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Time-Dependent Impairment of Inhibitory Avoidance Retention in Rats by Posttraining Infusion of a Mitogen-Activated Protein Kinase Kinase Inhibitor into Cortical and Limbic Structures
Neurobiology of Learning and Memory 73, 11–20 (2000) doi:10.1006/nlme.1999.3913, available online at http://www.idealibrary.com on
Time-Dependent Impairment of Inhibitory Avoidance Retention in Rats by Posttraining Infusion of a Mitogen-Activated Protein Kinase Kinase Inhibitor into Cortical and Limbic Structures Roger Walz,* Rafael Roesler,* Joa˜o Quevedo,* Ma´rcia K. Sant’Anna,* Marcelo Madruga,* Cleverson Rodrigues,* Carmen Gottfried,† Jorge H. Medina,‡ and Ivan Izquierdo* *Centro de Memo´ria, and †Laborato´rio de Fosforilac¸a˜o de Proteı´nas Cerebrais, Departamento de Bioquı´mica, ICBS, UFRGS, Ramiro Barcellos 2600, 90035-003 Porto Alegre, RS, Brazil; and †Laborato´rio de Neuroreceptores, Instituto de Biologı´a Celular, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 3er piso, Buenos Aires, Argentina
Mitogen-activated protein kinase (MAPK) is abundantly expressed in postmitotic neurons of the developed nervous system. MAPK is activated and required for induction of long-term potentiation (LTP) in the CA1 area of the hippocampus, which is blocked by the specific inhibitor of the MAPK kinase, PD 098059. Recently it was demonstrated that MAPK is activated in the hippocampus after training and is necessary for contextual fear conditioning learning. The present work tests the role of the MAPK cascade in step-down inhibitory avoidance (IA) retention. PD 098059 (50 mM) was bilaterally injected (0.5 ml/side) into the CA1 region of the dorsal hippocampus or entorhinal cortex at 0, 90, 180, or 360 min, or into the amygdala or parietal cortex at 0, 180, or 360 min after IA training in rats using a 0.4-mA foot shock. Retention testing was carried out 24 h after training. PD 098059 impaired retention when injected into the dorsal hippocampus at 180 min, but not 0, 90, and 360 min after training. When infused into the entorhinal cortex, PD 098059 was amnestic at 0 and 180 min, but not at 90 and 360 min after training. The MAPKK inhibitor also impairs IA retention when infused into the parietal cortex immediately after training, but not at 180 or 360 min. Infusions performed into amygdala were amnestic at 180 min, but not at 0 and 360 min after training. Our results suggest a time-dependent involvement of the MAPK cascade in the posttraining memory processing of IA; the time dependency is different in the hippocampus, amygdala, entorhinal cortex, or parietal cortex of rats. q 2000 Academic Press
This work was supported by PRONEX and Fundac¸a˙ VITAE, Brazil. Address correspondence and reprint request to Roger Walz, Centro de Memo´ria, Departamento de Bioquı´mica, Instituto de Cieˆncias Ba´sicas da Sau´de, UFRGS, Ramiro Barcelos 2600 (Anexo), 90035-003 Porto Alegre, RS, Brazil. Fax: 55 516 5535. E-mail: [email protected]. 11
1074-7427/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
12
WALZ ET AL.
Mitogen-activated protein kinase (MAPK) is a serine/threonine protein kinase that acts on cytoskeletal (e.g., tau, microtubule-associated protein types 1, 2, and 4, caldesmon), cytosolic (e.g., tyrosine hydroxylase, Raf-1, MAP2K, cPLA2), membranal (e.g., extracellular growth factor receptor, nerve growth factor receptor), and nuclear (e.g., immediateearly genes: ELK1, cFos, cMyc, ATF2, Ets2) targets. The MAPK cascade involves at least three sequential homologue kinases: MP3K (mitogen-activated protein kinase kinase kinase), MAP2K or MAPKK (mitogen-activated protein kinase kinase), and MAPK (Seger & Krebs, 1995). MAPK activation plays an important role in long-term-facilitation (LTF) of Aplysia (Martin, Michael, Rose, Barad, Casadio, Zhu, & Kandel, 1997; Bailey, Kaang, Chen, Martin, Lim, Casadio, & Kandel, 1997; Michael, Martin, Seger, Ning, Baston, & Kandel, 1998), and its activation (English & Sweatt, 1996; Baron, Benes, Van Tan, Fagard, & Roisin, 1996) is necessary (English & Sweatt, 1997; Atkins, Selcher, Petraitis, Trzaskos, & Sweatt, 1998) for the induction of long-term potentiation (LTP) in the hippocampus CA1 region. In rats, MAPK is activated in the hippocampus 1 h after fear conditioning training, and this form of learning is dose-dependently attenuated by the systemic pretraining administration of MAPK inhibitor SL327 (Atkins et al., 1998). These data strongly suggest that the MAPK cascade plays an important role in neural plasticity processes, including learning and memory. The present experiments were performed in order to investigate the involvement of MAPK cascade in the posttraining processing of step-down inhibitory avoidance (IA) in rats. This form of associative learning is known to involve several sensorial stimuli including spatial and visual perceptions, sensitivity to pain, and an emotional component modulated by the amygdala and stress hormones (Izquierdo, 1989; Izquierdo & Medina, 1997). The IA is ideal for studying posttraining processes initiated by training because it is usually acquired in a single trial, uncontaminated by prior or further trials, rehearsals, or retrievals (Gold, 1986; Izquierdo, 1989). We studied the amygdala (AMY), hippocampus (HIP), entorhinal cortex (EC), and parietal cortex (PC) because it was previously shown that IA induces several biochemical changes, including changes in the activity of protein kinases A and C (Izquierdo & Medina, 1997), related to activity of the MAPK cascade (Atkins, Selcher, Petraitis, Trzaskos, & Sweatt, 1998). Using specific pharmacological approaches we have demonstrated a time-dependent role for these enzymes for memory consolidation of the IA task in AMY, HIP, PC, and EC (Izquierdo, Medina, Bianchin, Walz, Zanatta, da Silva, Bueno e Silva, Ruschel, & Paczko, 1993; Izquierdo & Medina, 1997; Ardenghi, Barros, Izquierdo, Bevilaqua, Schro¨der, Quevedo, Rodrigues, Madruga, Medina, & Izquierdo, 1997; Bevilaqua, Ardenghi, Schro¨der, Bromberg, Schmitz, Schaeffer, Quevedo, Bianchin, Walz, Medina, & Izquierdo, 1997; Bernabeu, Bevilaqua, Ardenghi, Bromberg, Schmitz, Bianchin, Izquierdo, & Medina, 1997; Izquierdo, Quillfeldt, Zanatta, Quevedo, Schaeffer, Schmitz, & Medina, 1997). Here we infused the specific MAPKK inhibitor, PD 098059, into the AMY, CA1 region of HIP, EC, and PC at different times after IA training. As the role of MAPKK is basically to activate MAPK, the effect caused by the PD 098059 inhibitor is attributable to an inhibition of MAPK, activation (Alessi, Cuenda, Cohen, Dudley, & Saltiel, 1995).
