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BOX 2, cont'd (b) Fear-conditioning increases the slope and amplitude of CS-EPs, but unpaired training does not. Slope and amplitude of the negative-going potential are normalized as a percentage of the mean values before training (sessions 1 and 2). The normalized slope and amplitude of the evoked potentials were evaluated statistically with two-factor ANOVAs with group (conditioned, control) as the between-subjects factor and experimental session as within subject factor. A significant group-session interaction was observed for both measures (p < .05). Significant differences of post hoc analyses are indicated ( p < .05). Error bars, = s.e.m. Reproduced from Rogan, Staubli and LeDoux (56).

Thus, this led us to ask the question: does ERK activation occur in the hippocampus with hippocampus-dependent learning? If so, it is evidence that LTP has occurred in the hippocampus in association with memory formation. Coleen Atkins addressed this issue in an impressive series of studies that were part of her Ph.D. thesis project in my laboratory. Coleen, working with Joel Selcher, found that contextual fear conditioning results in the activation of ERK in the hippocampus (19). Coleen and Joel trained animals with a contextual fear-conditioning protocol, pairing five foot shocks with a novel context, and then assayed the hippocampus for changes in ERK phosphorylation 1 hour post-training. They observed a significant increase in hippocampal ERK2 activation in trained animals 1 hour after contextual fear conditioning, as measured using a phospho-site-specific antibody (see Figure 3).

Identical sham-training of the animals (by placing them in the fear-conditioning apparatus in the same manner as the trained animals, but without being shocked), resulted in no change in ERK2 activation. To control for potential changes in ERK2 activation due to the shock itself, animals were placed in the fear-conditioning box and immediately shocked. This protocol elicits no learning in response to the context, nor did it result in increased ERK activation. These control experiments indicate that the increase in ERK activation during training is not a nonspecific response to the context, handling, or foot shock alone.

To test whether another associative conditioning paradigm also led to ERK activation, Coleen and Joel paired a tone and shock three times in the unique context and then assayed for hippocampal ERK activation. This protocol resulted in robust associative conditioning to both the cue and context as well as a significant increase in ERK activation at 1 hour post-training, indicating that the ERK MAPK cascade is activated during cued-plus-contextual fear conditioning. Delivery of the tone alone in

FIGURE 3 Fear-conditioning results in associative learning and ERK MAPK activation. (A) Animals were trained using a contextual fear conditioning protocol, and pre-training baseline freezing behavior (baseline) was compared to freezing when the animals were placed in the context on testing day (context, n = 6, p < .001, Student's f-test). For the cue and contextual protocol, baseline freezing behavior (baseline; pre-tone) was compared to freezing behavior when placed in the context on testing day (context, n = 5, p < .001, ANOVA) or when placed in a novel context with tone presentation (tone, n = 5, p < .001, ANOVA).

(B) Representative western blots of MAPK phosphorylation levels and amounts from naïve and trained animals. This phospho-site-specific MAPK antibody detects the covalent modifications (phospho-Thr202 and Tyr204) that cause activation of MAPK.

(C) Densitometric analysis for MAPK activation 1 hour after training (contextual, n = 10 for p42, n = 11 for p44; cued and contextual, n = 14 for p42, n = 13 for p44), sham training (contextual, n = 6 for p42, n = 5 for p44; cued and contextual, n = 9 for both) and immediate shocking (IS, n = 8 for both). Phospho-p44 MAPK levels in trained animals were markedly lower relative to phospho-p42 MAPK levels, indicating a fairly selective activation of p42 MAPK (p42 MAPK = ERK2, p44 MAPK = ERK1. No changes in total MAPK amounts were observed 1 hour after training. *p < .05; **p < .01; ***p < .001. Data reproduced from Atkins et al. (19).

the unique context had no effect on ERK activation.

