amount of time as trace fear conditioning. After the final trial, the rat was returned to its home cage. The chambers were cleaned between the conditioning of each rat. 2.3.4. Memory testing The testing period was the same for all conditioned groups. We used a different chamber for testing because we wanted to test for association of the tone with the shock and not the context in which the rats were shocked. The light was off in the testing chamber and the isolation doors to the chamber were open. The testing chamber was covered with black and white stripes surrounding the walls and a black Plexiglas grid floor ð914
1114Þ was placed on top of the grid floor. Salient cues (small toys and individual rat bedding) were placed in the chamber before testing to contribute to the novelty of the testing chamber. There was also a food scent present in the chambers. Rats were tested for fear conditioning to tone 24 h after conditioning. Rats were allowed 90 s to acclimate to the testing chamber, which was followed by a 5-min tone presentation. The freezing responses were then measured during this time in 1-min increments. Rats were then returned to their home cage. 2.3.5. Data acquisition We measured freezing by using a method developed previously (for detailed explanation, see (Maren, 1998)). Briefly, each chamber rested on a load-cell platform that records chamber displacement in response to each ratÕs motor activity. The load cell amplifier output from each chamber was digitized and acquired on-line using Threshold Activity software (MED Associates, VT). The output activity was digitized at 5 Hz, which yields one observation per rat every 200 ms. Freezing was scored only if the animal was immobile for 1 s. We used an Excel template (kindly provided by Dr. Steven Maren, University of Michigan) that transforms load cell output activity into freezing percentages. This method has been used in a previous study in our laboratory (Villarreal, Dykes, & Barea-Rodriguez, 2004). 2.4. Western blot analysis 2.4.1. Protein extraction from hippocampal tissue At 45 or 90 min after injection, rats were anesthetized with Nembutal (.5–.7 ml of a 50 mg/ml solution) their brains were quickly removed and both hippocampi were dissected. Hippocampal tissue was homogenized by brief sonication in an extract buffer (500 ll) containing 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, protease inhibitor cocktail (Sigma, St. Louis, MO), and phosphatase inhibitor cocktail 1&2 (Sigma, St. Louis, MO) [adapted from (Einat et al., 2003)]. Homogenates were centri-

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fuged at 13,100g for 1 h at 4 C. The supernatant was removed and stored at 20 C until further use. An additional group of rats received either no training (n = 3 naı¨ve), trace fear conditioning (n = 3 trace trained), or pseudorandom fear conditioning (n = 3 pseudo trained). Immediately following trace or pseudorandom fear conditioning, hippocampi were extracted from all groups of rats and tissue was processed as above for Western blot analysis. 2.4.2. Western blot The Bradford method was used to determine protein concentration. We made a 1:10 dilution of each total protein sample and added 3 ll–1 ml of Bradford reagent. Samples were incubated for 10 min at room temperature. Samples were added to cuvettes and a spectrophotometer was used to measure the absorbance at 595 nm. Total hippocampal protein (25–275 lg) was separated on 10% polyacrylamide gels. Proteins were transferred from the gel to nitrocellulose membranes using a tank transfer apparatus filled with buffer containing 25 mM bicine, 25 mM Bis-Tris, and 20% methanol. To verify protein concentrations of samples, gels were silver stained. To control for protein loading, an antibody against a-tubulin (Abcam, Cambridge, MA) was used. For immunodetection of phosphorylated ERK, total ERK and CREB proteins, and a-tubulin, membranes were incubated in blocking buffer (1· TBS/0.1% Tween 20 with 5% nonfat dried milk) for 1 h at room temperature. An ERK polyclonal antibody (phospho-p44/42 MAP kinase (Thr202/Tyr204) or p44/42 MAP kinase at 1:1000 dilution) (Cell Signaling, Beverly, MA) or CREB antibody (CREB at 1:1000; Cell Signaling, Beverly, MA) was added to buffer solution (1· TBS/0.1% Tween 20 with 5% nonfat dried milk) and the membrane was incubated overnight at 4 C. The membrane was then washed several times in 1· TBS/0.1% Tween 20, and then incubated in HRP-conjugated anti-mouse or anti-rabbit IgG (secondary antibody, 1:2000; Cell Signaling, Beverly, MA) in blocking buffer for 1 h. Subsequently, membranes were washed with 1· TBS/0.1% Tween 20 several times. For immunodetection of phosphorylated CREB, membranes were washed twice with water and then were incubated in blocking buffer (1· PBS with 3% nonfat dried milk) for 1 h at room temperature with constant agitation. A CREB antibody (phospho-CREB at 1:500 dilution; Upstate, Waltham, MA) was added to the buffer solution (1· PBS with 3% nonfat dried milk) and the membrane was incubated overnight at 4 C. The membrane was then washed twice with water and then incubated in HRP-conjugated anti-rabbit IgG (secondary antibody, 1:1000; Cell Signaling, Beverly, MA) in blocking buffer for 1.5 h. The membrane was washed twice with water and then washed in 1· PBS/0.05% Tween 20 for 10 min. The membrane was rinsed with water 4–5 times. For enhanced chemilu-