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METHODS Animals and Surgery Adult male Wistar rats (250–320 g) were obtained from our own breeding colony. They were caged in group of 5 with free access to food and water and were maintained on a 12-h light-dark cycle (lights on at 7:00 AM), at a temperature of 23 6 18C. Animals were bilaterally implanted under thionembutal anesthesia (30 mg/kg, ip) with 30-g cannulae aimed 1 mm above area CA1 of the dorsal hippocampus, or amygdala, or entorhinal cortex, or parietal cortex. (Izquierdo et al., 1997; Bevilaqua et al., 1997; Ardenghi et al., 1997). Coordinates A 24.2, L 6 3.0, V 2.3 (HIP), A 22.3, L 6 4.5, V 5.0, (AMY), and A 26.7 L 6 5.0 V 5.8 (EC), A 22.8, L 6 1.0, V 3.0 (PC) were obtained from the atlas of Paxinos and Watson (1986). Animals received cloramphenicol (25 mg/ kg, ip) 20 min before anesthesia induction. Behavioral Test Procedures and Apparatus Animals were submitted to behavioral procedures 4 days after recovery from surgery. The inhibitory avoidance apparatus was a 50 3 25 3 25-cm acrylic box whose floor consisted of parallel 1.0-mm - caliber stainless-steel bars spaced 1.0 cm apart. A 7.0 cm wide, 2.5 cm high, 25.0 cm long platform was laid on the floor against the left wall. Four to 5 days after surgery animals were placed on the platform and their latency to step down on the grid with all four paws was measured with an automatic device. In the training session, immediately after stepping down, the animals received a 0. 4-mA, 3.0s scrambled foot shock. In test sessions no foot shock was administered and the stepdown latency (to a ceiling of 120 s) was used as a measure of retention. Behavioral procedures took place between 8:00 AM and 10:30 PM. Infusion Procedures and Drugs At the time of infusion, an infusion cannula was fitted into the guide cannula. The tip of the infusion cannula protruded 1.0 mm beyond the guide cannula and was aimed at the CA1 area of the HIP, AMY, and EC or 1 mm above the junction between posterior parietal cortex I and II (PC). Through the infusion cannulae animals received bilateral 0.5-ml infusions of vehicle (2.5% dimethyl sulfoxide in saline, pH 7.4) or PD 098059 (50 mM) over a 2-min period. Two minutes was allowed after infusion for diffusion of the drug. We used the concentration of 50 mM, because this is the concentration that attenuates LTP induction in hippocampus slices (English & Sweatt, 1997). The high degree of specificity of PD 098059 “in vitro and in vivo” is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two MAPKK homologues) (Alessi et al., 1995; Dudley et al., 1995). Drug infusions were performed between 8:00 AM and 3:00 PM. Cannulae Placements One or 2 days after the end of the behavioral procedures, 0.5 ml of a solution of 4% methylene blue in saline was infused through the cannulae and animals were killed by
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FIG. 1. (A and B) Drawings of planes A 24.3 and 2.3 of the atlas of Paxinos and Watson (1986) showing, stippled, the extent of the area reached by the infusion in the dorsal hippocampus and amygdala, respectively. (C) Ventral view of the brain showing the extent of infusions into the entorhinal cortex. (D) Lateral view of the brain (Ziles, 1985) showing the extent of infusions into the posterior parietal cortex.
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FIG. 2. Effects of posttraining infusions of PD 098059 into the dorsal hippocampus on retention of stepdown inhibitory avoidance task in rats. Data are median (interquartile range) of retention test latencies. N 5 10–13 animals per group. *Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .001). In this and following figures, training-test differences are significant in Wilcoxon tests at the p , .01 level, except in the groups marked by asterisks.
decapitation 10 min after. Brains were stored in formalin for at least 72 h and cannulae placements were verified by histological examination (Izquierdo et al., 1997; Ardenghi et al., 1997; Bevilaqua et al., 1997). Cannulae placements were found to be correct (within 0.8 mm3 of the intended sites) in 86 of 93 rats implanted into the dorsal HIP, and in 69 of 76 rats implanted into AMY, and in 64 of 71 animals implanted into PC, and 87 of 96 animals implanted into the EC. Only data from these animals were included in the final analysis. Injection placements are shown in Fig 1. Statistical Analysis Comparisons of both training and test session latencies between vehicle-treated and PD 098059-treated groups were performed using a Mann-Whitney U test, two-tailed. Comparisons between training and test sessions in individual groups were by a Wilcoxon test. In all comparisons, p , .05 was considered to indicate statistical significance. RESULTS When given into CA1 (Fig. 2), PD 098095 impaired retention when infused 180 but not 0, 90, or 360 min after training. There were significant differences between training
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FIG. 3. Effects of posttraining infusions of PD 098059 into the amygdala on retention of step-down inhibitory avoidance task in rats. Data are median (interquartile range) of retention test latencies. N 5 10–13 animals per group. *Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .001).
