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Different Rho GTPase-dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation.

Rex CS, Chen LY, Sharma A, Liu J, Babayan AH, Gall CM, Lynch G - J. Cell Biol. (2009)

Bottom Line: These experiments used this observation to uncover the synaptic processes that stabilize the potentiation effect.A search for the upstream origins of these effects showed that adenosine suppressed RhoA activity but only modestly affected Rac and Cdc42.A RhoA kinase (ROCK) inhibitor reproduced adenosine's effects on cofilin phosphorylation, spine actin polymerization, and LTP, whereas a Rac inhibitor did not.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Human Behavior, University of California, Irvine, CA 92697, USA. crex@uci.edu

ABSTRACT
The releasable factor adenosine blocks the formation of long-term potentiation (LTP). These experiments used this observation to uncover the synaptic processes that stabilize the potentiation effect. Brief adenosine infusion blocked stimulation-induced actin polymerization within dendritic spines along with LTP itself in control rat hippocampal slices but not in those pretreated with the actin filament stabilizer jasplakinolide. Adenosine also blocked activity-driven phosphorylation of synaptic cofilin but not of synaptic p21-activated kinase (PAK). A search for the upstream origins of these effects showed that adenosine suppressed RhoA activity but only modestly affected Rac and Cdc42. A RhoA kinase (ROCK) inhibitor reproduced adenosine's effects on cofilin phosphorylation, spine actin polymerization, and LTP, whereas a Rac inhibitor did not. However, inhibitors of Rac or PAK did prolong LTP's vulnerability to reversal by latrunculin, a toxin which blocks actin filament assembly. Thus, LTP induction initiates two synaptic signaling cascades: one (RhoA-ROCK-cofilin) leads to actin polymerization, whereas the other (Rac-PAK) stabilizes the newly formed filaments.

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Adenosine blocks phosphorylation of the F-actin–severing protein cofilin. Hippocampal slices receiving TBS or control stimulation to the Schaffer collaterals were processed for double pCofilin (pCof) and PSD95 immunofluorescence. 0.2 mM adenosine (Ado) or vehicle (veh) was locally applied for 4 min beginning 30 s after TBS. (A) The plot shows mean numbers (±SEM) of pCofilin/PSD95 double-labeled puncta in the zone of physiological recording (*, P < 0.05; **, P < 0.01 vs. vehicle; #, P < 0.05; ##, P < 0.01 vs. adenosine alone). (B) Results from A expressed as difference between treatment and TBS + treatment effects (TBSΔ; mean ± SEM; **, P < 0.01 vs. vehicle; #, P < 0.05 vs. adenosine). (C) Western blots from tissue treated with adenosine or vehicle for 5 min; plot of group mean (±SEM) band ODs shows that adenosine decreased pCofilin levels (*, P < 0.05). (D) Low frequency stimulation (LFS) blocked TBS-induced increases in numbers of pCofilin+ PSDs (mean ± SEM; *, P < 0.05 vs. control; #, P < 0.05 vs. TBS).
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fig3: Adenosine blocks phosphorylation of the F-actin–severing protein cofilin. Hippocampal slices receiving TBS or control stimulation to the Schaffer collaterals were processed for double pCofilin (pCof) and PSD95 immunofluorescence. 0.2 mM adenosine (Ado) or vehicle (veh) was locally applied for 4 min beginning 30 s after TBS. (A) The plot shows mean numbers (±SEM) of pCofilin/PSD95 double-labeled puncta in the zone of physiological recording (*, P < 0.05; **, P < 0.01 vs. vehicle; #, P < 0.05; ##, P < 0.01 vs. adenosine alone). (B) Results from A expressed as difference between treatment and TBS + treatment effects (TBSΔ; mean ± SEM; **, P < 0.01 vs. vehicle; #, P < 0.05 vs. adenosine). (C) Western blots from tissue treated with adenosine or vehicle for 5 min; plot of group mean (±SEM) band ODs shows that adenosine decreased pCofilin levels (*, P < 0.05). (D) Low frequency stimulation (LFS) blocked TBS-induced increases in numbers of pCofilin+ PSDs (mean ± SEM; *, P < 0.05 vs. control; #, P < 0.05 vs. TBS).

