Responses evoked by localized NMDA application in vitro are the result of activation of both synaptic and extrasynaptic receptors. We next carried out experiments in acute slices to confirm the presence of synaptic NMDAR-mediated

currents ex vivo. GluN2A-containing NMDARs have been shown to traffic to autaptic synapses in GluN2B null hippocampal cultures and in cultured cortical slices following knockdown of GluN2B (Tovar et al., 2000 and Barria and Malinow, 2002). However, their ability to traffic to GluN2B null synapses in vivo had not been tested. We recorded whole-cell currents from layer II/III cortical neurons in acute brain slices in response to presynaptic stimulation in layer IV of 2B→2A animals (P12–P16) (Figure 3). Voltage clamping at depolarized potentials to remove the Mg2+ block of NMDARs and electrically http://www.selleckchem.com/GSK-3.html stimulating afferent axons allowed us to record APV-sensitive currents in 2B→2A cortex (Figures 3A and 3B). The peak amplitude of the synaptic NMDAR-mediated current at +50mV was not significantly different in 2B→2A mice compared

AZD8055 order to controls (Figure 3B). Consistent with a pure population of GluN2A-containing receptors, these recordings revealed a lack of sensitivity to the GluN2B selective antagonist ifenprodil (Figure 3C) and to NMDAR-mediated currents that exhibited significantly faster decay times (decreased tau) (Figure 3D) (Vicini et al., 1998). Faster decay resulted in a slight, but statistically significant, decrease in integrated current measured at +50mV over the initial 200 ms

of the evoked response, but not over the initial 100 ms of the response (Figure 3D). These and data show that our genetic strategy was successful in removing GluN2B and driving precocious expression and synaptic incorporation of GluN2A-mediated NMDAR-receptors while recovering a significant amount of synaptic NMDAR-mediated current at cortical synapses in vivo. GluN2B and GluN2A interact with, and activate, distinct signaling cascades at excitatory synapses in order to control the composition and strength of synaptic contacts. The dominant way by which NMDARs regulate synaptic strength is through bidirectional trafficking of AMPARs. In light of this, we examined AMPAR-mediated currents in 2B→2A mice. For these experiments, cortical neuron cultures were prepared from both homozygous GluN2B knockout and 2B→2A embryos, as well as from WT littermate embryos. We isolated AMPAR-mediated miniature excitatory postsynaptic currents (mEPSCs) between 11 and 15 days in vitro (DIV) using 0.5 μM tetrodotoxin (TTX) + 50 μM picrotoxin.