and test session latencies in all groups except for the PD 098059 group injected at 180 min after training. The MAPKK inhibitor was amnestic when infused into AMY 180 min, but not 0 or 360 min posttraining (Fig. 3). There were significant differences between training and test session latencies in all groups except for the PD 098059 group injected into AMY at 180 min after training. Results of infusions into the EC are shown in Fig. 4. There were significant differences in retention test latencies between the PD 098059 and vehicle groups infused 0 and 180 min after training but not in those infused 90 and 360 min posttraining. Moreover, there were significant differences between training and test session latencies in all groups except those treated with PD 098059 at 0 and 180 min posttraining. Results of infusions into the parietal cortex are shown in Fig. 5. PD 098059 was amnestic when given into the PC immediately but not at 180 or 360 min after training. There were significant differences between training and test session latencies in all groups except the one treated with PD 098059 at 0 min after training. There were no significant differences between PD 098059 groups and their respective control groups in training session latencies (results not shown). DISCUSSION This is the first report of a time-dependent involvement of the MAPK cascade in posttraining memory processing by specific neocortical structures. Our data are in agreement with the study by Atkins et al. (1998), that suggested an involvement of MAPK in the HIP during contextual fear conditioning. They showed a delayed posttraining increase of MAPK activity in HIP, and an amnestic effect of a MAPK inhibitor given systemically. In view of our present findings, the effect they observed with the MAPK inhibitor given pretraining could have been due to influences on different brain sites at different times.
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FIG. 4. Effects of posttraining infusions of PD 098059 into the entorhinal cortex on retention of step-down inhibitory avoidance task in rats. Data are median (interquartile range) of retention test latencies. N 5 10–12 animals per group. *Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .001). **Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .05).
The amnestic effect of the specific MAPKK inhibitor used here could be due to interference with cytoskeletal, cytosolic, or membranal processes in the cortical and limbic structures studied, and/or to an impairment of IEGs or DRGs transcription (Finkbeiner & Greenberg, 1996; Segal & Greenberg, 1996). PD098059, like the protein kinase A inhibitor KT 5720 (Bernabeu et al., 1997; Izquierdo & Medina, 1997; Ardenghi et al., 1997), was amnestic when given into CA1 3 h after training, and when given into the EC at both 0 and 3 h after IA training. These pharmacological effects are coincident with an enhancement in cAMP and c-fos levels in both HIP and EC (Bernabeu et al., 1997; Izquierdo & Medina, 1997). The similarity of the posttraining time windows of effects of both the MAPKK and the PKA inhibitors in EC and CA1 raises the possibility of cross-talk between both cascades in these structures in relation to memory formation. Activation of MAPK by cAMP/PKA pathway stimulation has been demonstrated in the pheochromocytoma cell line PC12 (Vossler et al., 1997), in Aplysia (Bailey et al., 1997; Martin et al., 1997; Michael et al., 1998), and in mouse hippocampus (Michael et al., 1998; Martin et al., 1997). Recently it was also shown that cAMP can directly stimulate MAPK in neurons through a PKA-independent pathway (Kawasaki, Springett, Mochizuki, Toki, Nakaya, Matsuda, Housman, & Graybiel, 1998). The amnestic effect of the MAPKK inhibitor when infused in AMY is interesting because PKA inhibitors given into this structure have no effect on IA retention (Ardenghi et al., 1997; Bevilaqua et al., 1997). So it is possible that the MAPK cascade may be
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FIG. 5. Effects of posttraining infusions of PD 098059 into the parietal cortex on retention of step-down inhibitory avoidance task in rats. Data are median (interquartile range) of retention test latencies. N 5 10–12 animals per group. *Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .01).
more crucial to AMY function than the PKA cascade. Animals lacking the neuronalspecific guanine-nucleotide-exchange Ras-GRF, which are involved in MAPK cascade stimulation through the G protein Ras (Mattingly & Macara, 1996), have abnormal longterm potentiation in AMY and show an impairment of two-way active avoidance, inhibitory avoidance, and fear conditioning (Brambilla et al., 1997). The time window of the amnestic effect of the MAPKK inhibitor and the PKA inhibitor was not completely coincident in PC. The PKA inhibitor was amnestic when infused 0, 3, or 6 h after IA training (Ardenghi et al., 1997), but the MAPKK was effective only immediately after training. Thus, in some areas (PC, AMY) there is a discrepancy, and in others (CA1, EC) a coincidence, in the time windows and/or quality of the effect of the two enzymatic inhibitors. This suggests that in some areas (CA1, perhaps EC) there may be an interaction or a cross-talk between the two cascade systems, and in others not. In any case, these findings point once more to the need to examine more than one region of the brain when their involvement in a given learning process is being studied (Izquierdo & Medina, 1998). Further, these data support the view that the roles of the several kinases on memory processing vary according to the task, the procedure, the posttraining time, and the brain structure studied (Izquierdo & Medina, 1997). In LTP experiments, the MAPK cascade appears to be involved very early on. In the experiment
