Figure 3), and pulvinar nucleus (Figure 4) of the tree shrew by combining immunohistochemical labeling for synapsin I or synapsin II with immunohistochemical labeling for vGLUT2 (a marker for both tectothalamic and retinogeniculate terminals; [9,10,18]) or vGLUT1 (a marker for corticothalamic terminals; [39,40,41]). As illustrated in Figure 3, we found that retinogen- iculate terminals (RL profiles labeled with the vGLUT2 antibody) do not contain either synapsin I or synapsin II (as was previously reported in the mouse; [36]). We also found that corticothalamic terminals in the dLGN and pulvinar nucleus (RS profiles labeled with the vGLUT1 antibody) contain synapsin I but not synapsin II (Figures 3–4). Finally, we observed that tectopulvinar terminals (RM profiles labeled with the vGLUT2 antibody) contained both synapsin I and synapsin II (Figure 4). We found a similar distribution of synapsins in vGLUT1- and vGLUT2-labeled terminals in the rat dLGN and lateral posterior nucleus (unpublished data). Because tecopulvinar terminals form dense clusters, and individual terminals are difficult to distinguish at the light microscopic level, we used ImageJ software to quantify the labeling patterns by calculating the number of white pixels (double labeled areas) as a percentage of the total area labeled with the vGLUT antibodies (white + green pixels). For each image, we only analyzed areas that included both synapsin and vGLUT labeling to control for differences in antibody penetration. Three images of Tectopulvinar Synapses and Synaptic Potentials each antibody combination in the dLGN, Pd and Pc were analyzed. This analysis confirmed our qualitative assessment. There was very little overlap of vGLUT2 in the dLGN (retinogeniculate terminals) with either synapsin I (1.6260.40%) or synapsin II (1.9960.43%). For vGLUT1 staining in the dLGN (corticogen- iculate terminals) there was significant double-labeling with synapsin I (33.7466.12%) but not with synapsin II (3.8560.84%). This was also the case for vGLUT1 staining in the Pd and Pc (corticopulvinar terminals; Pd vGLUT1 29.3661.54% overlap with synapsin I and 3.8361.29% overlap with synapsin II; Pc vGLUT1 38.5265.51% overlap with synapsin I and 3.2461.29% overlap with synapsin II). In constrast, for vGLUT2 staining in the Pd and Pc (tectopulvinar terminals) there was significant double-labeling with both synapsins (Pd vGLUT2 57.8662.45% overlap with synapsin I and 48.7961.31% overlap with synapsin II; Pc vGLUT2 34.8465.49% overlap with synapsin I and 47.8364.25% overlap with synapsin II). In vitro recording of tectopulvinar synaptic potentials In 400 mm thick parasagittal sections, whole cell recordings were obtained in the pulvinar nucleus and the rostral SC was stimulated to evoke EPSPs. The pulvinar EPSPs evoked by SC stimulation can be identified as tectopulvinar EPSPs because the cortical input to the dorsal (Pd) and central (Pc) pulvinar Figure 3. Distribution of synapsins and vGLUTs in the dLGN. Tree shrew dLGN tissue was labeled with antibodies against synapsin I or II (purple), and vGLUT1 or 2 (green). Profiles that are labeled with two antibodies appear white. Representative confocal images (single 0.2 mm scan with a 100x objective) are illustrated. Corticogeniculate terminals (RS profiles labeled with the vGLUT1 antibody) contain synapsin I (A), but not synapsin II (C). Retinogeniculate terminals (RL profiles labeled with the vGLUT2 antibody) do not contain either synapsin I (B) or synapsin II (D). Scale = 10 mm. doi:10.1371/journal.pone.0023781.g003 PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23781