Journal: The Journal of Cell Biology

Article Title: Different Rho GTPase–dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation

doi: 10.1083/jcb.200901084

Figure Lengend Snippet: Activity-induced PAK phosphorylation is not blocked by adenosine. (A) Deconvolution images show total PAK3 ir (left) and the colocalization of pPAK1/2/3 and PSD95 (right panels) in proximal CA1 str. radiatum. The arrows indicate a double-labeled element. (B) Quantification (mean ± SEM) of pPAK + PSDs shows that adenosine (Ado) significantly increased the numbers of pPAK + PSDs ( n = 7–11; *, P < 0.05 vs. control). TBS followed by adenosine 30 s later increased pPAK + PSDs above values in adenosine alone slices ( n = 11; ***, P < 0.001 vs. control; ##, P < 0.01 vs. adenosine). (C) Results from B presented as the difference between TBS + treatment and treatment alone group values (TBSΔ; mean ± SEM). (D) Western blots from slices treated with adenosine or vehicle (veh) for 5 min (same slices as in ). (E) Plots show raw band ODs (left) or the same measure normalized to β-actin (right; means ± SEM; *, P = 0.02). The latter measure exposed an adenosine-induced increase in levels of pPAK. Bars: (A [left]) 10 µm; (A [right]) 5 µm.

Article Snippet: Fixed slices were sectioned (20 µm), slide mounted, and processed using a primary antisera cocktail of mouse anti-PSD95 (Thermo Fisher Scientific) with rabbit antisera to pPAK1/2/3 S141 (Invitrogen), pCofilin S3 (Abcam), PAK3 (Millipore), or cofilin (Cytoskeleton, Inc.) and secondary antisera, including Alexa Fluor 488 anti–mouse and Alexa Fluor 594 anti–rabbit (Invitrogen; ).

Techniques: Activity Assay, Labeling, Western Blot

Journal: The Journal of Cell Biology

Article Title: Different Rho GTPase–dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation

doi: 10.1083/jcb.200901084

Figure Lengend Snippet: Inhibition of Rac-PAK signaling extends the period over which actin filament assembly is required for LTP. Stimulation was applied to Schaffer collateral afferents to hippocampal field CA1b. (A) Slices labeled for F-actin after 4-min local infusion of 0.2 µM Lat A. (left) Images show that Lat A blocked increases in phalloidin-labeled spines when applied 30 s but not 10 min after TBS. (middle) The plot shows F-actin–labeled spine counts (mean ± SEM) from slices treated with vehicle (veh) at 30 s after TBS or with Lat A initiated at the time points indicated (*, P < 0.05 vs. control). (right) The plot shows mean LTP magnitude (percent fEPSP slope measured 80–90 min after TBS vs. baseline) from experiments in which slices were treated locally with Lat A or vehicle for 4 min beginning at the time points indicated (**, P < 0.01 vs. respective control pathways). Measures for untreated, unstimulated (control [con]) slices are also shown. The dashed line indicates baseline level. (B) Prolonged Lat A bath infusion (open horizontal line), timed to hit slices at 10 min after TBS (arrow), did not disrupt LTP when applied alone (with vehicle) but blocked the stable maintenance of LTP in the presence of the Rac inhibitor NSC23766 (closed horizontal bar). A second control pathway (triangles) not receiving TBS was unaffected by drug manipulations. (top) Representative fEPSP traces collected at time points indicated in the plot. (C) Western blots from slices incubated with 2 µM of the Group I PAK–specific inhibitor IPA-3 for 1 h and probed for pPAK S141 or pPAK T423 . β-Actin bands shown were generated from the stripped PAK T423 blot. Plots show group mean (±SEM) band densities as a fraction of vehicle controls (*, P < 0.05 vs. vehicle). (D) Slices incubated for 50 min with 2 µM IPA-3 (closed horizontal bar) before TBS (arrow; TBS pathway shown as closed circles) exhibited stable LTP of typical magnitude (dashed line), but Lat A washed in at 30 min after TBS caused potentiation to decay to baseline levels within 2 h. The unstimulated control pathway (open triangles) was unaffected by drug manipulations. (top) Representative fEPSP traces from the time points indicated. (B and D) Vertical bar, 1 mV; horizontal bar, 10 ms. Bar, 5 µm.

Article Snippet: Fixed slices were sectioned (20 µm), slide mounted, and processed using a primary antisera cocktail of mouse anti-PSD95 (Thermo Fisher Scientific) with rabbit antisera to pPAK1/2/3 S141 (Invitrogen), pCofilin S3 (Abcam), PAK3 (Millipore), or cofilin (Cytoskeleton, Inc.) and secondary antisera, including Alexa Fluor 488 anti–mouse and Alexa Fluor 594 anti–rabbit (Invitrogen; ).