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SL327-treated rats showed significantly lower levels of freezing throughout the 5-min tone testing when compared to DMSO-treated rats (Fig. 2). A two-way repeated measures ANOVA showed significant main effects of drug (F (1, 14) = 34.24, p < .001), time period (F (5, 70) = 34.59, p < .001), and drug · time period interaction (F (1, 70) = 16.08). Post hoc comparisons with Tukey tests showed the effects of the drug were significant (p < .05) for each time period except baseline.

minescence of all membranes, an equal volume of DuoLox Chemiluminescent Substrate with peroxidase converter solution was mixed and membranes were incubated for 1 min. Membranes were imaged using a Kodak Image Station 2000R (Kodak Scientific Imaging Systems, New Haven, CT) and exposure time was 1–15 min. Densitometry analysis was performed using the Quantity One software (Bio-Rad Laboratories, Hercules, CA). 2.4.3. Statistical analysis For behavioral experiments, a two-way repeated measure ANOVA using the post hoc Tukey test was performed. For Western blot analyses, the StudentÕs t test or one way ANOVA using the post hoc Tukey test was performed.

3.3. Effects of SL327 on memory are reversible To control for long lasting detrimental effects of SL327, rats that received 100 mg/kg SL327 were retrained without drug 24 h after they were first tested. The following day, they were retested for fear to the tone in the testing chamber. When retested, the rats that had failed to associate the tone and the shock the first time now exhibited high levels of freezing similar to the DMSO-treated rats (Fig. 3). A two-way repeated measures ANOVA revealed no significant main effects of drug (F (1, 14) = 2.58, p = .13) and time period (F (5, 70) = 1.24, p = 1.25).

3. Results 3.1. SL327-treated rats show no impairment in acquisition of trace fear conditioning Forty-five minutes after injection, both DMSO-treated (n = 8) and SL327-treated (n = 8) rats received 10 trace trials in the conditioning chamber. Fig. 1 shows mean freezing percentages obtained from baseline and tone, trace, shock, and ITI of the 10th trial of trace fear conditioning. There were no significant differences in freezing levels between both groups of rats on the last training trial, which was reflected across all 10 training trials (data not shown).

3.4. ERK phosphorylation is increased after trace fear conditioning Additional groups of young rats received either no training (n = 3 naı¨ve), trace fear conditioning (n = 3), or pseudorandom conditioning (n = 3). Immediately after training, the hippocampi were removed from all groups of rats. A one way ANOVA (F (2, 6) = 17.89; p = .003 for p42 and F (2, 6) = 9.06; p = .015 for p44) revealed that levels of hippocampal phosphorylated ERK were significantly increased in trace trained rats (p < .05; Tukey test) as compared to naı¨ve and pseudo trained rats (Fig. 4A). There were no significant differences in

3.2. SL327-treated rats show deficits in trace fear memory Twenty-four hours after trace fear conditioning, rats were tested for fear to the tone in the testing chamber.