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of Atkins et al. (1998) on contextual fear conditioning the involvement of the MAPK cascade in HIP appears to be before the one we report here for IA. In summary, the MAPK cascade plays an important role in the posttraining memory processing of IA, at different times, in cortical and limbic structures of rats. Taking into account that in humans there are as many as 2000 conventional protein kinase genes and 1000 phosphatase genes (Hunter, 1995), possible cross-talks between the PKA and MAPK pathways or other protein kinase pathways in HIP, EC, and PC deserve further study. Different cross-talks between (or among) protein kinase cascades may explain the differences in timing of the influence of one or other task or situation and would certainly be expected from the vastness of the repertory of kinases and phosphatases. REFERENCES Alessi, R. D., Cuenda, A., Cohen, P., Dudley, D. T., & Saltiel, A. R. (1995). PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. Journal of Biological Chemistry, 276, 27489–27494. Ardenghi, P., Barros, D., Izquierdo, L., Bevilaqua, L., Schro¨der, N., Quevedo, J., Rodrigues, C., Madruga, M., Medina, J. H., & Izquierdo, I. (1997). Late and prolonged post-training memory modulation in entorhinal and parietal cortex by drugs acting on the cAMP/protein kinase A signalling pathway. Behavioural Pharmacology, 8, 745–751. Atkins, C. M., Selcher, J. J., Petraitis, J. J., Trzaskos, J. M., & Sweatt, J. D. (1998). The MAPK cascade is required for mammalian associative learning. Nature Neuroscience, 1, 602–609. Bailey, C. H., Kaang, B. K., Chen, M., Martina, K. C., Lim, C. S., Casadio, A., & Kandel, E. R. (1997). Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apCAM in Aplysia sensory neurons. Neuron, 18, 913–924. Baron, C., Benes, C., Van Tan, H., Fagard, R., & Roisin, M. (1996). Potassium chloride pulse enhances mitogenactivated protein kinase activity in rat hippocampal slices. Journal of Neurochemistry, 66, 1005–1010. Bernabeu, R., Bevilaqua, L., Ardenghi, P., Bromberg, E., Schmitz, P., Bianchin, M., Izquierdo, I., & Medina, J. H. (1997). Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats. Proceedings of the National Academy of Sciences USA, 94, 7041–7046. Bevilaqua, L., Ardenghi, P., Scho¨der, N., Bromberg, E., Schmitz, P. K., Schaffer, E., Quevedo, J., Bianchin, M., Walz, R., Medina, J. H., & Izquierdo, I. (1997). Drugs acting upon the cyclic adenosine monophosphate/ protein kinase A signalling pathway modulate memory consolidation when given after training into rat hippocampus but not amygdala. Behavioural Pharmacology, 8, 331–338. Brambilla, R., Gnesutta, N., Minichiello, L., Whit, G., Roylance, A. J., CHerron, C. E., Ramsey, M., Wolfer, D. P., Cestari, V., Rossi-Arnaud, C., Grant, S. G. N., Champman, P. F., Lipp, H., Sturani, E., & Klein, R. (1997). A role for the Ras signalling pathway in synaptic transmission and long-term memory. Nature, 390, 281–286. English, J. D., & Sweat, J. D. (1996). Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. Journal of Biological Chemistry, 271, 24329–24332. English, J. D., & Sweat, J. D. (1997). A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. Journal of Biological Chemistry, 272, 19103–19106. Finkbeiner, S., & Greenberg, M. E. (1996). Ca11-dependent routs to Ras: Mechanisms for neuronal survival, differentiation, and plasticity. Cell, 16, 233–236. Gold, P. E. (1986). The use of avoidance training in studies of modulation of memory storage. Behavioral and Neural Biology, 46, 87–98. Hunter, T. (1995). Protein kinases and phosphatase: The yin and yang of protein phosphoriylation and signaling. Cell, 80, 225–236.
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Izquierdo, I. (1989). Different forms of post-training memory processing. Behavioral and Neural Biology, 51, 171–202. Izquierdo, I., Medina, J. H., Bianchin, M., Walz, R., Zanatta, M. S., da Silva, R. C., Bueno e Silva, M., Ruschel, A. C., & Paczko, N. (1993). Memory processing by the limbic system: Role of specific neurotransmitter systems. Behavioural Brain Research, 58, 91–98. Izquierdo, I., Quillfeldt, J. A., Zanatta, M. S., Quevedo, J., Schaeffer, E., Schmitz, P. K., & Medina, J. H. (1997). Sequential involvement of hippocampus, amygdala, entorhinal and parietal cortex in the memory formation and expression of memory for inhibitory avoidance in rats. European Journal of Neuroscience, 9, 786–793. Izquierdo, I., & Medina, J. H. (1997). Memory formation: The sequence of biochemical events in the hippocampus and its connection to activity in other brain structures. Neurobiology of Learning and Memory, 68, 285–316. Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E., & Graybiel, A. M. (1989). A family of cAMP-binding proteins that directly activate Rap1. Science, 282, 2275–2279. Mattingly, R. R., & Macara, I. G. (1996). Phosphorylation-dependent activation of the Ras-GRF/CD25Mm exchange factor by muscarinic receptors and G-protein bg subunits. Nature, 382, 268–272. Martin, K. C., Michael, D., Rose, J. C., Barad, M., Casadio, A., Zhu, H., & Kandel, E. R. (1997). MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia. Neuron, 18, 899–912. Michael, D., Martin, C. K., Seger, R., Ning, M. M., Baston, R., & Kandel, E. R. (1998). Repeated pulses of serotonin required for long-term facilitation activate mitogen-activated protein kinase in sensory neurons of Aplysia. Proceedings of the National Academy of Sciences USA, 95, 1864–1869. Paxinos, G., & Watson, C. (1992). The rat brain in stereotaxic coordinates. Sydney: Academic Press. Segal, R. A., & Greenberg, M. E. (1996). Intracellular signaling pathways activated by neurotrophic factors. Annual Review of Neuroscience, 19, 463–489. Seger, R., & Krebs, E. G. (1995). The MAPK signaling cascade. FASEB Journal, 9, 726–735. Vossler, M. R., Yao, H., York, R. D., Pan, M., Rim, C. S., & Stork, P. J. S. (1997). cAMP activates MAP kinase ELK-1 through a B-Raf- and Rap-1 dependent pathway. Cell, 89, 73–82. Zilles, W. (1985). The cortex of rat: A stereotaxic atlas. Berlin:Springer.
