We first used AMPA/NMDA ratios to ensure that similar synaptic defects were present in the hippocampus of NexCnih2−/− mice. Because CNIH-2 has no effect on NMDAR-eEPSCs, a change in this ratio should be an accurate reflection of synaptic AMPAR content. AMPA/NMDA ratios were reduced by half in CA1 pyramidal neurons lacking CNIH-2 (Figure 5A). We also observed similar reductions in dentate granule neurons and layer 2/3 pyramidal neurons in barrel cortex (Figure 5A). Interestingly, no change in the ratio was found in the heterozygous (NexCnih2+/−) mice (Figure 5A) despite a 30–50% reduction in total CNIH-2 expression (Figure S6A), thus providing further evidence that CNIH-2 is expressed in excess in CA1 pyramidal neurons and that all available CNIH-2 binding sites on AMPARs are occupied or “saturated”. In paired recordings from slice cultures from NexCnih2−/− mice, transfection of CA1 pyramidal neurons with CNIH-2 fully rescued AMPAR-eEPSCs (Figure 5B). No change in the NMDAR-eEPSC was observed (Figure 5C). As previously shown, CNIH-2 overexpression in wild-type neurons has no effect on AMPAR- or NMDAR-eEPSCs (Figures S6B–C) (Shi et al., 2010) again indicating saturation of CNIH binding sites on native AMPARs.