These data demonstrate that the ERK cascade is activated with fear conditioning, just as it is with LTP-inducing stimulation in hippocampal area CA1 (20, 21). Is this learning-associated effect NMDA receptor-dependent as it is in LTP? Administration of the NMDA receptor antagonist MK801 to animals prior to training resulted in an attenuation of learning with both protocols, and also attenuated ERK activation when the hippocampi were assayed 1 hour after training with the contextual fear conditioning protocol or the cued and contextual fear conditioning protocol (19). These data indicate a necessity for NMDA receptor activation for the learning-associated ERK activation and are consistent with the hypothesis that LTP or a similar phenomenon is causing the stimulation of hippocampal ERK.

Having observed activation of ERK in response to behavioral associative conditioning using the fear-conditioning paradigm, it was of interest to determining if a blockade of ERK activation could cause a blockade of learning in this situation. Again, this would be reminiscent of the necessity of ERK activation for LTP to be triggered (21). Fortunately, the MEK inhibitor SL327 can be administered intraperitoneally and will achieve effective concentrations in the CNS. Administration of SL327 either before or immediately after behavioral training led to a blockade of learning, (i.e., the animals exhibited essentially no contextual or cued fear conditioning) (19). These data strongly support the hypothesis that ERK activation is a necessary component of the biochemical cascades utilized to establish behavioral plasticity and are consistent with the general hypothesis that LTP is involved in contextual memory formation.

One limitation to the experiments described above using SL327 is that the drug is administered intraperitoneally and thus inhibits MEK throughout the animal. In a significant refinement, Schafe et al. (22)

and Ohno et al. (23) provided additional strong evidence that MEK activation is required for contextual fear conditioning. These investigators infused MEK inhibitors into the CNS and observed selective blockade of long-term but not short-term fear conditioning. Also, Walz et al. (24, 25), using a similar approach of cortical and limbic infusion of PD98059, observed significant effects of MEK inhibition on step-down inhibitory avoidance, a learning paradigm with similarities to associative fear conditioning. Overall these studies, combined with those by Atkins et al, (19), provide convincing evidence of a necessity for ERK activation in mammalian contextual learning.

Learning in the Morris water maze is also associated with hippocampal ERK activation. In pioneering studies, Pramod Dash's laboratory (26) nicely demonstrated ERK activation in the hippocampus when animals underwent Morris maze training, directly demonstrating that ERK activation occurs in the hippocampus during spatial learning (Figure 4). These same investigators used MEK inhibitor infusion into the hippocampus to demonstrate a necessity for hippocampal ERK activation for spatial memory formation. This necessity for ERK activation later was confirmed by Selcher et al. (27) using SL327 in mice (see Figure 5). The effects of MEK inhibition were selective for the hidden platform test, while no effect was seen in the visible platform; these data indicate that the MEK inhibition did not nonspecifically interfere with the animal's physical ability to execute the task. Thus, similar to fear conditioning, spatial learning in rodents involves ERK activation in the CNS, an effect that is necessary for the formation of lasting memories.

These data directly investigating ERK activation with spatial learning are a specific example of a variation on the "measure" experiment. ERK activation is triggered with LTP in an NMDA receptor-dependent fashion, and the same molecular event occurs with spatial learning in the animal. These observations are consistent with the

FIGURE 4 Behavioral training in the Morris water maze increases MAPK phosphorylation in the CA1/CA2 subfields of the dorsal hippocampus. Representative photomicrographs for phospho-MAPK immunoreactivity in the CA1/CA2 subfields of dorsal hippocampi (1.0 mm from the dorsal tip) from (A) naive and (B) 5-minutes post-training animals. Training to criterion increases the number of immunopositive cells for phospho-MAPK as compared with naive controls. (C) Laser confocal image, indicating that the phospho-MAPK-positive cells (red) are also immunoreactive for a neuron-specific nuclear antigen (green). Areas with overlapping immunofluores-cences appear yellow. Representative photomicrographs for MAPK immunoreactivity from (D) a naive and (E) a 5-minutes post-training animal. (F) Summary showing percentage of neurons staining positive for phos-phorylated MAPK in the dorsal hippocampus from the control and experimental groups. The data are represented as the mean ± SEM (n = 5 for each group). (G) Representative laser confocal images from a randomized platform and an animal trained to criterion and killed 5 minutes later, showing increased numbers of CA1/CA2 pyramidal neurons with nuclear staining for phospho-MAPK as a result of training. The open and closed arrows indicate neurons with weak or strong phospho-MAPK nuclear immunoreactivity, respectively. The bar graph shows the nuclear to cytoplasmic ratio for phospho-MAPK immunofluroscence. *p < .05; p.t.c., posttraining to criterion. Data and figure reproduced from Blum, Moore, Adams, and Dash (26).

