Mitogen-activated protein kinase regulates early phosphorylation and delayed expression of Ca2+/calmodulin-dependent protein kinase II in long-term potentiation

Abstract

Activation of mitogen-activated protein kinase (MAPK) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are required for numerous forms of neuronal plasticity, including long-term potentiation (LTP). We induced LTP in rat hippocampal area CA1 using theta-pulse stimulation (TPS) paired with beta-adrenergic receptor activation [isoproterenol (ISO)], a protocol that may be particularly relevant to normal patterns of hippocampal activity during learning. This stimulation resulted in a transient phosphorylation of p42 MAPK, and the resulting LTP was MAPK dependent. In addition, CaMKII was regulated in two, temporally distinct ways after TPS-ISO: a transient rise in the fraction of phosphorylated CaMKII and a subsequent persistent increase in CaMKII expression. The increases in MAPK and CaMKII phosphorylation were strongly colocalized in the dendrites and cell bodies of CA1 pyramidal cells, and both the transient phosphorylation and delayed expression of CaMKII were prevented by inhibiting p42/p44 MAPK. These results establish a novel bimodal regulation of CaMKII by MAPK, which may contribute to both post-translational modification and increased gene expression.

Fig. 1.

MAPK participates in TPS–ISO-induced LTP. A, Nissl-stained unstimulated hippocampal slice showing the positioning of the stimulating (left) and recording (right) electrodes and the cuts used to excise the CA1 region. Thearrows indicate the boundaries of the CA1 region, for the purpose of counting cell bodies. B, MAPK activity is required for LTP induced by TPS–ISO. Isoproterenol (1 μm) was applied to the bath for 10 min, followed by theta pulse stimulation of the Schaffer collaterals (TPS; 150 pulses at 10 Hz). TPS–ISO-induced LTP persisted for at least 60 min after the end of stimulation (○; n = 5). In slices preincubated with PD98059 (50 μm), the maintenance of TPS–ISO LTP was blocked (●; n = 6).Asterisks indicate group differences with p < 0.05 (Newman–Keuls multiple comparison test). The superimposed traces show representative field EPSPs before TPS–ISO and 60 min after stimulation. Additional experiments showed a similar inhibition of LTP by pretreatment with 30 μm PD98059 [LTP at 60 min: 142 ± 27 in controls (n = 3) and 117 ± 13 in treated slices (n = 3); p< 0.05], in contrast to the previous findings with HFS-induced LTP (Liu et al., 1999). Calibration: 0.5 mV, 5 msec. C, TPS–ISO increases p42 MAPK phosphorylation in area CA1 of the hippocampus. The bar graph summarizes immunoblot data and shows the levels of phospho-(Thr202/Tyr204)-p42 MAPK normalized to total MAPK in each CA1 region and expressed as percentage of paired, untreated control slices. 2, 15, and60 indicate minutes after stimulation. Significant differences from sham-stimulated slices are indicated by theasterisks (p < 0.05). PD98059 (PD) was applied at 50 μm in the maintenance chamber, and aminophosphonovaleric acid (APV) was applied in the recording chamber at 100 μm for 10 min before stimulation). ISOindicates slices exposed to 1 μm isoproterenol alone for 10 min. The inset shows the time course of MAPK phosphorylation after TPS–ISO stimulation. An immunoblot, from a single experiment, of phospho-p42 MAPK (top panel) and total-p42 MAPK (bottom panel) is shown below. D, TPS–ISO does not phosphorylate Erk5. The immunoblot, probed for Erk5 immunoreactivity, was run with homogenates from hippocampal area CA1 (4 lanes on the left) and HeLa cells (4 lanes on the right). Within each type of tissue, the two lanes on the left are from untreated controls, whereas the two lanes on theright are from stimulated tissue (TPS–ISO, 15 min after stimulation for CA1 and 15 min of 1 ng/ml EGF for HeLa cells). Erk5 from EGF-stimulated HeLa cells shows a clear mobility shift, indicative of increased phosphorylation, but no shift occurred in stimulated hippocampus.

Fig. 2.

The hippocampal distribution of phosphorylated MAPK after TPS–ISO. A–C, Phospho-MAPK was visualized using DAB staining. The top images show entire hippocampal slices, with the boxed region (areaCA1) digitally expanded in the bottom images. A shows a sham-treated slice,B shows a slice treated with 1 μmisoproterenol alone and harvested 15 min after treatment, andC shows a TPS–ISO slice 15 min after treatment. Note the prominent staining in stratum pyramidale (s.p.) and stratum radiatum (s.r.) of CA1, which was consistently seen in TPS–ISO-treated slices. In this slice, stratum oriens (s.o.) also showed strong staining, which was observed only in a minority of slices treated with TPS–ISO. Scale bars:top traces, 1 mm; bottom traces, 500 μm. D, Summary of the quantitative analysis performed on DAB-positive pyramidal cell bodies in areas CA1 (left panel) and CA2–CA4 (right panel). DAB-positive cell bodies were counted for CA1 (roughly corresponding to the bottom panels ofA–C) (Fig. 1A) and for the remainder of the pyramidal layer (CA2–CA4). In area CA1, only TPS–ISO-treated slices, harvested at either 2 or 15 min after stimulation, showed an increase in DAB-positive cells (*p < 0.01; ^#p < 0.05). No significant effects were observed in CA2–CA4.E, A laser confocal immunofluorescent image of phospho-MAPK immunoreactivity in area CA1 of a TPS–ISO-treated slice harvested 15 min after treatment. Labeling was observed throughout the dendrites of stratum radiatum, in the perinuclear region, and in the nucleus. Scale bar, 25 μm. F, G, DAB-labeled images within area CA1 of sham-stimulated (F) and TPS–ISO (G) slices, harvested 15 min after stimulation. The entire apical dendritic tree of CA1 pyramidal neurons is shown. s.o., Stratum oriens; s.p., stratum pyramidale; s.r., stratum radiatum; s.l.m., stratum lacunosum-moleculare. Scale bar, 100 μm.

