Figure 6.

Levels of GluN2B subunit-containing NMDA receptors are increased in juvenile Cst-1 KO mice. A, Representative averaged traces of evoked AMPA and NMDA receptor-mediated responses recorded in the same CA1 pyramidal cell at −70 mV (inward currents) and at +30 mV (outward currents) from a WT and Cst-1 KO mouse. Note that traces are aligned horizontally. Histograms for the ratio of the AMPA to NMDA receptor-mediated response. To minimize the contribution from AMPA receptors, the NMDA receptor-mediated current was measured 80 ms after stimulation. B, Scaling of traces reveals a slowing in the decay kinetics of the NMDA but not the AMPA receptor-mediated responses in Cst-1 KO mice. Histograms show pooled data. C, Pharmacologically isolated NMDA EPSCs exposed to NMDA receptor subunit-specific antagonists shows control current, current remaining after application of an GluN2B subunit antagonist (Ro25–6981; 3 μm) and residual current in the presence of antagonists for both GluN2A (NVP-AAM; 0.1 μm) and GluN2B (Ro25–6981) subunits. Histograms show pooled data. D, Protein levels for GluN2B and for GluN2A quantified by determining the ratio of GluN2B/GAPDH and GluN2A/GAPDH in hippocampal tissue extracts. E, Quantification shows a significant increase in GluN2B but not GluN2A. F, Western blots of recycling endosomes, identified by Rab11 co-IP, from whole brains of WT juvenile mice (n = 3). Note the strong co-IP for NMDA receptors in the R140-enriched fraction, a marker for calsyntenin-1-positive recycling endosomes. Recycling endosomes are also positive for syntaxin-13 (Stx13), a general marker for endosomes.