FIGURE 4 Behavioral training in the Morris water maze increases MAPK phosphorylation in the CA1/CA2 subfields of the dorsal hippocampus. Representative photomicrographs for phospho-MAPK immunoreactivity in the CA1/CA2 subfields of dorsal hippocampi (1.0 mm from the dorsal tip) from (A) naive and (B) 5-minutes post-training animals. Training to criterion increases the number of immunopositive cells for phospho-MAPK as compared with naive controls. (C) Laser confocal image, indicating that the phospho-MAPK-positive cells (red) are also immunoreactive for a neuron-specific nuclear antigen (green). Areas with overlapping immunofluores-cences appear yellow. Representative photomicrographs for MAPK immunoreactivity from (D) a naive and (E) a 5-minutes post-training animal. (F) Summary showing percentage of neurons staining positive for phos-phorylated MAPK in the dorsal hippocampus from the control and experimental groups. The data are represented as the mean ± SEM (n = 5 for each group). (G) Representative laser confocal images from a randomized platform and an animal trained to criterion and killed 5 minutes later, showing increased numbers of CA1/CA2 pyramidal neurons with nuclear staining for phospho-MAPK as a result of training. The open and closed arrows indicate neurons with weak or strong phospho-MAPK nuclear immunoreactivity, respectively. The bar graph shows the nuclear to cytoplasmic ratio for phospho-MAPK immunofluroscence. *p < .05; p.t.c., posttraining to criterion. Data and figure reproduced from Blum, Moore, Adams, and Dash (26).

FIGURE 5 Administration of the MEK inhibitor SL327 significantly impaired spatial learning performance in the Morris water maze task. Mice treated with SL327 failed to exhibit a spatial strategy when searching for the platform during probe trials. A selective search strategy is one in which the subjects spend significantly more time searching in the trained quadrant than in the other three quadrants. The subjects must also cross the area where the platform had been during the training sessions significantly more often than they cross the corresponding areas in the other quadrants. (A) During the probe trials on days 4 and 5, mice treated with vehicle (n = 13) spent significantly more time searching in the trained quadrant and crossed the platform area in the trained quadrant more frequently than in any of the alternate quadrants. However, mice injected with an inhibitor of ERK MAP kinase activation (30 mg/kg SL327, n = 11) did not exhibit a selective search strategy. There was no significant difference between the time spent in the trained quadrant and platform crossings as compared with the other quadrants. (B) The drug treatment was switched on day 6; animals who had received SL327 during the first 5 days now received vehicle and vice versa. The vehicle-trained mice (n = 11) still exhibited a selective search strategy after injection with SL327 on day 6. SL327-trained mice (n = 9) who were administered vehicle on day 6, did not display a selective search strategy. (***) Significantly larger (p < .01) than all three of the other quadrants. Panels C and D. (C) Representative probe trial of a vehicle-treated mouse. The swim path trace shown here provides an excellent example of a selective search. This particular subject was trained with the platform located in the northeast quadrant. During the probe trial, this mouse spent 56% of the time in the correct quadrant and crossed the exact area where the platform had been nine times. (D) Representative probe trial of an SL327-treated mouse. This trace does not represent a selective search. This mouse was trained with the platform in the northwest quadrant, but during the probe trial, the subject crossed this platform area only once and spent 32% of the time in this quadrant (vs. 34% in the opposite quadrant). Adapted from Selcher et al. (27).

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