24 ± 1.51 Hz; n =

5) significantly reduced thalamocortical neurotransmission in comparison to WT mice (amplitude: 11.84 ± 0.84 pA; frequency: 10.47 ± 2.14 Hz; n = 3). We did not detect any thalamocortical synaptic response at P4–P6 in ThVGdKO mice (n = 4) and detected only very weak response in some slices at P13–P15 that was similar in amplitude and frequency to that observed at P9–P11 and much smaller than that observed in control littermates (p < 0.001; Figures 1F and 1G). These results indicate that Vglut1 and Vglut2 can both contribute to glutamatergic neurotransmission at thalamocortical synapses, and elimination of both Vglut1 and Vglut2 in ThVGdKO mice nearly completely abolishes thalamocortical neurotransmission. We confirmed these results using in vivo electrophysiological techniques in P9–P12 mice (Figures 1H and 1I). Local field potentials (LFPs) recorded with extracellular multisite silicon array electrodes in somatosensory S3I-201 cortex in response to peripheral whisker stimulation typically produce brief multiphasic events that

are dominated by an initial negative-going waveform with greatest amplitude in L4 (Quairiaux et al., 2007). Stimulus-triggered waveform averages in control (Vglut1−/−;Vglut2fl/−) mice showed robust evoked LFPs ( Figure 1I, left; maximum negative amplitudes of 207 μV and 209 μV; maxima at 38 ms and 51 ms, check details respectively, after stimulus onset; waveform widths at half maximum were 23 ms and 31 ms). The same experimental procedure in ThVGdKO mice failed to elicit evoked potentials (n = 4). Indeed, the stimulus-triggered

waveform averages revealed no stimulus-related activity in the LFPs at all ( Figure 1I, right). Histology confirmed that the recording probes were placed in similar locations within somatosensory cortex in both groups of mice (data not shown). Together, these results indicate that glutamatergic neurotransmission at thalamocortical synapses in ThVGdKO somatosensory cortex was largely, if not completely, abolished. over Barrels in the somatosensory cortex of mice are composed of clusters of thalamocortical axon arbors in L4 surrounded by rings of spiny stellate neuron somata whose dendrites are oriented toward the center of the barrel to synapse with thalamocortical afferents relaying information from a single whisker (Li and Crair, 2011). We used cytochrome oxidase (CO) histochemistry and Nissl staining to examine whether cortical barrel formation was dependent on thalamocortical glutamatergic neurotransmission. In flattened tangential sections through somatosensory cortex, clear CO barrel patterns were present in Vglut1−/−,Vglut2fl/− and all other control mice, while a barrel pattern was not detectable in ThVGdKO mice ( Figure 2A, top; Figure S1A available online). This suggests that thalamocortical afferents fail to cluster into barrels in ThVGdKO mice.