DMSO (n=8) SL327 (n=8)

100 90

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80 70 60 50 40 30 20 10 0 BASELINE

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Fig. 1. Both DMSO (2 ml/kg 100% DMSO) and SL327 (100 mg/kg/2 ml dissolved in 100% DMSO) treated rats showed similar levels of freezing during baseline and the last tone, trace, shock, and ITI (10th trial) presentation of trace fear conditioning. Data are expressed as mean percent freezing (±SEM).

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Fig. 2. SL327-treated rats show significantly (*p < .001) lower levels of freezing as compared to DMSO-treated rats during the 5-min tone testing 24 h after training. Data are expressed as mean percent freezing (±SEM) and the 5-min tone is expressed in 1 min (M) intervals.

DMSO (n=8) RETRAINED SL327 (n=8)

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Fig. 3. When SL327-treated are retrained without the presence of the drug, there are no significant differences in freezing levels between both groups. Data are expressed as mean percent freezing (±SEM) and the 5-min tone is expressed in 1 min (M) intervals.

the levels of total ERK (Fig. 4B) among the three groups. 3.5. SL327 inhibits ERK but not CREB phosphorylation at 45 and 90 min post-injection Additional groups of rats received SL327 (100 mg/kg; n = 4) or 100% DMSO (2 ml/kg; n = 3) IP injections and the hippocampi were removed either 45 or 90 min after injection. Based on our behavioral studies, we chose to analyze the effect of the drug at 45 min after injection because we wanted to investigate the levels of ERK phosphorylation at the start of training. In Fig. 5A, levels of phosphorylated ERK from hippocampal protein extracts of SL327-treated rats were significantly de-

creased at 45 min post-injection (StudentÕs t test, p < .05). However, total levels of ERK were unchanged in both groups (Fig. 5B). There were no significant differences in the levels of phosphorylated and total CREB from hippocampal protein extract of DMSO and SL327treated rats (Fig. 6). We chose the 90-min time point because we wanted to investigate the level of ERK phosphorylation at the end of training. In Fig. 7A, levels of phosphorylated ERK from hippocampal protein extracts remained significantly decreased in SL327-treated rats (StudentÕs t test, p < .05). Again, total levels of ERK were unchanged in both groups (Fig. 7B). There were no significant differences in the levels of phosphorylated and total CREB from hippocampal protein extract of DMSO- and SL327-treated rats (Fig. 8).

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A

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phospho-ERK 44 NAIVE (n=3) TRACE TRAINED (n=3) PSEUDO TRAINED (n=3)

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Fig. 4. (A) (Left) Hippocampal protein extracts from trace trained rats show significantly (*p < .05) higher levels of phosphorylated ERK as compared to naı¨ve and pseudo trained rats. Data are expressed as intensity units/area (±SEM). (Right) Representative Western blots for phosphorylated ERK from naı¨ve, trace trained, and pseudo trained rats. (B) (Left) There were no significant differences in levels of total ERK from hippocampal protein extracts of naı¨ve, trace trained, and pseudo trained rats. Data are expressed as intensity units/area (±SEM). (Right) Representative Western blots for total ERK from naı¨ve, trace trained, and pseudo trained rats.

3.6. SL327 inhibits ERK but not CREB phosphorylation after trace fear conditioning Additional control groups of rats received SL327 (100 mg/kg) or 100% DMSO (2 ml/kg) IP injections 45 min prior to trace fear conditioning. Immediately after training, hippocampi were removed from both groups of rats. Fig. 9A shows that levels of phosphorylated ERK from hippocampal protein extract remained decreased in trained SL327-treated rats as compared to DMSO-treated rats (StudentÕs t test, p < .05). Total levels of ERK were unchanged in both groups (Fig. 9B). There were no significant differences in the levels of phosphorylated and total CREB from hippocampal protein extract of trained DMSO and SL327-treated rats (Fig. 10).