Time-Dependent Impairment of Inhibitory Avoidance Retention in Rats by Posttraining Infusion of a Mitogen-Activated Protein Kinase Kinase Inhibitor into Cortical and Limbic Structures Roger Walz,* Rafael Roesler,* Joa˜o Quevedo,* Ma´rcia K. Sant’Anna,* Marcelo Madruga,* Cleverson Rodrigues,* Carmen Gottfried,† Jorge H. Medina,‡ and Ivan Izquierdo* *Centro de Memo´ria, and †Laborato´rio de Fosforilac¸a˜o de Proteı´nas Cerebrais, Departamento de Bioquı´mica, ICBS, UFRGS, Ramiro Barcellos 2600, 90035-003 Porto Alegre, RS, Brazil; and †Laborato´rio de Neuroreceptores, Instituto de Biologı´a Celular, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, 3er piso, Buenos Aires, Argentina
Mitogen-activated protein kinase (MAPK) is abundantly expressed in postmitotic neurons of the developed nervous system. MAPK is activated and required for induction of long-term potentiation (LTP) in the CA1 area of the hippocampus, which is blocked by the specific inhibitor of the MAPK kinase, PD 098059. Recently it was demonstrated that MAPK is activated in the hippocampus after training and is necessary for contextual fear conditioning learning. The present work tests the role of the MAPK cascade in step-down inhibitory avoidance (IA) retention. PD 098059 (50 mM) was bilaterally injected (0.5 ml/side) into the CA1 region of the dorsal hippocampus or entorhinal cortex at 0, 90, 180, or 360 min, or into the amygdala or parietal cortex at 0, 180, or 360 min after IA training in rats using a 0.4-mA foot shock. Retention testing was carried out 24 h after training. PD 098059 impaired retention when injected into the dorsal hippocampus at 180 min, but not 0, 90, and 360 min after training. When infused into the entorhinal cortex, PD 098059 was amnestic at 0 and 180 min, but not at 90 and 360 min after training. The MAPKK inhibitor also impairs IA retention when infused into the parietal cortex immediately after training, but not at 180 or 360 min. Infusions performed into amygdala were amnestic at 180 min, but not at 0 and 360 min after training. Our results suggest a time-dependent involvement of the MAPK cascade in the posttraining memory processing of IA; the time dependency is different in the hippocampus, amygdala, entorhinal cortex, or parietal cortex of rats. q 2000 Academic Press
This work was supported by PRONEX and Fundac¸a˙ VITAE, Brazil. Address correspondence and reprint request to Roger Walz, Centro de Memo´ria, Departamento de Bioquı´mica, Instituto de Cieˆncias Ba´sicas da Sau´de, UFRGS, Ramiro Barcelos 2600 (Anexo), 90035-003 Porto Alegre, RS, Brazil. Fax: 55 516 5535. E-mail: [email protected]. 11
1074-7427/00 $35.00 Copyright q 2000 by Academic Press All rights of reproduction in any form reserved.
12
WALZ ET AL.
Mitogen-activated protein kinase (MAPK) is a serine/threonine protein kinase that acts on cytoskeletal (e.g., tau, microtubule-associated protein types 1, 2, and 4, caldesmon), cytosolic (e.g., tyrosine hydroxylase, Raf-1, MAP2K, cPLA2), membranal (e.g., extracellular growth factor receptor, nerve growth factor receptor), and nuclear (e.g., immediateearly genes: ELK1, cFos, cMyc, ATF2, Ets2) targets. The MAPK cascade involves at least three sequential homologue kinases: MP3K (mitogen-activated protein kinase kinase kinase), MAP2K or MAPKK (mitogen-activated protein kinase kinase), and MAPK (Seger & Krebs, 1995). MAPK activation plays an important role in long-term-facilitation (LTF) of Aplysia (Martin, Michael, Rose, Barad, Casadio, Zhu, & Kandel, 1997; Bailey, Kaang, Chen, Martin, Lim, Casadio, & Kandel, 1997; Michael, Martin, Seger, Ning, Baston, & Kandel, 1998), and its activation (English & Sweatt, 1996; Baron, Benes, Van Tan, Fagard, & Roisin, 1996) is necessary (English & Sweatt, 1997; Atkins, Selcher, Petraitis, Trzaskos, & Sweatt, 1998) for the induction of long-term potentiation (LTP) in the hippocampus CA1 region. In rats, MAPK is activated in the hippocampus 1 h after fear conditioning training, and this form of learning is dose-dependently attenuated by the systemic pretraining administration of MAPK inhibitor SL327 (Atkins et al., 1998). These data strongly suggest that the MAPK cascade plays an important role in neural plasticity processes, including learning and memory. The present experiments were performed in order to investigate the involvement of MAPK cascade in the posttraining processing of step-down inhibitory avoidance (IA) in rats. This form of associative learning is known to involve several sensorial stimuli including spatial and visual perceptions, sensitivity to pain, and an emotional component modulated by the amygdala and stress hormones (Izquierdo, 1989; Izquierdo & Medina, 1997). The IA is ideal for studying posttraining processes initiated by training because it is usually acquired in a single trial, uncontaminated by prior or further trials, rehearsals, or retrievals (Gold, 1986; Izquierdo, 1989). We studied the amygdala (AMY), hippocampus (HIP), entorhinal cortex (EC), and parietal cortex (PC) because it was previously shown that IA induces several biochemical changes, including changes in the activity of protein kinases A and C (Izquierdo & Medina, 1997), related to activity of the MAPK cascade (Atkins, Selcher, Petraitis, Trzaskos, & Sweatt, 1998). Using specific pharmacological approaches we have demonstrated a time-dependent role for these enzymes for memory consolidation of the IA task in AMY, HIP, PC, and EC (Izquierdo, Medina, Bianchin, Walz, Zanatta, da Silva, Bueno e Silva, Ruschel, & Paczko, 1993; Izquierdo & Medina, 1997; Ardenghi, Barros, Izquierdo, Bevilaqua, Schro¨der, Quevedo, Rodrigues, Madruga, Medina, & Izquierdo, 1997; Bevilaqua, Ardenghi, Schro¨der, Bromberg, Schmitz, Schaeffer, Quevedo, Bianchin, Walz, Medina, & Izquierdo, 1997; Bernabeu, Bevilaqua, Ardenghi, Bromberg, Schmitz, Bianchin, Izquierdo, & Medina, 1997; Izquierdo, Quillfeldt, Zanatta, Quevedo, Schaeffer, Schmitz, & Medina, 1997). Here we infused the specific MAPKK inhibitor, PD 098059, into the AMY, CA1 region of HIP, EC, and PC at different times after IA training. As the role of MAPKK is basically to activate MAPK, the effect caused by the PD 098059 inhibitor is attributable to an inhibition of MAPK, activation (Alessi, Cuenda, Cohen, Dudley, & Saltiel, 1995).