Mentions: We tested whether the TBS-induced pCofilin effect is modified by adenosine. Brief (4 min) adenosine infusions to control slices reduced the number of pCofilin+ synapses in CA1 str. radiatum by ∼30%, and this effect was completely blocked by the A1R antagonist DPCPX (Fig. 3 A). Moreover, adenosine applied at 30 s after TBS profoundly reduced the increase in pCofilin+ synapses normally found after TBS (72 ± 15% vs. 273 ± 39% of control for adenosine vs. vehicle; n = 11 and 14; P < 0.0001). The potent block of activity-driven cofilin phosphorylation by adenosine was eliminated by DPCPX (Fig. 3 A) but was unaffected by 10 µM of the adenosine A2a receptor antagonist MSX3 (117 ± 24% of control + adenosine; P = 0.52). Because adenosine depressed basal pCofilin levels, we recalculated the effects of TBS as the percent change from the appropriate (adenosine treated or untreated) control group mean (Fig. 3 B). By this measure, TBS increased the number of pCofilin+ PSDs by 173 ± 39% in untreated slices but only by 44 ± 16% in slices treated with adenosine (P = 0.001). The full effect of TBS was restored in adenosine-treated slices by preincubation with DPCPX (156 ± 64% of adenosine + DPCPX–treated control slices). Western blot analyses confirmed that adenosine infusion reduced basal pCofilin levels in control slices (vehicle vs. adenosine: 39.8 ± 4.2 vs. 26.3 ± 3.0 × 103 OD units; n = 19; P = 0.01; Fig. 3 C). Similar results were obtained when normalized to β-actin.


Different Rho GTPase-dependent signaling pathways initiate sequential steps in the consolidation of long-term potentiation.

Rex CS, Chen LY, Sharma A, Liu J, Babayan AH, Gall CM, Lynch G - J. Cell Biol. (2009)