4. Discussion Our studies provide evidence that pretraining treatment with the MEK inhibitor SL327 causes trace fear

memory deficits in young F344 rats. From our training results, SL327-treated rats showed no differences in freezing levels as compared to the DMSO-treated rats by the last training trial of trace fear conditioning, which supports previous findings that inhibiting ERK phosphorylation does not affect acquisition (Blum et al., 1999; Martin et al., 1997; Runyan et al., 2004; Schafe et al., 2000). However, 24 h later, SL327-treated rats showed significantly lower levels of freezing to the tone as compared to the DMSO-treated rats, which indicates memory deficits in this task. These memory deficits were reversible. The SL327-treated rats were retrained without the presence of drug and were able to show similar levels of freezing to the tone as the DMSO-treated rats during retesting. A study by Atkins et al. (1998) demonstrated that the compound SL327 has no profound effects on the physiology or general behavior of the rat, which may contribute to memory deficits. Shock threshold level tests revealed no differences between DMSO-treated and SL327-treated rats in the shock amplitude that elicited first flinch response. Testing for hot plate sensitivity

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A

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Fig. 5. (A) (Left) At 45 min post-injection, hippocampal protein extracts from SL327-treated rats show significantly (*p < .05) lower levels of phosphorylated ERK (p42/44) as compared to DMSO-treated rats. Data are expressed as intensity units/area (±SEM). (Right) Representative Western blots for phosphorylated ERK from DMSO- and SL327-treated rats. (B) (Left) At 45 min post-injection, there were no significant differences in levels of total ERK from hippocampal protein extracts of DMSO- and SL327-treated rats. Data are expressed as intensity units/area (±SEM). (Right) Representative Western blots for total ERK from DMSO- and SL327-treated rats.

revealed no differences in latency to lick the hind paw between the two groups. Open-field tests revealed no evidence of anxiolytic effects of SL327 nor were there any differences in total distance traveled, average speed of movements, total time spent moving, or total number of movements among DMSO and SL327-treated rats. SL327 was also found to selectively inhibit MEK and thus, affect only ERK phosphorylation, because SL327 had no effects on other kinases such as PKC, a-CaMKII, or PKA (Atkins et al., 1998; Selcher et al., 1999). Therefore, we have no reason to believe that SL327 would cause different effects in our studies; thus, the memory deficits we saw in our studies were not due to any general effects of the compound but were correlated with inhibition of ERK phosphorylation. Our results are consistent with earlier evidence supporting the involvement of ERK in other associative learning paradigms including Aplysia (Martin et al., 1997), Hermissenda (Crow et al., 1998) rats and mice

(Atkins et al., 1998; Schafe et al., 2000; Selcher et al., 1999). The use of SL327 in our experiments also reproduces previous findings of its effect on ERK phosphorylation in rats (Atkins et al., 1998). Additionally, our results showing trace fear memory deficits in SL327treated rats strengthen the involvement of phosphorylated ERK in other hippocampally dependent tasks such as the Morris water maze. Selcher et al. (1999) demonstrated that SL327-treated mice show impairments in escape latency as well as long-term spatial memory deficits. Bilateral dorsal hippocampi infusions of the MEK inhibitor PD098059 in rats also produced similar long-term spatial memory deficits in the water maze (Blum et al., 1999). Taken together, our data corroborate nicely with the existing evidence supporting the importance of ERK phosphorylation in different learning and memory tasks. Recent studies by Runyan et al. (2004) used posttraining bilateral intrahippocampal infusions of the

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total CREB

Fig. 6. (Top) Representative Western blots for phosphorylated and total CREB from DMSO- and SL327-treated rats. (Bottom) At 45 min postinjection, there were no significant differences in levels of phosphorylated or total CREB from hippocampal protein extracts of DMSO- and SL327treated rats. Data expressed as intensity units/area (±SEM).