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METHODS Animals and Surgery Adult male Wistar rats (250–320 g) were obtained from our own breeding colony. They were caged in group of 5 with free access to food and water and were maintained on a 12-h light-dark cycle (lights on at 7:00 AM), at a temperature of 23 6 18C. Animals were bilaterally implanted under thionembutal anesthesia (30 mg/kg, ip) with 30-g cannulae aimed 1 mm above area CA1 of the dorsal hippocampus, or amygdala, or entorhinal cortex, or parietal cortex. (Izquierdo et al., 1997; Bevilaqua et al., 1997; Ardenghi et al., 1997). Coordinates A 24.2, L 6 3.0, V 2.3 (HIP), A 22.3, L 6 4.5, V 5.0, (AMY), and A 26.7 L 6 5.0 V 5.8 (EC), A 22.8, L 6 1.0, V 3.0 (PC) were obtained from the atlas of Paxinos and Watson (1986). Animals received cloramphenicol (25 mg/ kg, ip) 20 min before anesthesia induction. Behavioral Test Procedures and Apparatus Animals were submitted to behavioral procedures 4 days after recovery from surgery. The inhibitory avoidance apparatus was a 50 3 25 3 25-cm acrylic box whose floor consisted of parallel 1.0-mm - caliber stainless-steel bars spaced 1.0 cm apart. A 7.0 cm wide, 2.5 cm high, 25.0 cm long platform was laid on the floor against the left wall. Four to 5 days after surgery animals were placed on the platform and their latency to step down on the grid with all four paws was measured with an automatic device. In the training session, immediately after stepping down, the animals received a 0. 4-mA, 3.0s scrambled foot shock. In test sessions no foot shock was administered and the stepdown latency (to a ceiling of 120 s) was used as a measure of retention. Behavioral procedures took place between 8:00 AM and 10:30 PM. Infusion Procedures and Drugs At the time of infusion, an infusion cannula was fitted into the guide cannula. The tip of the infusion cannula protruded 1.0 mm beyond the guide cannula and was aimed at the CA1 area of the HIP, AMY, and EC or 1 mm above the junction between posterior parietal cortex I and II (PC). Through the infusion cannulae animals received bilateral 0.5-ml infusions of vehicle (2.5% dimethyl sulfoxide in saline, pH 7.4) or PD 098059 (50 mM) over a 2-min period. Two minutes was allowed after infusion for diffusion of the drug. We used the concentration of 50 mM, because this is the concentration that attenuates LTP induction in hippocampus slices (English & Sweatt, 1997). The high degree of specificity of PD 098059 “in vitro and in vivo” is indicated by its failure to inhibit 18 protein Ser/Thr kinases (including two MAPKK homologues) (Alessi et al., 1995; Dudley et al., 1995). Drug infusions were performed between 8:00 AM and 3:00 PM. Cannulae Placements One or 2 days after the end of the behavioral procedures, 0.5 ml of a solution of 4% methylene blue in saline was infused through the cannulae and animals were killed by
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FIG. 1. (A and B) Drawings of planes A 24.3 and 2.3 of the atlas of Paxinos and Watson (1986) showing, stippled, the extent of the area reached by the infusion in the dorsal hippocampus and amygdala, respectively. (C) Ventral view of the brain showing the extent of infusions into the entorhinal cortex. (D) Lateral view of the brain (Ziles, 1985) showing the extent of infusions into the posterior parietal cortex.
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FIG. 2. Effects of posttraining infusions of PD 098059 into the dorsal hippocampus on retention of stepdown inhibitory avoidance task in rats. Data are median (interquartile range) of retention test latencies. N 5 10–13 animals per group. *Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .001). In this and following figures, training-test differences are significant in Wilcoxon tests at the p , .01 level, except in the groups marked by asterisks.
decapitation 10 min after. Brains were stored in formalin for at least 72 h and cannulae placements were verified by histological examination (Izquierdo et al., 1997; Ardenghi et al., 1997; Bevilaqua et al., 1997). Cannulae placements were found to be correct (within 0.8 mm3 of the intended sites) in 86 of 93 rats implanted into the dorsal HIP, and in 69 of 76 rats implanted into AMY, and in 64 of 71 animals implanted into PC, and 87 of 96 animals implanted into the EC. Only data from these animals were included in the final analysis. Injection placements are shown in Fig 1. Statistical Analysis Comparisons of both training and test session latencies between vehicle-treated and PD 098059-treated groups were performed using a Mann-Whitney U test, two-tailed. Comparisons between training and test sessions in individual groups were by a Wilcoxon test. In all comparisons, p , .05 was considered to indicate statistical significance. RESULTS When given into CA1 (Fig. 2), PD 098095 impaired retention when infused 180 but not 0, 90, or 360 min after training. There were significant differences between training
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FIG. 3. Effects of posttraining infusions of PD 098059 into the amygdala on retention of step-down inhibitory avoidance task in rats. Data are median (interquartile range) of retention test latencies. N 5 10–13 animals per group. *Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .001).
