The Journal of Neuroscience, May 16, 2007, ():
Dysregulated Metabotropic Glutamate Receptor-Dependent Translation of AMPA Receptor and Postsynaptic Density-95 mRNAs at Synapses in a Mouse Model of Fragile X Syndrome
J. Neurosci. Muddashetty et al.
27: 5338
Supplemental Data
Files in this Data Supplement:
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Supplemental Table 1
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Supplemental Table 2
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Fig. S1. FISH detection of dendritic mRNAs in the hippocampal CA1 region of wild type and Fmr1 knockout mice.
(a and b) Coronal brain slices from wild type (a) and Fmr1 knockout mice (b) were hybridized with digoxigenin-labeled antisense riboprobes specific for CaMKII?, GluR1, GluR2, PSD-95 and ?-Tubulin mRNAs (as indicated above), detected with fluorescein-based tyramide signal amplification and analyzed by confocal microscopy. Shown are photomicrographs from the pyramidal cell layer and the stratum radiatum of the CA1 region. Strong CaMKII? mRNA specific signals can be detected throughout the entire stratum radiatum. In situ hybridizations for GluR1, GluR2 and PSD-95 likewise display a significant, but weaker dendritic staining that can be detected even in distal parts of apical dendrites of CA1 pyramidal cells (highlighted by arrows). In contrast, in situ hybridization signals for ?-Tubulin mRNA are restricted to the pyramidal cell layer. No obvious difference in mRNA localization between wild type and Fmr1 knockout mice could be detected. (c) Control in situ hybridizations with sense riboprobes produced only low background signals. Exposure times and image processing were identical for each sample.
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Fig. S2. FISH detection of dendritic mRNAs in the dentate gyrus of wild type and Fmr1 knockout mice.
(a and b) Coronal brain slices from wild type (a) and Fmr1 knockout mice (b) were treated as described in Figure S1. Shown are the granule cell layer and molecular layer of the dentate gyrus. CaMKII? mRNA specific signals can be detected throughout the entire length of the dendrites within the molecular layer. In situ hybridizations for GluR1 and GluR2 mRNAs also show considerable dendritic staining that is, however, significantly weaker than the CaMKII? signal. In contrast, strong PSD-95 mRNA specific signals can be detected throughout the entire dendritic layer of the dentate gyrus. Fluorescent in situ hybridization intensities for ?-Tubulin mRNA are robust in the granule cell layer, but can barely be detected in the dendrites. No obvious differences in mRNA localization between wild type and Fmr1 knockout mice were observed. (c) Control in situ hybridizations with sense riboprobes produced only low background signals. Exposure times and image processing were identical for each sample.
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Fig. S3. FISH using oligo probes detects PSD-95 mRNA localization in dendrites of the cerebral cortex, hippocampus and striatum. (b and c) PSD-95 mRNA expression revealed by FISH and confocal microscopy is largely seen in neurons and apical dendrites of the cerebral cortex in coronal sections; their adjacent sections were used for Nissl staining (a). Localization of PSD-95 mRNA in dendrites is indicated by arrows. Note that some dendritic labeling extends over 40?m (arrowheads) in single optical sections. (e) PSD-95 mRNA in the dendrites of hippocampal neurons (arrows). Arrowheads track the length of fluorescence signal into proximal dendrites. This image was taken from an approximate area of the white box in (d) that shows Nissl stain. (g) Another example of PSD-95 mRNA detection in dendrites was observed in striatum (arrows). The white box in (f) represents the area where the image was taken.
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Fig. S4. Oligonucleotide FISH detection of GluR1 mRNA localization in dendrites of brain sections. (a, b) Fluorescence signal for GluR1 mRNA, detected by confocal microscopy, was frequently detected in dendrites from neurons across cortical layers. Note that distinctive labeling of apical dendrites was observed (arrows). In some neurons, the dendritic labeling extended over 40?m (arrowheads in (a)) and dendrites with fluorescence signal could occasionally be tracked for longer distances in these single optical sections (arrowheads in (b)). (c, c' and c'') Z-series of confocal images were taken from an area overlapping with (a). Three optical sections are shown at 1?m apart. Arrows indicate the neuronal cell body where the apical dendrites originate; arrowheads track the extent of distal signal along apical dendrites within each optical section. Neurons were frequently positive for GluR1 mRNA labeling in at least one of serial optical planes.
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Fig. S5. Oligonucleotide FISH detection of tubulin mRNA was restricted to cell bodies. (a) Nissl stain of cerebral cortex in brain section. (b) Fluorescence signal for tubulin mRNA was largely restricted to cell bodies and not detectable in dendrites, as we had observed for PSD95 and GluR1 mRNA. (c) Sense probe showed uniform nonspecific staining across the section.
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Fig. S6. Expression of specific proteins in WT and FMR1 KO synaptoneurosomes. Western blots of protein extracts from cortex synaptoneurosome preparations of WT and Fmr1 KO mice showed an obvious lack of FMRP in the KO (a), but did not show any differences in protein levels for the homologs FXR1P (doublet). There were no differences in levels for PSD-95 (b), CaMKII? (c,d) or NMDAR (c).
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Fig. S7. Polyribosome association of mRNAs from wt and Fmr1 KO synaptoneurosomes upon DHPG stimulation for 5 min.
a) Polysomal incorporation of mRNAs was compared between wild type at basal level (wt) or after stimulation (wt+) with DHPG (50µM for 5 minutes). Upon DHPG stimulation CaMKII? and GluR2 mRNAs had significantly higher polysomal incorporation b) In Fmr1 knockout, stimulation with DHPG induced an opposite effect on polysomal incorporation of synaptoneurosomal mRNAs. There was a significant decrease in the polysomal incorporation of CaMKII?, PDS-95 and GluR1 mRNAs in Fmr1 KO compared to wild type (* indicates P<0.05, ** for P<0.01, *** for P<0.005 n=3, unpaired T-test).