MEK inhibitor U0126 to determine the importance of ERK in trace fear conditioning. These studies showed that inhibiting ERK phosphorylation in the hippocampus after trace fear conditioning caused memory deficits 24 h later. They also showed that hippocampal phosphorylated ERK is increased 1 h after training. This is in contrast to our results showing increases in hippocampal phosphorylated ERK immediately after training. This inconsistency could be due to the fact that our trace fear conditioning protocols differed and different strains of rats were used. However, they did not show that preventing this increase in hippocampal ERK phosphorylation at 1 h after training disrupted fear memory 24 h later. The difference in our studies is that pretraining treatment of SL327 did not affect the freezing levels during training, which suggests that ERK phosphorylation is not required for acquisition of the task. Our data also showed that ERK phosphorylation was increased immediately after training as compared to naı¨ve rats. Levels of phosphorylated ERK were not increased in rats that received pseudorandom fear conditioning, in which the same CS and US were used as in trace fear conditioning but given in a random order. Therefore, the increase in the levels of hippocampal phosphorylated ERK was due to trace fear conditioning and any consolidation processes that may be occurring during this time. Because pretraining treatment of SL327 caused levels of hippocampal phosphorylated

ERK to be decreased immediately after training, then we can conclude that inhibiting hippocampal ERK phosphorylation at this time produced memory deficits in trace fear conditioning 24 h later. Although the hippocampus was a reasonable brain structure to investigate the effects of inhibiting ERK phosphorylation on trace fear conditioning, a caveat to our findings is that it was the only brain structure studied. Future studies would include examining other structures such as the amygdala and prefrontal cortex that are known to be involved in trace fear conditioning. To determine whether ERK phosphorylation was inhibited in the hippocampus by SL327, we investigated levels of phosphorylated ERK from hippocampal protein extracts of rats at 45 and 90 min post-injection. ERK phosphorylation was significantly inhibited at 45 and 90 min in SL327-treated rats when compared to DMSO-treated rats, regardless of training. Levels of total ERK at both time points in hippocampal protein extracts remained the same for both groups, which suggests that inhibition of ERK phosphorylation is not due to a decrease in total levels of ERK. Thus, we can conclude that decreases in ERK phosphorylation before and after trace fear conditioning correlates with trace fear memory deficits 24 h later. The fact that acquisition was not affected indicates that the SL327 compound did not produce any performance deficits because both SL327 and DMSO-treated rats showed similar freezing levels during

J.S. Villarreal, E.J. Barea-Rodriguez / Neurobiology of Learning and Memory 85 (2006) 44–57

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Fig. 7. (A) (Left) At 90 min post-injection, hippocampal protein extracts from SL327-treated rats show significantly (p < .05) lower levels of phosphorylated ERK (p42/44) as compared to DMSO-treated rats. Data are expressed as intensity units/area (±SEM). (Right) Representative Western blots for phosphorylated ERK from DMSO- and SL327-treated rats. (B) (Left) At 90 min post-injection, there were no significant differences in levels of total ERK from hippocampal protein extracts of DMSO- and SL327-treated rats. Data are expressed as intensity units/area (±SEM). (Right) Representative Western blots for total ERK from DMSO- and SL327-treated rats.

