These experiments help define limits on the role of intrinsic factors in cortical development and establish a role for extrinsic, presumably activity-dependent factors on cortical columnar, laminar, and neuronal morphological development. To examine the role of thalamocortical neurotransmission in cortical development, we generated mice in which glutamatergic release is disrupted in thalamocortical Cytoskeletal Signaling inhibitor neurons using a Cre/loxP recombination approach. We focused on vesicular glutamate transporters, of which there are three known

genetic forms in mice (Vglut1–3). Vglut3 is expressed weakly and sporadically in the brain, while Vglut2 and Vglut1 have strong and largely complementary expression patterns ( Fremeau et al., 2004), with Vglut2

robustly expressed in the thalamus and Vglut1 to a lesser extent. Because Vglut2 null mice die at www.selleckchem.com/products/AZD6244.html birth ( Moechars et al., 2006), we crossed floxed Vglut2 mice (Vglut2fl/fl; Hnasko et al., 2010) with the Sert-Cre driver line ( Zhuang et al., 2005) to delete Vglut2 from thalamocortical projection neurons. Somewhat to our surprise, thalamocortical neurotransmission in these mice was indistinguishable from that in control mice ( Figures 1A–1E). Reasoning that Vglut1 may compensate for the absence of Vglut2 in thalamic neurons, we generated mice that lacked Vglut1 and Vglut2 in the thalamus by crossing Sert-Cre mice with Vglut1+/−;Vglut2fl/fl mice to generate Vglut1 and Vglut2 double knockout mice (Sert-Cre+/−;Vglut1−/−;Vglut2fl/fl, or ThVGdKO). ThVGdKO mice had severely disrupted thalamocortical neurotransmission, whereas all littermate control mice, even those with just a single copy of Vglut1 or Vglut2, had thalamocortical neurotransmission that was grossly indistinguishable from that in wild-type (WT) mice ( Figure 1). almost We measured the effect

of Vglut deletion on thalamocortical neurotransmission in two ways. First, we used in vitro electrophysiological techniques to examine miniature excitatory postsynaptic current (mini-EPSC) amplitude and frequency in thalamocortical brain slices ( Crair and Malenka, 1995) across a range of ages (postnatal days 4–15, P4–P15). Mini-EPSCs were measured using whole-cell patch-clamp recordings from layer 4 (L4) neurons following thalamic stimulation after replacing Ca2+ with Sr2+ in the extracellular medium to desynchronize neurotransmitter release ( Iwasato et al., 2008). In 5 of 11 ThVGdKO mice at P9–P11, we could not evoke a measurable thalamocortical response. In the remaining six ThVGdKO mice at P9–P11 ( Figures 1C–1E), evoked mini-EPSC amplitude (3.9 ± 0.18 pA) and frequency (0.28 ± 0.24 Hz) were much smaller in comparison to littermate controls (p < 0.01). Neither single knockout of Vglut1 (Vglut1−/−;Vglut2fl/−; amplitude: 8.44 ± 1.78 pA; frequency: 8.0 ± 1.18 Hz; n = 6) nor thalamic deletion of Vglut2 (Sert-Cre+/−;Vglut1+/−;Vglut2fl/fl, amplitude: 12.92 ± 0.