Techniques: Inhibition, Labeling, Western Blot, Incubation, Generated

Journal: The Journal of Cell Biology

Article Title: Different Rho GTPase–dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation

doi: 10.1083/jcb.200901084

Figure Lengend Snippet: Activity-induced PAK phosphorylation is not blocked by adenosine. (A) Deconvolution images show total PAK3 ir (left) and the colocalization of pPAK1/2/3 and PSD95 (right panels) in proximal CA1 str. radiatum. The arrows indicate a double-labeled element. (B) Quantification (mean ± SEM) of pPAK + PSDs shows that adenosine (Ado) significantly increased the numbers of pPAK + PSDs ( n = 7–11; *, P < 0.05 vs. control). TBS followed by adenosine 30 s later increased pPAK + PSDs above values in adenosine alone slices ( n = 11; ***, P < 0.001 vs. control; ##, P < 0.01 vs. adenosine). (C) Results from B presented as the difference between TBS + treatment and treatment alone group values (TBSΔ; mean ± SEM). (D) Western blots from slices treated with adenosine or vehicle (veh) for 5 min (same slices as in ). (E) Plots show raw band ODs (left) or the same measure normalized to β-actin (right; means ± SEM; *, P = 0.02). The latter measure exposed an adenosine-induced increase in levels of pPAK. Bars: (A [left]) 10 µm; (A [right]) 5 µm.

Article Snippet: Samples were normalized by Bio-Rad protein assay and processed for Western blot analysis (12% SDS-PAGE) using rabbit antisera to pCofilin S3 , pPAK1/2/3 S141 (see Immunocytochemistry), or pPAK1/2/3 T423 (Cell Signaling Technology) and the ECL Plus detection system (GE Healthcare).

Techniques: Activity Assay, Labeling, Western Blot

Journal: The Journal of Cell Biology

Article Title: Different Rho GTPase–dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation

doi: 10.1083/jcb.200901084

Figure Lengend Snippet: Inhibition of Rac-PAK signaling extends the period over which actin filament assembly is required for LTP. Stimulation was applied to Schaffer collateral afferents to hippocampal field CA1b. (A) Slices labeled for F-actin after 4-min local infusion of 0.2 µM Lat A. (left) Images show that Lat A blocked increases in phalloidin-labeled spines when applied 30 s but not 10 min after TBS. (middle) The plot shows F-actin–labeled spine counts (mean ± SEM) from slices treated with vehicle (veh) at 30 s after TBS or with Lat A initiated at the time points indicated (*, P < 0.05 vs. control). (right) The plot shows mean LTP magnitude (percent fEPSP slope measured 80–90 min after TBS vs. baseline) from experiments in which slices were treated locally with Lat A or vehicle for 4 min beginning at the time points indicated (**, P < 0.01 vs. respective control pathways). Measures for untreated, unstimulated (control [con]) slices are also shown. The dashed line indicates baseline level. (B) Prolonged Lat A bath infusion (open horizontal line), timed to hit slices at 10 min after TBS (arrow), did not disrupt LTP when applied alone (with vehicle) but blocked the stable maintenance of LTP in the presence of the Rac inhibitor NSC23766 (closed horizontal bar). A second control pathway (triangles) not receiving TBS was unaffected by drug manipulations. (top) Representative fEPSP traces collected at time points indicated in the plot. (C) Western blots from slices incubated with 2 µM of the Group I PAK–specific inhibitor IPA-3 for 1 h and probed for pPAK S141 or pPAK T423 . β-Actin bands shown were generated from the stripped PAK T423 blot. Plots show group mean (±SEM) band densities as a fraction of vehicle controls (*, P < 0.05 vs. vehicle). (D) Slices incubated for 50 min with 2 µM IPA-3 (closed horizontal bar) before TBS (arrow; TBS pathway shown as closed circles) exhibited stable LTP of typical magnitude (dashed line), but Lat A washed in at 30 min after TBS caused potentiation to decay to baseline levels within 2 h. The unstimulated control pathway (open triangles) was unaffected by drug manipulations. (top) Representative fEPSP traces from the time points indicated. (B and D) Vertical bar, 1 mV; horizontal bar, 10 ms. Bar, 5 µm.

Article Snippet: Samples were normalized by Bio-Rad protein assay and processed for Western blot analysis (12% SDS-PAGE) using rabbit antisera to pCofilin S3 , pPAK1/2/3 S141 (see Immunocytochemistry), or pPAK1/2/3 T423 (Cell Signaling Technology) and the ECL Plus detection system (GE Healthcare).

Techniques: Inhibition, Labeling, Western Blot, Incubation, Generated