Ca2+ -stimulated adenylyl cyclases regulate ERK-dependent activation of MSK1 during fear conditioning

Abstract

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca(2+)-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory.

Figure 1

MAPK is selectively and transiently activated in the CA1 subregion of the hippocampus after contextual fear conditioning (A) Immunohistochemistry for pERK in CA1, CA3 and dentate gyrus (DG) from sections representative of naïve, unpaired, context and paired mice 30 minute after training. Neuropil staining was heaviest in the CA3 mossy fibers (MF), moderate in the inner (iml) and outer (oml) layers of DG, and mild to moderate in the dendritic layers of Ammon’s horn. Only pERK staining in the pyramidal cell layer (sp) and stratum radiatum (sr) in CA1 increased with training. Scale bar, 100 μm. (B) Effect of training on 6 hour memory for contextual fear conditioning. n = 9–12 mice per group. (C) Effect of fear conditioning on the number of pERK+ cells in the main hippocampal subregions (30 min). (D) Time course of pERK+ cell numbers in CA1, normalized to unpaired animals.

Figure 2. Training for contextual memory triggers MAPK activation selectively in CA1 pyramidal neurons

Images are three dimensional reconstructions of confocal z-stacks. (A) First row, pERK+ cell bodies co-localized with the neuronal marker NeuN. Second row, pERK labeling was found in cell bodies and apical dendrites immunoreactive to MAP2, but not in GABAergic interneurons stained with GAD67, or in astrocytes stained with GFAP. (B) Fear conditioning increased the percentage of CA1 pyramidal neurons stained for pERK. Scale bars, 20 μm.

Figure 3. Nuclear and synaptic activation of MAPK

(A) Average line scans of pERK staining in paired (n = 42 neurons, pERK+ subset) and unpaired (n = 36 neurons) mice (30 min); x-axis represents relative position across the soma. (B) Double-labeling for pERK and CaMKIIα in a CA1 pyramidal neuron after fear conditioning. Arrowheads indicate dendritic branches of the neuron shown in which pERK staining was absent. (C) Apical dendritic branches (occupying areas devoid of synaptophysin labeling) were decorated with pERK+ puncta, which were often juxtaposed to synaptophysin boutons (arrows). Scale bar, 5 μm. (D) Example of a single confocal section subfield included in the formal quantitative analysis. Green regions with white margins represent pERK+ puncta that met criteria of size and labeling intensity after thresholds were applied. Raw image is in red. Yellow-outlined objects represent overlaid synaptophysin-labeled puncta obtained after similar processing. pERK+ and synaptophysin+ loci showed partial overlap (white arrows) or were juxtaposed to one another (blue arrows). Scale bar, 1 μm. (E) Quantification of the gray scale signal intensity of pERK+ puncta associated with synaptophysin in unpaired and paired mice.

Figure 4. Co-localized activation of MAPK and PKA signaling in CA1 pyramidal neurons

(A) Acute infusion of Sp-cAMP into the CA1 region increased the immunoreactivity for phosphorylated PKA substrates (pPKA-s), which was blocked by Rp-cAMP but not by SL327. (B) Frequency distribution of the levels of pPKA-s labeling in CA1 somata in paired and unpaired mice (30 min after training). (C) A representative optical section from CA1 showing higher levels of pPKA-s labeling in pERK+ neurons (arrows). Scale bar, 10 μm. Right, summary data. Each dot pair represents the mean gray scale intensity of pPKA-s in pERK+ and pERK- somata for a given mouse. (D) Single-cell line scans showing the correlation between the nuclear levels of pERK and pPKA-s from two neurons that were centered at the same plane along the z-axis. Scale bar, 20 μm. (E) Average line scans of pPKA-s staining in paired (n = 42 cells, pERK+ subset) and unpaired (n = 36 cells) mice;x-axis represents relative position across the soma.

Figure 5. Impaired activation of MAPK and PKA following contextual fear conditioning in DKO mice

(A) Western blot analysis of hippocampal pERK in WT and DKO mice after training (5–10 min). Blot shows pooled samples; the graph shows mean data from individually run samples. U, unpaired; P, paired. (B) Immunohistochemical analysis of pERK in DKO mice after training (30 min). Representative images of the CA1 region. Scale bar, 50 μm. (C) Western blot analysis of total ERK expression in WT and DKO hippocampi. (D) Average intensity levels of pPKA-s in pERK- and pERK+ CA1 somata from WT and DKO hippocampi after training (30 min). (E) Ratio of the pERK labeling intensity in the nucleus relative to the cytoplasm in WT and DKO mice.

Figure 6. Activation of MAPK/PKA and downstream CREB kinases following contextual fear conditioning

Images are three dimensional reconstructions of confocal z-stacks. (A, B) Immunodetection of pERK and pMSK1 (A) or pERK and pRSK1/2 (B) in the CA1 layer of paired and unpaired mice (30 min). Arrows indicate co-localized signals, arrowheads indicate segregated signals. Scale bar, 20 μm. (C) First row, immunodetection of pPKA-s and pMSK1. Arrows indicate neurons heavily labeled for both kinases. Second row, immunodetection of pCREB and pERK. Third row, immunodetection of pCREB and pMSK1. Low-to-moderate pERK or pMSK1 staining (indicated by arrowheads) was not associated with high pCREB staining, whereas high pERK or pMSK1 staining (indicated arrows) was. Images were taken from the CA1 layer of paired mice (30 min). Scale bar, 20 μm. (D) Summary data of the relative pCREB levels as a function of the quartile intensity for pERK or pMSK1.

Figure 7

Calcium-stimulated adenylyl cyclase and MAPK regulate MSK1 and CREB activation following contextual fear conditioning. (A) Infusion of Sp-cAMP into the CA1 region increased the immunoreactivity for phosphorylated MSK1, which was blocked by Rp-cAMP and SL327. (B) Post-training inhibition of MEK1/2 prevents the co-localized phosphorylation of MSK1 and MAPK in CA1 nuclei. Images are three dimensional reconstructions of confocal z-stacks. Scale bar, 20 μm. Summary data is shown on the right. (C) Nuclear extracts from wild type and DKO hippocampi were prepared from context-exposed or paired mice and analyzed for relative levels of pCREB, pMSK1 or pRSK2. Blots show individually run samples. Summary data is shown on the right.