training. Therefore, we can speculate that consolidation of the trace fear memory is affected in SL327-treated rats. Although the time course of SL327Õs effect on ERK phosphorylation has not been shown in rats, Selcher et al. (1999) determined the time course of the compoundÕs effect on ERK phosphorylation in the mouse using a 30 mg/kg concentration. Their results showed that SL327 is most effective in blocking ERK phosphorylation between 30 min and 2 h after injection but seemed to be returning to baseline levels at 3 h, which was the latest time point tested (Selcher et al., 1999). Our own results demonstrate that at least 1.5 h after injection, ERK phosphorylation remained significantly inhibited. Further studies are needed to determine the time course of the compound at 100 mg/kg in the rat and whether or not injecting SL327 at a later time point would disrupt long-term memory. Because the SL327 compound is selective in the inhibition of MEK, it does not differentiate between the p42

ERK and p44 ERK isoforms. Our results showed that phosphorylation levels of both p42 and p44 ERK isoforms are decreased in the hippocampus of SL327-treated rats. Consequently, our studies cannot demonstrate whether one isoform is more important than the other. However, previous studies have shown that ERK1 knockout mice are viable, fertile, and develop normally (Selcher, Nekrasova, Paylor, Landreth, & Sweatt, 2001) while ERK 2 knockouts are embryonic lethal (Hatano et al., 2003). The ERK 1 knockout mice also showed normal acquisition and retention of contextual and cue fear conditioning, which did not differ from the wildtype control (Selcher et al., 2001). Additionally, studies have demonstrated increases in ERK 2 phosphorylation with LTP and contextual fear conditioning (Atkins et al., 1998; English & Sweatt, 1996) with no change in ERK 1 phosphorylation. In contrast, Mazzucchelli et al. (2002) showed that ERK 1 knockout mice showed an enhancement of learning and long-term memory in

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Fig. 8. (Top) Representative Western blots for phosphorylated and total CREB from DMSO- and SL327-treated rats. (Bottom) At 90 min postinjection, there were no significant differences in levels of phosphorylated or total CREB from hippocampal protein extracts of DMSO- and SL327treated rats. Data expressed as intensity units/area (±SEM).

active and passive avoidance tasks as well as an enhancement of LTP in the nucleus accumbens. These findings suggest that selective activation of ERK 1 and ERK 2 may play a role in long-term synaptic plasticity and memory formation mechanisms. Interestingly, there were no significant differences in the levels of phosphorylated CREB from hippocampal protein extract from DMSO and SL327-treated rats at any time point and regardless of training. Previous studies have shown that CREB is a downstream target of ERK in hippocampal area CA1 following PKA and PKC activation and a plausible mechanism coupling ERK to CREB is through Rsk2 (Impey et al., 1998; Roberson et al., 1999; Xing, Ginty, & Greenberg, 1996). Impey et al. (1998) has demonstrated that Ca2+induced CREB phosphorylation was inhibited by the MEK inhibitor PD98059 in PC12 cells, in cultured hippocampal neurons, and in rat hippocampal slices. In similar findings, Roberson et al. (1999) also demonstrates a key role of ERK as a regulator of CREB phosphorylation. Other studies have found that application of the MEK inhibitor U0126 and forskolin or phorbol diacetate to rat hippocampal slices decreased CREB phosphorylation in area CA1 (Adams, Roberson, English, Selcher, & Sweatt, 2000). Based on these previous findings, we hypothesized that CREB phosphorylation would be affected by inhibiting ERK phosphorylation

with the SL327 compound. On the contrary, our results show that CREB phosphorylation was not affected by decreased levels of phosphorylated ERK at the 45- or 90-min time point. This may be due to other kinases (PKA and PKC) that are available to phosphorylate CREB [as reviewed in (Silva et al., 1998)]. Also, the time points that were studied may not reflect any decreases in CREB phosphorylation that may have occurred with the inhibition of phosphorylated ERK by SL327. Further studies are necessary to determine whether direct inhibition of CREB phosphorylation would affect trace fear memory. The association of the CS and US over time may be recruiting and activating different kinases, such as ERK, in the hippocampus in order for consolidation processes to occur. Once ERK becomes activated, it in turn activates downstream nuclear and cytosolic substrates through phosphorylation. Evidence has shown that consolidation of long-term memory is dependent on new RNA and protein synthesis (Bailey, Kim, Sun, Thompson, & Helmstetter, 1999; Davis & Squire, 1984; Maren, Ferrario, Corcoran, Desmond, & Frey, 2003; Schafe & LeDoux, 2000; Silva et al., 1998). The substrate molecules of ERK include nuclear proteins such as transcription factors (Grewal, York, & Stork, 1999; Seger & Krebs, 1995; Valjent, Caboche, & Vanhoutte, 2001); thus, ERK phosphorylation may play a

