Figure 1. Genetic knockout of GluN2B from principal cortical neurons in vivo.
(A) Conditional ‘floxed’ GluN2B knockout mice were crossed with NEX-Cre animals to ablate GluN2B from principal cortical neurons in vivo (2BΔCtx). Genotyping strategy and data, generated using tail tissue DNA samples, are shown. (B) Western blots, normalized to actin, and quantification for cortical and brainstem lysates from animals at P10 demonstrate cortex-restricted suppression of GluN2B protein in 2BΔCtx mice (PFC = prefrontal cortex). Control lysates from P0 cortices of global GluN2B KO and WT animals run on the same blots served as positive and negative controls. Western blots and quantification of protein expression in control and 2BΔCtx cortex in vivo from P50–P70 animals shows selective decrease in GluN2B and no change in GluN1 and GluN2A. (C) Example images from NEX-Cre expressing DsRed flox-stop-flox GFP reporter mouse brain slices. Top, coronal section showing restricted cortical expression pattern of NEX-Cre (GFP+ = Cre+). Bottom, parasagittal slice showing strong Cre expression in both dorsal and ventral hippocampus and quantification, v = ventral, d = dorsal, scale bars = 500 µm. (D) Example traces recorded at +50 and −65 mV overlaid show the current response of layer II/III pyramidal neurons in control and 2BΔCtx slices from P18 to P21 animals in response to intracortical stimulation. Traces at +50 mV from control and 2BΔCtx slices demonstrate loss of ifenprodil sensitivity and faster decay kinetics of NMDAR-mediated current in 2BΔCtx neurons. (E) Combined analysis revealed a significant increase in the AMPAR to NMDAR-mediated current ratio in 2BΔCtx neurons, no change in overall peak current at +50 mV, a significant increase in the ifenprodil insensitive current at +50 mV, and a significant decrease in decay tau of the NMDAR-mediate current. Data values are means ± sem. *p < 0.05; **p < 0.01; ***p < 0.001 t test with respect to control; n.c. = no significant change.