Infusion of CA1 neurons with pep-OPHN1Endo had no effect on basal

Infusion of CA1 neurons with pep-OPHN1Endo had no effect on basal synaptic transmission (Figure 6B). These findings indicate that the actions of pep-OPHN1Endo are rapid and corroborate our results obtained with the OPHN1Endo mutant. When pep-OPHN1Hom was included in the pipette, mGluR-LTD and baseline EPSC amplitudes were comparable to those of the control peptide (Figures

6C and 6D). Bafilomycin A1 cell line Of note, the lack of an effect on basal synaptic transmission upon short-term disruption of the OPHN1/Homer 1b/c interaction with pep-OPHN1Hom is consistent with previous findings that prolonged, but not short-term, knockdown of OPHN1 reduces basal synaptic transmission (Nadif Kasri et al., 2009). Together, our data indicate that OPHN1 plays a crucial role in mediating mGluR-LTD, and that OPHN1′s interaction with Endo2/3, but not Homer 1b/c proteins, is critical for this event. Previous studies have shown that activation of group I mGluRs leads to persistent decreases in surface AMPAR expression levels that mediate LTD (Snyder et al., 2001 and Waung et al., 2008). Since the OPHN1-Endo2/3 interaction is critical for mGluR-LTD, we directly tested whether it is important for mGluR-induced changes in surface AMPAR expression www.selleckchem.com/products/INCB18424.html and endocytosis. To quantify

AMPAR surface levels and the degree of AMPAR internalization, we employed a biochemical method to crosslink surface-only AMPAR subunits. Acute slices of hippocampal area CA1 were preincubated with no peptide, pep-contEndo or pep-OPHN1Endo. The CA1 slices were then treated with DHPG or control vehicle (for 10 min), and 50 min later incubated with the membrane-impermeant cross-linking reagent bis (sulfosuccinimidyl) suberate (BS3). Western blotting with an anti-GluR1 antibody revealed a decrease in cell-surface GluR1 expression and an increase in internalized GluR1 levels

1 hr after DHPG treatment in the no peptide and control peptide preincubated CA1 slices (Figures S7A and S7B). The DHPG-induced decrease in cell-surface GluR1 expression and increase in Rolziracetam internal GluR1 levels were, however, significantly attenuated in CA1 slices that were preincubated with pep-OPHN1Endo (Figures S7A and S7B). Of note, the pep-OPHN1Endo peptide did not affect basal levels of surface GluR1 (Figures S7A and S7B). Similar results were obtained for the GluR2 AMPAR subunit (data not shown). To corroborate these findings, we undertook an immunofluorescence approach to measure AMPAR surface levels. Cultured hippocampal neurons, preincubated with no peptide, pep-contEndo or pep-OPHN1Endo, were treated with DHPG or control vehicle (for 10 min), and 1 hr after treatment labeled with an N-terminal directed anti-GluR1 antibody.

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