These authors contributed equally to this study.
Dopamine D1 receptor-mediated enhancement of NMDA receptor trafficking requires rapid PKC-dependent synaptic insertion in the prefrontal neurons
Version of Record online: 31 MAR 2010
© 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry
Journal of Neurochemistry
Volume 114, Issue 1, pages 62–73, July 2010
How to Cite
Li, Y.-C., Liu, G., Hu, J.-L., Gao, W.-J. and Huang, Y.-Q. (2010), Dopamine D1 receptor-mediated enhancement of NMDA receptor trafficking requires rapid PKC-dependent synaptic insertion in the prefrontal neurons. Journal of Neurochemistry, 114: 62–73. doi: 10.1111/j.1471-4159.2010.06720.x
- Issue online: 8 JUN 2010
- Version of Record online: 31 MAR 2010
- Received January 20, 2010; revised manuscript received March 23, 2010; accepted March 24, 2010.
Vol. 124, Issue 6, 894, Version of Record online: 3 MAR 2013
Appendix S1. Supplementary Materials and Methods.
Figure S1. Control experiments for recording of NMDA mEPSCs under zero- Mg2+ condition. In zero-Mg2+ extracellular solution, the Mg2+ blockade of NMDA receptor channels was gradually relieved. Therefore, it is possible that the potentiation of mEPSCs was induced not by D1 activation but by the gradual release of Mg2+ block for NMDA receptor channels when cortical slices were incubated in zero-Mg2+ solution. To exclude this possibility, we analyzed the changes of mEPSCs for 15 min without application of SKF-81297. Under this condition, both frequency and amplitude of mEPSCs appeared to be stable, without significance (n = 4; p > 0.05 for both frequency and amplitude), suggesting the reliability of the recording and SKF-81297 effects.
Figure S2. Non-specificity because of secondary antibodies is negligible in immunostaining processes. (a–c) Mouse anti-NR2B antibody was included whereas anti-PSD95 was omitted in the primary antibody incubation; FITC conjugated goat-antimouse and Cy3-conjugated goat-anti-rabbit antibodies were included in the subsequent incubations. Specific staining of NR2B (a) and negligible non-specific staining from Cy3-conjugated goat-antirabbit antibody (b) were distinguished. In panels (d–f) however, only rabbit anti-PSD95 was included in the primary incubation whereas NR2B was omitted. Under this condition, the non-specific staining derived from FITC-conjugated goat anti-mouse antibody was minimal whereas the staining of PSD95 was strong. Scale bar = 10 μm.
Figure S3. The AMPA receptor-mediated currents were not affected by D1 agonist. Sample traces (a) and summary histograph (b) showing that neither frequency nor amplitude of mEPSCs recorded under −60 mV was changed by co-application SKF-81297 (1 μM) with APV (50 μM), TTX (1 μM), and picrotoxin (50 μM), indicating an unaltered pre-synaptic glutamate release (n = 6, p > 0.05).
Figure S4. D1 activation increases NMDA receptor NR1 clusters but not PSD95 in total staining. (a–i) Neurons treated with dimethyl sulfoxide (DMSO, 0.1%) as vehicle control (a, d, and g), D1 agonist SKF-81297 (b, e, and h), or SKF-81297 + D1 antagonist SCH-23390 (c, f, and i) were immunostained with NR1 and PSD95 antibodies. Scale bar = 10 μm. (j) Quantitative analysis of D1 effect on NR1 clusters and fluorescence intensity. Neurons treated with SKF81297 had significantly higher number of NR1 puncta and significantly greater fluorescence intensity (**p < 0.01 for both cluster and fluorescence intensity). Neurons treated with SKF81297 + SCH23390, however, exhibited no significant change in either puncta number or fluorescent intensity compared with the vehicle-treated controls (p > 0.05). (k) Quantitative analysis shows that D1 activation did not change PSD95 total staining, neither on PSD95 clusters nor fluorescence intensity (p > 0.05).
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