CaMKIIα mRNA dendritic localization and translatability.

<p><b>A,B </b><i>In situ</i> hybridization for CaMKIIα mRNA. <b>A,</b> On a horizontal section, CaMKIIα mRNA is visible in multiple regions of the brain, particularly in the hippocampus (HC), cortex and olfactory bulb (OB). Scale bar: 500 µm. <b>B,</b> Higher magnification of the OB shows strong CaMKIIα mRNA expression in the granule cell layer (GCL) and in a diffuse staining of the external plexiform layer (EPL), where GCs extend their apical dendrites. Gl: glomerular layer. Scale bar: 25 µm. <b>C,</b> Quantification of mRNA levels in synaptosomes (SN) by quantitative PCR. CaMKIIα and PSD95 transcripts are highly elevated (7 fold) at the synaptic level, as compared to HPRT mRNA, a transcript restricted to the soma. “I” is the index of synaptic localization and represents the ratio of mRNA present in the SN fraction over the total quantity present in the OB homogenate, normalized to HPRT mRNA. Error bars represent sem (n = 3). <b>D,</b> SN were metabolically labeled with a mixture of ³⁵S-Met and ³⁵S-Cys with (lane +) or without (lane -) stimulation by 10 µM glutamate and 50 µM NMDA. Newly-synthesized proteins were detected by autoradiography (left panel) or blotted with an anti-CaMKIIα antibody (right panel). <b>Ea,b</b> Electron micrograph of a dendrodendritic synapse between a granule cell dendrite (GCd) and a mitral/tufted cell dendrite (M/Td). Ribosomes (r, arrows) are located in the GCd, at the base of, or in close proximity to, a spine. The inset displays a magnification of the boxed region showing polyribosomes with a characteristic rosette morphology. The GC is colorized in pink in b, and the blue arrow in the granule cell spine head (GSh) indicates the synapse formed onto the mitral/tufted cell dendrite (M/Td). Scale bar: 0.5 µm.</p

CaMKIIα expression in the OB.

<p><b>A,B,C</b> Immunostaining for CaMKIIα (red) on olfactory bulb (OB) sections. <b>A:</b> CaMKIIα immunoreactivity is present in the Granule Cell Layer (GCL) and in the External Plexiform Layer (EPL), where granule cells (GCs) extend their apical dendrites. The glomerular layer (Gl) appears unlabeled. Scale bar: 50 µm. <b>B,</b> Higher magnification of the GCL shows staining of some GCs in their cell bodies (arrow) and in their dendrites extending towards the EPL (arrowheads). Mitral cells do not express the protein (star). Scale bar: 25 µm. <b>C,</b> Magnification of the boxed region in B. Some GCs express CaMKIIα in the thin rim of cytoplasm surrounding their nuclei (arrows). Nuclei are counterstained with DAPI. Scale bar: 10 µm.</p

CaMKIIα mRNA synaptic localization is regulated by olfactory activity.

<p><b>A,</b> Mice were presented with a tea-ball containing either garlic and tarragon (“enriched”) or nothing (“basal”) for various durations. 30 min of this olfactory enrichment raises the levels of CaMKIIα mRNA in synaptosomes (SN) by 3.4-fold as compared to basal levels. No increase is observed after 15 min of exposure, and mRNA levels return to baseline (index ratio of 1) 60 min after exposure. “I” is the index of synaptic localization and represents the ratio of mRNA present in the SN fraction over the total quantity present in the OB homogenate. Error bars represent sem. (n = 3 for 15 min and 30 min, n = 2 for 60 min; *p = 0.005, **p<0.02, t-test) <b>B,</b> CaMKIIα index of synaptic localization rises from 7 in basal conditions to 26 upon 30 min of olfactory enrichment (n = 3; *p<0.02, t-test). <b>C, D </b><i>In situ</i> hybridization for CaMKIIα mRNA of control or 30 min enriched mice shows an increase of the EPL staining upon enrichment Scale bar: 50 µm. Quantification of the signal showed that it was increased by 2.3 upon enrichment (n = 2, p = 0.006). <b>E,F</b> Western blot of SN shows a 1.5 increase in CaMKIIα protein in enriched conditions versus basal conditions (n = 4, p<0.05). The results were normalized over β-actin signal.</p

Altered CaMKIIα mRNA and protein synaptic localization disrupts olfactory associative learning.

<p><b>A, </b><i>In situ</i> hybridization for CaMKIIα mRNA in wild-type mice (WT, upper panels) and 3′UTR mutants (3′UTR, lower panels) shows a dramatic decrease of the dendritic staining (stars) in the mutant hippocampus (HC, left panels) and olfactory bulb (OB, right panels). EPL: external plexiform layer Scale bars: 200 µm and 50 µm, in HC and OB respectively. <b>B,</b> Quantification of the index of synaptic localization (I) of CaMKIIα and PSD95 mRNAs normalized to HPRT mRNA in WT and 3′UTR mutants. CaMKIIα mRNA index is significantly decreased in 3′UTR mutants as compared to WT (n = 3, p = 0.026) <b>C,</b> Behavioral habituation/dishabituation paradigm: both WT and 3′UTR mutants mice habituated to the habituation odor, as shown by decreased investigation time across the four trials (Hab1-Hab4) (WT: F(3,27) = 30.452, p<0.001; 3′UTR: F(3,27) = 20.469, p<0.001; ANOVA, trial effect) and discriminated. Upon presentation of the test odor (Test), the investigation time increased when compared to Hab4, indicating that the animals could discriminate the test odor from the habituation odor (WT: *p<0.00005; 3′UTR: *p<0.00005, t-test). <b>D,</b> Olfactory associative learning: mice were conditioned to associate an odor stimulus to a food reward. Success rate results are presented as the percentage of correct choice across consecutive blocks of trials (1 block = 4 trials). Conditioned WT mice learned the task as indicated by the increase in correct choice throughout blocks (F(5,49) = 3.391, p<0.01; ANOVA, block effect). In contrast, conditioned 3′UTR mutants did not show an increase in success rate across trials (F(5,54) = 1.078, p>0.1; ANOVA, block effect), showing that impaired CaMKIIα mRNA dendritic localization disrupts olfactory-associative learning. <b>E,</b> Latencies: conditioned WT mice showed a decrease in latency confirming that they learned the task (F(5,49) = 4.037, p<0.005; ANOVA, block effect). In contrast, latencies of conditioned 3′UTR mutants did not significantly change (F(5,54) = 0.635, p>0.5; ANOVA, block effect), indicating that the mutants were not able to associate odor and reward.</p

CaMKIIα mRNA synaptic localization is regulated by NMDAR.

<p><b>A,</b> NMDAR antagonist: 30 min prior to the 30 min olfactory enrichment protocol, mice were injected with CPP. Upon 30 min of olfactory enrichment, the synaptic localization index remains unchanged as compared to basal conditions. NMDA receptor blockade thus prevents the increase in CaMKIIα mRNA observed upon olfactory enrichment (compare with B) (n = 3; p = 0.6, t-test). <b>B,</b> NMDAR agonist: mice were injected with D-cycloserine or saline. 30 min after injection, the index of synaptic localization is increased 3.4-fold as compared to saline, thus recapitulating the effect of olfactory enrichment on CaMKIIα mRNA localization (compare with B) (n = 2 for saline, n = 3 for D-cycloserine; *p = 0.02, t-test).</p