and test session latencies in all groups except for the PD 098059 group injected at 180 min after training. The MAPKK inhibitor was amnestic when infused into AMY 180 min, but not 0 or 360 min posttraining (Fig. 3). There were significant differences between training and test session latencies in all groups except for the PD 098059 group injected into AMY at 180 min after training. Results of infusions into the EC are shown in Fig. 4. There were significant differences in retention test latencies between the PD 098059 and vehicle groups infused 0 and 180 min after training but not in those infused 90 and 360 min posttraining. Moreover, there were significant differences between training and test session latencies in all groups except those treated with PD 098059 at 0 and 180 min posttraining. Results of infusions into the parietal cortex are shown in Fig. 5. PD 098059 was amnestic when given into the PC immediately but not at 180 or 360 min after training. There were significant differences between training and test session latencies in all groups except the one treated with PD 098059 at 0 min after training. There were no significant differences between PD 098059 groups and their respective control groups in training session latencies (results not shown). DISCUSSION This is the first report of a time-dependent involvement of the MAPK cascade in posttraining memory processing by specific neocortical structures. Our data are in agreement with the study by Atkins et al. (1998), that suggested an involvement of MAPK in the HIP during contextual fear conditioning. They showed a delayed posttraining increase of MAPK activity in HIP, and an amnestic effect of a MAPK inhibitor given systemically. In view of our present findings, the effect they observed with the MAPK inhibitor given pretraining could have been due to influences on different brain sites at different times.
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FIG. 4. Effects of posttraining infusions of PD 098059 into the entorhinal cortex on retention of step-down inhibitory avoidance task in rats. Data are median (interquartile range) of retention test latencies. N 5 10–12 animals per group. *Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .001). **Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .05).
The amnestic effect of the specific MAPKK inhibitor used here could be due to interference with cytoskeletal, cytosolic, or membranal processes in the cortical and limbic structures studied, and/or to an impairment of IEGs or DRGs transcription (Finkbeiner & Greenberg, 1996; Segal & Greenberg, 1996). PD098059, like the protein kinase A inhibitor KT 5720 (Bernabeu et al., 1997; Izquierdo & Medina, 1997; Ardenghi et al., 1997), was amnestic when given into CA1 3 h after training, and when given into the EC at both 0 and 3 h after IA training. These pharmacological effects are coincident with an enhancement in cAMP and c-fos levels in both HIP and EC (Bernabeu et al., 1997; Izquierdo & Medina, 1997). The similarity of the posttraining time windows of effects of both the MAPKK and the PKA inhibitors in EC and CA1 raises the possibility of cross-talk between both cascades in these structures in relation to memory formation. Activation of MAPK by cAMP/PKA pathway stimulation has been demonstrated in the pheochromocytoma cell line PC12 (Vossler et al., 1997), in Aplysia (Bailey et al., 1997; Martin et al., 1997; Michael et al., 1998), and in mouse hippocampus (Michael et al., 1998; Martin et al., 1997). Recently it was also shown that cAMP can directly stimulate MAPK in neurons through a PKA-independent pathway (Kawasaki, Springett, Mochizuki, Toki, Nakaya, Matsuda, Housman, & Graybiel, 1998). The amnestic effect of the MAPKK inhibitor when infused in AMY is interesting because PKA inhibitors given into this structure have no effect on IA retention (Ardenghi et al., 1997; Bevilaqua et al., 1997). So it is possible that the MAPK cascade may be
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FIG. 5. Effects of posttraining infusions of PD 098059 into the parietal cortex on retention of step-down inhibitory avoidance task in rats. Data are median (interquartile range) of retention test latencies. N 5 10–12 animals per group. *Significant difference from the vehicle-treated group (Mann-Whitney U test, p , .01).
more crucial to AMY function than the PKA cascade. Animals lacking the neuronalspecific guanine-nucleotide-exchange Ras-GRF, which are involved in MAPK cascade stimulation through the G protein Ras (Mattingly & Macara, 1996), have abnormal longterm potentiation in AMY and show an impairment of two-way active avoidance, inhibitory avoidance, and fear conditioning (Brambilla et al., 1997). The time window of the amnestic effect of the MAPKK inhibitor and the PKA inhibitor was not completely coincident in PC. The PKA inhibitor was amnestic when infused 0, 3, or 6 h after IA training (Ardenghi et al., 1997), but the MAPKK was effective only immediately after training. Thus, in some areas (PC, AMY) there is a discrepancy, and in others (CA1, EC) a coincidence, in the time windows and/or quality of the effect of the two enzymatic inhibitors. This suggests that in some areas (CA1, perhaps EC) there may be an interaction or a cross-talk between the two cascade systems, and in others not. In any case, these findings point once more to the need to examine more than one region of the brain when their involvement in a given learning process is being studied (Izquierdo & Medina, 1998). Further, these data support the view that the roles of the several kinases on memory processing vary according to the task, the procedure, the posttraining time, and the brain structure studied (Izquierdo & Medina, 1997). In LTP experiments, the MAPK cascade appears to be involved very early on. In the experiment