J.S. Villarreal, E.J. Barea-Rodriguez / Neurobiology of Learning and Memory 85 (2006) 44–57

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Fig. 9. (A) (Left) At 90 min post-injection, hippocampal protein extracts from trained SL327-treated rats show significantly (p < .05) lower levels of phosphorylated ERK (p42/44) as compared to DMSO-treated rats. Data are expressed as intensity units/area (±SEM). (Right) Representative Western blots for phosphorylated ERK from trained DMSO- and SL327-treated rats. (B) (Left) At 90 min post-injection, there were no significant differences in levels of total ERK from hippocampal protein extracts of trained DMSO- and SL327-treated rats. Data are expressed as intensity units/ area (±SEM). (Right) Representative Western blots for total ERK from trained DMSO- and SL327-treated rats.

role in controlling gene expression during memory formation. Previous studies have reported translocation of phosphorylated ERK to the nucleus, where it is able to activate other kinases and transcription factors necessary for gene expression (Impey et al., 1998; Martin et al., 1997). Inhibition of ERK phosphorylation may prevent transcription and translation of certain genes that are involved in consolidating memory. Because transcription factors such as Elk1 that are directly activated by ERK may be affected, more experiments would be necessary to investigate the levels of these other transcription factors and their role in memory (Grewal et al., 1999; Seger & Krebs, 1995; Valjent et al., 2001). Some cytosolic and cytoskeleton proteins (such as MAP2, neurofilaments, phospholipase A2, ApCAM) are also activated by ERK. Inhibiting ERK phosphorylation may prevent activation of these proteins, which may prevent

the synaptic structural changes associated with longterm memory formation [as reviewed in (Grewal et al., 1999)]. It is interesting to speculate that this could be underlying the trace fear memory deficits we see in young SL327-treated rats. However, we do know that the effects of SL327 are not long lasting because retraining without the presence of the compound does not produce memory deficits 24 h later. In summary, the present study has shown that pretraining administration of the MEK inhibitor SL327 produces long-term memory deficits in trace fear conditioning. These memory deficits can be reversed, which indicates that SL327 is not long-lasting and does not cause any permanent damage. Also, hippocampal phosphorylated ERK was increased immediately after training. Inhibiting this increase with pretraining treatment of SL327 was correlated with memory deficits 24 h later.

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phospho-CREB

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Fig. 10. (Top) Representative Western blots for phosphorylated and total CREB from trained DMSO- and SL327-treated rats. (Bottom) At 90 min post-injection, there were no significant differences in levels of phosphorylated or total CREB from hippocampal protein extracts of trained DMSOand SL327-treated rats. Data expressed as intensity units/area (±SEM).

However, there were no decreases in phosphorylated CREB with SL327 injections at those same time points. These findings support a role for ERK phosphorylation in the formation of trace fear memories.

Acknowledgments We would especially like to thank Dr. Steve Maren from the University of Michigan for kindly allowing us to use his Excel file for calculating freezing percentages in our behavioral analyses. We would especially like to thank Bristol-Myers Squibb Co. for providing the SL327 compound. This research was supported by 1 R03 AG023373-01A1, NIA to J.S.V., G12RR1346-02RCMI NCRR, NIH to E.J.B.R., SCORE GM08194, and NIA Dissertation Pilot Student Study to J.S.V.

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