(2 mA positive current for 15–30 minutes) into the central and/or lateral SC. After a 7-day survival period, the tree shrews were given an overdose of ketamine (600 mg/kg) and xylazine (130 mg/kg) and were perfused through the heart with Tyrode solution, followed by a fixative solution of 4% paraformaldehyde. The BDA was revealed by incubating sections in a 1:100 dilution of avidin and biotinylated horseradish peroxidase (ABC; Vector Laboratories, Burlingame, CA) in phosphate-buffered saline (0.01 M PB with 0.9% NaCl, pH 7.4; PBS) with 1% normal goat serum (NGS) overnight at 4uC. The sections were subsequently rinsed, reacted with nickel-intensified 3,39-diaminobenzidine (DAB) for 5 minutes, and washed in PB. For confocal microscopy, the BDA was revealed by incubating sections in a 1:100 dilution of streptavidin conjugated to Alexa Fluor 546 (Molecular Probes, Eugene, OR). Immunohistochemistry Three adult tree shrews were given an overdose of sodium pentobarbital (tree shrew) were perfused through the heart with Tyrode solution, followed by a fixative solution of 4% parafor- maldehyde in 0.1 M PB. The brain was removed from the skull, sectioned into 50- mm-thick slices using a vibratome (Leica VT100E, Leica Microsystems, Bannockburn, IL) and collected in a solution of 0.1 M PB. The sections were incubated at 4uC overnight with one of the following antibodies: guinea pig monoclonal anti-vGLUT1 or anti-vGLUT2 (1:5000, Chemicon Temecula, CA; catalogue #’s AB5905 and AB2251), rabbit polyclonal anti-synapsin I (1:1000, Millipore, Billerica, MA; catalogue #AB1543P) or rabbit monoclonal anti-synapsin II (1:500, Abcam, Cambridge, MA; catalogue #AB76494). The following day the sections were rinsed in PB and incubated for 1 hour in an anti-guinea pig antibody conjugated to Alexafluor-488 (1:100, Invitrogen/Molecular Probes, Carlsbad, CA; catalogue # Tectopulvinar Synapses and Synaptic Potentials A11073) or anti-rabbit antibody conjugated to Alexafluor-546 (1:100, Invitrogen/Molecular Probes, Carlsbad, CA; catalogue # A11010). The sections were subsequently rinsed in PB and mounted on slides for confocal microscopic examination. Ultrastructural Analysis Electron micrographs of diffuse and specific tectopulvinar terminals labeled by the anterograde transport of BDA injected into the SC, which were collected in a previous study [9], were re- analyzed. As described above, following BDA injections in the central and/or lateral SC, ‘‘diffuse’’ terminals were labeled in the Pd and ‘‘specific’’ terminals were labeled in the Pd. Resin-embedded sections were first examined with a light microscope to select ‘‘specific’’ terminals in the Pc and ‘‘diffuse’’ terminals in the Pd for electron microscopic analysis. Selected areas of the Pc or Pd were mounted on blocks, ultrathin sections (70–80 nm, silver-gray interference color) were cut using a diamond knife, and every fifth section was collected on Formvar-coated nickel slot grids. One in four of the collected sections was subsequently stained for the presence of gamma amino butyric acid (GABA) using gold particles, and examined using an electron microscope. Within each examined section, all labeled terminals involved in a synapse were photographed. The section spacing ensured that no single terminal was examined more than once. In the current study, we used ImageJ software (Rasband, W.S., U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2009.) to measure the length of each synaptic contact, and the area of each presynaptic BDA-labeled terminal, within the previously collected images. Statistical Analysis Student t-tests were used to test for statistical significance. Quantitative data are expressed as means 6 SD. The significance level was set at p,0.05 for all statistical comparisons. Figure 1. Convergence of tectopulvinar terminals. Injections of biotinylated dextran amine (BDA) in the SC labels tectopulvinar axons that form widespread (‘‘diffuse’’) axons and boutons as well as more discrete clustered (‘‘specific’’) boutons. Representative images of BDA-labeled ‘‘diffuse’’ (A) and ‘‘specific’’ (B) axons in 50 mm thick sections are illustrated using transmitted light, a 40x objective, and Nomarski optics. C–G) Confocal images (single 0.1 mm scan with a 100x objective) illustrate tectopulvinar terminals labeled with BDA (purple) and tectopulvinar terminals labeled with antibodies against the type 2 vesicular glutamate transporter (vGLUT2, green). Terminals labeled with both BDA and vGLUT2 appear white. Clusters of vGLUT2-stained terminals contain at most 1 bouton contributed by ‘‘diffuse’’ axons (F), while ‘‘specific’’ axons contributed several boutons to each cluster (C-E, G) Scale in B = 10 mm and applies to A. Scale in D = 10 mm and applies to C, F and G. Scale in E = 2 mm. doi:10.1371/journal.pone.0023781.g001 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e23781