Adenosine blocks phosphorylation of the F-actin–severing protein cofilin. Hippocampal slices receiving TBS or control stimulation to the Schaffer collaterals were processed for double pCofilin (pCof) and PSD95 immunofluorescence. 0.2 mM adenosine (Ado) or vehicle (veh) was locally applied for 4 min beginning 30 s after TBS. (A) The plot shows mean numbers (±SEM) of pCofilin/PSD95 double-labeled puncta in the zone of physiological recording (*, P < 0.05; **, P < 0.01 vs. vehicle; #, P < 0.05; ##, P < 0.01 vs. adenosine alone). (B) Results from A expressed as difference between treatment and TBS + treatment effects (TBSΔ; mean ± SEM; **, P < 0.01 vs. vehicle; #, P < 0.05 vs. adenosine). (C) Western blots from tissue treated with adenosine or vehicle for 5 min; plot of group mean (±SEM) band ODs shows that adenosine decreased pCofilin levels (*, P < 0.05). (D) Low frequency stimulation (LFS) blocked TBS-induced increases in numbers of pCofilin+ PSDs (mean ± SEM; *, P < 0.05 vs. control; #, P < 0.05 vs. TBS).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig3: Adenosine blocks phosphorylation of the F-actin–severing protein cofilin. Hippocampal slices receiving TBS or control stimulation to the Schaffer collaterals were processed for double pCofilin (pCof) and PSD95 immunofluorescence. 0.2 mM adenosine (Ado) or vehicle (veh) was locally applied for 4 min beginning 30 s after TBS. (A) The plot shows mean numbers (±SEM) of pCofilin/PSD95 double-labeled puncta in the zone of physiological recording (*, P < 0.05; **, P < 0.01 vs. vehicle; #, P < 0.05; ##, P < 0.01 vs. adenosine alone). (B) Results from A expressed as difference between treatment and TBS + treatment effects (TBSΔ; mean ± SEM; **, P < 0.01 vs. vehicle; #, P < 0.05 vs. adenosine). (C) Western blots from tissue treated with adenosine or vehicle for 5 min; plot of group mean (±SEM) band ODs shows that adenosine decreased pCofilin levels (*, P < 0.05). (D) Low frequency stimulation (LFS) blocked TBS-induced increases in numbers of pCofilin+ PSDs (mean ± SEM; *, P < 0.05 vs. control; #, P < 0.05 vs. TBS).
Mentions: We tested whether the TBS-induced pCofilin effect is modified by adenosine. Brief (4 min) adenosine infusions to control slices reduced the number of pCofilin+ synapses in CA1 str. radiatum by ∼30%, and this effect was completely blocked by the A1R antagonist DPCPX (Fig. 3 A). Moreover, adenosine applied at 30 s after TBS profoundly reduced the increase in pCofilin+ synapses normally found after TBS (72 ± 15% vs. 273 ± 39% of control for adenosine vs. vehicle; n = 11 and 14; P < 0.0001). The potent block of activity-driven cofilin phosphorylation by adenosine was eliminated by DPCPX (Fig. 3 A) but was unaffected by 10 µM of the adenosine A2a receptor antagonist MSX3 (117 ± 24% of control + adenosine; P = 0.52). Because adenosine depressed basal pCofilin levels, we recalculated the effects of TBS as the percent change from the appropriate (adenosine treated or untreated) control group mean (Fig. 3 B). By this measure, TBS increased the number of pCofilin+ PSDs by 173 ± 39% in untreated slices but only by 44 ± 16% in slices treated with adenosine (P = 0.001). The full effect of TBS was restored in adenosine-treated slices by preincubation with DPCPX (156 ± 64% of adenosine + DPCPX–treated control slices). Western blot analyses confirmed that adenosine infusion reduced basal pCofilin levels in control slices (vehicle vs. adenosine: 39.8 ± 4.2 vs. 26.3 ± 3.0 × 103 OD units; n = 19; P = 0.01; Fig. 3 C). Similar results were obtained when normalized to β-actin.

Bottom Line: These experiments used this observation to uncover the synaptic processes that stabilize the potentiation effect.A search for the upstream origins of these effects showed that adenosine suppressed RhoA activity but only modestly affected Rac and Cdc42.A RhoA kinase (ROCK) inhibitor reproduced adenosine's effects on cofilin phosphorylation, spine actin polymerization, and LTP, whereas a Rac inhibitor did not.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Human Behavior, University of California, Irvine, CA 92697, USA. crex@uci.edu

ABSTRACT
The releasable factor adenosine blocks the formation of long-term potentiation (LTP). These experiments used this observation to uncover the synaptic processes that stabilize the potentiation effect. Brief adenosine infusion blocked stimulation-induced actin polymerization within dendritic spines along with LTP itself in control rat hippocampal slices but not in those pretreated with the actin filament stabilizer jasplakinolide. Adenosine also blocked activity-driven phosphorylation of synaptic cofilin but not of synaptic p21-activated kinase (PAK). A search for the upstream origins of these effects showed that adenosine suppressed RhoA activity but only modestly affected Rac and Cdc42. A RhoA kinase (ROCK) inhibitor reproduced adenosine's effects on cofilin phosphorylation, spine actin polymerization, and LTP, whereas a Rac inhibitor did not. However, inhibitors of Rac or PAK did prolong LTP's vulnerability to reversal by latrunculin, a toxin which blocks actin filament assembly. Thus, LTP induction initiates two synaptic signaling cascades: one (RhoA-ROCK-cofilin) leads to actin polymerization, whereas the other (Rac-PAK) stabilizes the newly formed filaments.

Show MeSH
Related in: MedlinePlus