Fig. 3.

The MAPK pathway is required for TPS–ISO-induced phosphorylation of CaMKII in area CA1. The bar graph summarizes immunoblot data and shows phospho-CaMKII levels normalized to total CaMKII and expressed as percentage of paired, untreated control slices. Only TPS–ISO slices harvested 15 min after stimulation showed a significant increase in CaMKII phosphorylation (*p< 0.05). This effect was completely blocked by treatment with 50 μm PD98059. The inset shows the time course of phospho-CaMKII after TPS–ISO stimulation. The representative Western immunoblot, which was taken from a single experiment, includes the treatments summarized in the graph. The top panelshows phosphorylated CaMKII, and the bottom panel shows total CaMKII.

Fig. 4.

The MAPK requirement for LTP does not involve PP1 inhibition or spike modulation. A, PD98059 blocks LTP induced by TPS in CA1 neurons injected with 10 μmthiophosphorylated inhibitor-1 (I-1-P). In control slices (n = 5) pretreated with 0.1% DMSO, a stable LTP was obtained after TPS (150 pulses at 10 Hz), but LTP was absent in slices pretreated with 50 μm PD98059 (n = 5). The traces show superimposed sample intracellular EPSPs obtained during the baseline period and 40 min after TPS (arrowhead). Calibration: 5 mV, 10 msec. B, PD98059 does not affect the pattern of spiking during TPS in cells recorded with thiophosphorylated I-1. The intracellular potential was sampled every 30th pulse during TPS. Sample trace series and the summary data are shown. The groups did not differ significantly at any sample time. Data are from the same cells as in A. Calibration: 20 mV, 10 msec.

Fig. 5.

Phospho-MAPK and phospho-CaMKII are colocalized in CA1 pyramidal neurons after TPS–ISO. Laser confocal images were obtained from slices that were double labeled using antibodies specific for phospho-MAPK and phospho-CaMKII. Phospho-MAPK labeling is indicated by green (A), phospho-CaMKII labeling is indicated by red (B), and combined labeling is indicated by yellow-orange(C, D). The panels on theleft are from sham-stimulated controls, and those on theright are from slices harvested 15 min (A–C) or 2 min (D) after TPS–ISO. A–C, At 15 min after stimulation, TPS–ISO increased both phospho-MAPK and phospho-CaMKII in dendrites of stratum radiatum (s.r.) and in cell bodies of stratum pyramidale (s.p.). The digitally combined signals (C) show that phospho-MAPK and phospho-CaMKII were colocalized in dendrites and in the perinuclear region, but only phospho-MAPK translocated to the nucleus. Scale bar, 50 μm. D, As early as 2 min after TPS–ISO, phospho-MAPK had already translocated to the nucleus. Scale bar, 20 μm.

Fig. 6.

TPS–ISO produces a delayed increase in total CaMKII that requires MAPK activation and protein synthesis.A, A MEK inhibitor prevents the increase in CaMKII expression after TPS–ISO. Western immunoblots were prepared from CA1 regions, using antibody probes for total CaMKII and for actin. The bar graph shows summary data, with total CaMKII level normalized to actin from the same CA1 sample and expressed as percentage change from unstimulated controls. Total CaMKII was significantly increased only at 60 min after TPS–ISO (*p < 0.05). Atright is a representative immunoblot, which was run with CA1 homogenates from slices harvested at 60 min. PD= 50 μm PD98059. B, LTP measured at 60 min after TPS–ISO is inhibited by actinomycin-D or anisomycin. Both drugs were applied in the bath starting 30 min before TPS–ISO. The graph summarizes EPSP slopes at 60 min after stimulation, expressed as percentage of baseline. TPS–ISO-induced LTP measured 31.1 ± 6.7% above baseline (n = 5) and was blocked by 40 μm actinomycin-D (Act-D) or 20 μm anisomycin (Aniso) [+9.7 ± 11.7% (n = 3) and −1.1 ± 7.3% (n = 3), respectively; both pvalues < 0.05 vs TPS–ISO]. C, The increase in total CaMKII by TPS–ISO is blocked by actinomycin-D and anisomycin. Slices were harvested 60 min after TPS–ISO or at the equivalent time for sham-stimulated controls. The graph indicates total CaMKII levels normalized to actin for each band and expressed as percentage change from the sham-stimulated slices. TPS–ISO produced an increase in total CaMKII (+33.9 ± 16.6%; n = 5), which was prevented by 40 μm actinomycin-D and 20 μmanisomycin [+9.7 ± 6% (n = 3) and −24.2 ± 5.7% (n = 3), respectively; both pvalues < 0.05 vs TPS–ISO]. Anisomycin reduced total CaMKII below sham-stimulated controls (p < 0.05). Sample immunoblots are shown for CA1 homogenates from slices treated with actinomycin-D (top blot) and anisomycin (bottom blot).