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PNAS 99 (1): 467-472

Copyright © 2002 by the National Academy of Sciences.

A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus

Shao Jun Tang*, Gerald Reis*, Hyejin Kang*, Anne-Claude Gingras{dagger}, Nahum Sonenberg{dagger}, and Erin M. Schuman*,{ddagger}

*California Institute of Technology, Howard Hughes Medical Institute, Division of Biology 216-76, Pasadena, CA 91125; and {dagger}Department of Biochemistry, McGill University, Montreal, QC, Canada H3G 1Y6


Figure 1
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Figure 1 Western blot analysis of eIF-4E, 4E-BP1, 4E-BP2, and mTOR proteins in hippocampal lysates. Total lysates of the rat adult hippocampus were used for Western blot analysis, with antibodies specific for eIF-4E, 4E-BP1, 4E-BP2, and mTOR (see Material and Methods for details). The size of protein markers is labeled on the left of each blot. Note the doublet bands detected by anti-eIF-4E, 4E-BP1, and 4E-BP2 (arrowheads).

 

Figure 2
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Figure 2 Spatial distribution of eIF-4E, 4E-BP1, 4E-BP2, and mTOR in hippocampal slices. Shown are confocal images of hippocampal slices stained with primary antibodies against purified rabbit IgG (control staining), eIF4-E, 4E-BP1, 4E-BP2, or mTOR proteins, and FITC-conjugated secondary antibodies. Note the staining signals for eIF-4E, 4E-BP1, 4E-BP2, and mTOR in cell bodies and dendrites. Because mTOR is mainly a membrane protein, little signal for this protein is detected in the cytoplasm of the cell body (asterisk). c, cell body; d, dendrites; s, stratum radiatum; m, molecular layer.

 

Figure 3
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Figure 3 Synaptic localization of eIF-4E, 4E-BP1, 4E-BP2, and mTOR. (A) Dissociated hippocampal neurons (P2; 14 DIV) were double-stained with components of the mTOR pathway (first column) including anti-eIF-4E (a), 4E-BP1 (d), 4E-BP2 (g), mTOR (j), or PSD-95 (m) antibodies and anti-synapsin I antibodies (second column) (b, e, h, and k). Signals from eIF-4E, 4E-BP1, 4E-BP2, mTOR, and PSD-95, and signals from the corresponding anti-synapsin staining on the same cells were overlaid in c, f, i, l, and o, respectively. In the overlaid images, green signals were generated by anti-eIF-4E, 4E-BP1, 4E-BP2, mTOR, or PSD-95 staining, red signals were generated by anti-synapsin staining, and yellow signals resulted from the overlapping of green and red signals. Insets in c, f, i, l, and o are the higher magnification images for the dendritic regions marked by boxes. (B) Double-labeling of eIF-4E (a), 4E-BP1 (d), mTOR (g) with PSD-95 (b, e, h). Signals from eIF-4E, 4E-BP1, and mTOR and signals from the corresponding PSD-95 staining on the same cells were overlaid in c, f, l, and i, respectively. Insets in c, f, and i are the higher magnification images for the dendritic regions marked by boxes (green signals resulted from eIF-4E, 4E-BP1, or mTOR staining; red signals from PSD-95 staining; yellow signals from the overlapping of green and red signals). Arrows indicate the synaptic regions that are double-stained. (Bar = 50 µm.)

 

Figure 4
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Figure 4 Rapamycin decreases the magnitude of late-phase LTP in hippocampal slices. Shown are ensemble averages for all DMSO, FK506, and rapamycin experiments (n = 6, 9, and 8, respectively). All drugs were applied in the ACSF at least 30 min before the first tetanus. Mean DMSO control fEPSP slope values were 0.20 ± 0.01 mV/msec before LTP induction, and 0.35 ± 0.03 mV/msec 220–240 min after LTP induction. Mean FK506-treated fEPSP slope values were 0.21 ± 0.02 mV/msec before LTP induction, and 0.30 ± 0.09 mV/msec 220–240 min after LTP induction. Mean rapamycin-treated fEPSP slope values were 0.24 ± 0.05 mV/msec before LTP induction, and 0.31 ± 0.07 mV/msec 220–240 min after LTP induction. The control pathways for FK506 and rapamycin experiments are symbolized by the filled inverted triangle and the open circles, respectively.

 

Figure 5
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Figure 5 Rapamycin prevents BDNF-induced synaptic potentiation in hippocampal slices. Shown are ensemble averages for all control and rapamycin experiments. BDNF (50 ng/ml) was applied in the ACSF at the time indicated by the bar. Mean control fEPSP slope values were 0.13 ± 0.03 mV/msec before BDNF application and 0.23 ± 0.04 mV/msec after BDNF application. Mean rapamycin-treated fEPSP slope values were 0.16 ± 0.01 mV/msec before BDNF application and 0.18 ± 0.01 mV/msec after BDNF application.

 


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