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- supplemental material - Figure S1. Labeling of the intracellular protein PICK1 is detected in permeabilized, but not intact neurons Labeling of a nonpermeabilized (b) and permeabilized (d) neuron with an anti-PICK1 antibody. (a,c) Brightfield images of the neurons illustrated in b and d, respectively. These findings indicate that that the neuron is indeed nonpermeabilized under conditions used for labeling of surface AMPARs. Scale bar, 20 �m.
- supplemental material - Figure S2. GluR1 and GluR2 mRNA fluorescence are not saturated in unadjusted (�raw�) images (a,b) Enlarged view of the cell bodies of neurons shown in Figure 1b,e, respectively. Images show GluR2 (a) and GluR1 (b) mRNA signal in cell soma without adjustments for brightness or contrast (�raw images�). GluR2 mRNA appears as densely packed granules or �packets� implicated in RNA transport in neurons.
- supplemental material - Figure S3. GluR2 mRNA is not detectable in axons of hippocampal neurons or astrocytes (a-f) Neurofilament-H (NF-H) protein (a) and GluR2 mRNA (b) exhibit little or no overlap within axons of hippocampal neurons (merge, c). GluR2 mRNA clusters localize to dendritic shafts and spine-like protrusions (b, high magnification). The axon filament marked by NF-H appears to wrap around the dendritic shaft (d,f). Representative images from 3 independent experiments (n = 12). Scale bars, a-c, 20 �m; d-f, 2 �m. (g-i) GFAP protein (g) and GluR2 mRNA (h) exhibit little or no overlap in cultured cortical astrocytes (merge, i). Some nonspecific GluR2 mRNA labeling is apparent at low levels in the nuclei. Representative images from 3 independent experiments (n = 12). Scale bar, g-i, 30 �m.
- supplemental material - Figure S4. GluR2 mRNA localizes dendrites of hippocampal neurons, assessed by alkaline phosphatase (a,b) Localization of GluR2 mRNA in hippocampal neurons visualized by non-isotopic in situ hybridization with alkaline phosphatase labeling. GluR2 mRNA hybridization signal was present in the soma and throughout dendritic processes. In processes, GluR2 mRNA was organized as large clusters or granules. (c,d) Hybridization with a randomized GluR2 oligonucleotide probe showed little or no labeling in the soma or neurites. (e,f) Hybridization with an oligonucleotide probe directed to the Lac Z mRNA showed or no labeling in the soma and neuronal processes. (b,d,f) Enlarged view of the boxed segments in a,c,e. Scale bars, a,c,e, 20 �m; b,d,f, 10 �m.
- supplemental material - Figure S5. NMDA treatment does not compromise neuronal viability NMDA treatment (50 �M; 60 sec) did not significantly compromise neuronal viability of cultured cortical neurons. a,c: phase-contrast images of cortical neurons in culture. b,d: DAPI labeling of neurons shown in a,c. Scale bar, 50 �m.
- supplemental material - Figure S6. Scheme of bidirectional regulation of GluR2 mRNA trafficking in neuronal dendrites See text for details.