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of Atkins et al. (1998) on contextual fear conditioning the involvement of the MAPK cascade in HIP appears to be before the one we report here for IA. In summary, the MAPK cascade plays an important role in the posttraining memory processing of IA, at different times, in cortical and limbic structures of rats. Taking into account that in humans there are as many as 2000 conventional protein kinase genes and 1000 phosphatase genes (Hunter, 1995), possible cross-talks between the PKA and MAPK pathways or other protein kinase pathways in HIP, EC, and PC deserve further study. Different cross-talks between (or among) protein kinase cascades may explain the differences in timing of the influence of one or other task or situation and would certainly be expected from the vastness of the repertory of kinases and phosphatases. REFERENCES Alessi, R. D., Cuenda, A., Cohen, P., Dudley, D. T., & Saltiel, A. R. (1995). PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. Journal of Biological Chemistry, 276, 27489–27494. Ardenghi, P., Barros, D., Izquierdo, L., Bevilaqua, L., Schro¨der, N., Quevedo, J., Rodrigues, C., Madruga, M., Medina, J. H., & Izquierdo, I. (1997). Late and prolonged post-training memory modulation in entorhinal and parietal cortex by drugs acting on the cAMP/protein kinase A signalling pathway. Behavioural Pharmacology, 8, 745–751. Atkins, C. M., Selcher, J. J., Petraitis, J. J., Trzaskos, J. M., & Sweatt, J. D. (1998). The MAPK cascade is required for mammalian associative learning. Nature Neuroscience, 1, 602–609. Bailey, C. H., Kaang, B. K., Chen, M., Martina, K. C., Lim, C. S., Casadio, A., & Kandel, E. R. (1997). Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apCAM in Aplysia sensory neurons. Neuron, 18, 913–924. Baron, C., Benes, C., Van Tan, H., Fagard, R., & Roisin, M. (1996). Potassium chloride pulse enhances mitogenactivated protein kinase activity in rat hippocampal slices. Journal of Neurochemistry, 66, 1005–1010. Bernabeu, R., Bevilaqua, L., Ardenghi, P., Bromberg, E., Schmitz, P., Bianchin, M., Izquierdo, I., & Medina, J. H. (1997). Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats. Proceedings of the National Academy of Sciences USA, 94, 7041–7046. Bevilaqua, L., Ardenghi, P., Scho¨der, N., Bromberg, E., Schmitz, P. K., Schaffer, E., Quevedo, J., Bianchin, M., Walz, R., Medina, J. H., & Izquierdo, I. (1997). Drugs acting upon the cyclic adenosine monophosphate/ protein kinase A signalling pathway modulate memory consolidation when given after training into rat hippocampus but not amygdala. Behavioural Pharmacology, 8, 331–338. Brambilla, R., Gnesutta, N., Minichiello, L., Whit, G., Roylance, A. J., CHerron, C. E., Ramsey, M., Wolfer, D. P., Cestari, V., Rossi-Arnaud, C., Grant, S. G. N., Champman, P. F., Lipp, H., Sturani, E., & Klein, R. (1997). A role for the Ras signalling pathway in synaptic transmission and long-term memory. Nature, 390, 281–286. English, J. D., & Sweat, J. D. (1996). Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation. Journal of Biological Chemistry, 271, 24329–24332. English, J. D., & Sweat, J. D. (1997). A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. Journal of Biological Chemistry, 272, 19103–19106. Finkbeiner, S., & Greenberg, M. E. (1996). Ca11-dependent routs to Ras: Mechanisms for neuronal survival, differentiation, and plasticity. Cell, 16, 233–236. Gold, P. E. (1986). The use of avoidance training in studies of modulation of memory storage. Behavioral and Neural Biology, 46, 87–98. Hunter, T. (1995). Protein kinases and phosphatase: The yin and yang of protein phosphoriylation and signaling. Cell, 80, 225–236.
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Izquierdo, I. (1989). Different forms of post-training memory processing. Behavioral and Neural Biology, 51, 171–202. Izquierdo, I., Medina, J. H., Bianchin, M., Walz, R., Zanatta, M. S., da Silva, R. C., Bueno e Silva, M., Ruschel, A. C., & Paczko, N. (1993). Memory processing by the limbic system: Role of specific neurotransmitter systems. Behavioural Brain Research, 58, 91–98. Izquierdo, I., Quillfeldt, J. A., Zanatta, M. S., Quevedo, J., Schaeffer, E., Schmitz, P. K., & Medina, J. H. (1997). Sequential involvement of hippocampus, amygdala, entorhinal and parietal cortex in the memory formation and expression of memory for inhibitory avoidance in rats. European Journal of Neuroscience, 9, 786–793. Izquierdo, I., & Medina, J. H. (1997). Memory formation: The sequence of biochemical events in the hippocampus and its connection to activity in other brain structures. Neurobiology of Learning and Memory, 68, 285–316. Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D. E., & Graybiel, A. M. (1989). A family of cAMP-binding proteins that directly activate Rap1. Science, 282, 2275–2279. Mattingly, R. R., & Macara, I. G. (1996). Phosphorylation-dependent activation of the Ras-GRF/CD25Mm exchange factor by muscarinic receptors and G-protein bg subunits. Nature, 382, 268–272. Martin, K. C., Michael, D., Rose, J. C., Barad, M., Casadio, A., Zhu, H., & Kandel, E. R. (1997). MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia. Neuron, 18, 899–912. Michael, D., Martin, C. K., Seger, R., Ning, M. M., Baston, R., & Kandel, E. R. (1998). Repeated pulses of serotonin required for long-term facilitation activate mitogen-activated protein kinase in sensory neurons of Aplysia. Proceedings of the National Academy of Sciences USA, 95, 1864–1869. Paxinos, G., & Watson, C. (1992). The rat brain in stereotaxic coordinates. Sydney: Academic Press. Segal, R. A., & Greenberg, M. E. (1996). Intracellular signaling pathways activated by neurotrophic factors. Annual Review of Neuroscience, 19, 463–489. Seger, R., & Krebs, E. G. (1995). The MAPK signaling cascade. FASEB Journal, 9, 726–735. Vossler, M. R., Yao, H., York, R. D., Pan, M., Rim, C. S., & Stork, P. J. S. (1997). cAMP activates MAP kinase ELK-1 through a B-Raf- and Rap-1 dependent pathway. Cell, 89, 73–82. Zilles, W. (1985). The cortex of rat: A stereotaxic atlas. Berlin:Springer.