arrangements, referred to as ‘‘diffuse’’ and ‘‘specific’’ [9,37]. The specific projections are topographically arranged and distributed throughout both the dorsal (Pd) and central (Pc) pulvinar nucleus. The diffuse projections are nontopographic projections that only innervate the Pd. Our recent tract tracing studies indicate that the Pd and Pc project to the temporal cortex and striatum, and the Pd additionally projects to the amygdala [6,38]. Thus, we have suggested that the specific projections relay topographic visual information from the SC to the cortex and striatum to aid in guiding precise movements, while the diffuse projection relays nontopographic visual information from the SC to the amygdala to alert the animal to potentially dangerous visual images. In the current study, we characterized the length of synaptic contacts made by diffuse and specific tectopulvinar terminals, the synapsin content of RS, RM and RL profiles in the pulvinar nucleus and dorsal lateral geniculate nucleus (dLGN), and the properties of tectopulvinar EPSPs. We conclude that the short- term synaptic plasticity of RM profiles is distinct from that of RS and from that of RL profiles because each terminal type contains a unique distribution of synapsins. Furthermore, we suggest that the diffuse and specific tectopulvinar projections display subtle differences in short-term plasticity due to differences in glutamate release probability. Materials and Methods A total of 16 tree shrews (Tupaia belangeri); 7 adults (more than 3 months old) and 9 juveniles (3 weeks old), were used for these experiments. Twelve tree shrews were used for in vitro physiology experiments, 1 tree shrew received injections of biotinylated dextran amine (BDA) in the SC, and tissue from 3 tree shrews was used for immunocytochemisty. In addition, we analyzed material from 2 tree shrews used in a previous study [9]. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Louisville (Animal Welfare Assurance number A358601). Slice preparation Thalamic slices (400 mm) were prepared from 9 juvenile and 3 adult male and female tree shrews using procedures previously described [22]. Briefly, the animals were deeply anesthetized with carbon dioxide and decapitated, the brain was excised and a block of tissue containing the thalamus was removed and placed in an ice-cold oxygenated solution of modified artificial cerebrospinal fluid (ACSF) containing (in mM) 206 sucrose, 2.5 KCl, 1 CaCl2, 1 MgSO4, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3 and 10 glucose at a pH of 7.4 and equilibrated with 95% O2/5% CO2. Parasagittal slices were cut on a vibratome (Leica, VT 1000E, Deerfield, IL) at a thickness of 400 mm and transferred into a holding chamber with ACSF containing (in mM) 124 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 1.25 NaH2PO4, 26 NaHCO3 and 10 glucose at a pH of 7.4 and equilibrated with 95% O2/5% CO2, where they incubated for at least 2 hours before recording. Electrophysiology Whole cell recordings were obtained from the pulvinar nucleus. All recordings were carried out in the recording chamber maintained at 33uC with ACSF (with 10 mM bicuculline and 2.5 mM CGP55845 added; Tocris; Ellisville, MO) continually superfused at a rate of 2.0 ml/min. Pipettes were pulled from borosilicate glass (Sutter Instrument, Novato, CA) and had a tip resistance of 4–6 MV when filled with a solution containing (in mM) 115 K-gluconate, 2 MgCl2, 10 HEPES, 10 sodium phosphocreatine, 2 Na2-ATP, 20 KCl, 0.3 Na2-GTP with pH Tectopulvinar Synapses and Synaptic Potentials adjusted to 7.2 with KOH (osmolarity 290–295 mOsm). Biocytin (0.5%) was added to allow morphological reconstruction of the recorded neurons. Current-clamp recording were made with an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA); the bridge was continually monitored and adjusted as needed. Data were digitized and stored on an IBM-compatible computer for off- line analyses. Only recordings showing a stable resting membrane potential more negative than -50 mV and over shooting action potentials were included in this study (n = 46). To stimulate the tectothalamic fibers a multipolar stimulation electrode (matrix microelectrode; FHC, Bowdoin, ME) was placed in the superficial layers of the superior colliculus (SC). Stimulating electrodes were at least 2 mm away from the recording electrode. Once a stable whole-cell recording was obtained, paired-pulse or train stimula- tion was produced by using any two adjacent electrodes (115 mm apart) in the arrays until the best response was achieved. Histochemistry A subset of neurons was filled with 0.5% biocytin by diffusion from the pipette during recording (20 recovered). At the end of each recording, slices were fixed at 4uC overnight in 4% paraformaldehyde and rinsed several times in 0.1 M phosphate buffer (PB). Slices were then incubated in 10% methanol in PB with 3% hydrogen peroxide to react with the endogenous peroxidase activity of red blood cells. After several rinses in PB, slices were incubated overnight at 4uC under agitation in a 1% solution of avidin and biotinylated-horseradish peroxidase (ABC Kit Standard, Vector Laboratories) prepared in 0.3% Triton X- 100. The slices were subsequently rinsed, reacted with nickel- intensified 3,39-diaminobenzidine (DAB) for 5 min, and washed in PB. After rinses in phosphate buffer, slices were mounted onto slides and reconstructed with a Neurolucida system (Micro Bright Field Inc., USA). In some cases, biocytin-filled neurons were revealed by incubating slices in a 1:100 dilution of streptavidin conjugated to Alexa Fluor 546 (Molecular Probes, Eugene, OR) and confocal images of the cell were obtained using an Olympus Fluoview laser scanning microscope (BX61W1). Tracer injections As described in detail in our previous study [9], single tracer injections in the SC label dense topographic clusters of terminals (‘‘specific’’ terminals); the most medial SC projects to the Pd, while the central and lateral regions of the SC project to the Pc. In contrast, the ‘‘diffuse’’ terminals are nontopographic; injections in any part of the SC label sparsely-distributed terminals in the Pd [37]. Therefore, when BDA is injected into the medial SC, tracer- labeled diffuse and specific terminals overlap within the Pd. However, when BDA is injected into the central or lateral SC, diffuse terminals are labeled in the Pd, while specific terminals are labeled in the Pc, allowing analysis of these segregated populations. Therefore we placed injections into the central and/or lateral regions of the SC to enable us to examine the isolated ‘‘diffuse’’ and ‘‘specific’’ projections in the Pd and Pc respectively. Tree shrews that received BDA (3,000 MW; Molecular Probes, Eugene, OR) injections were initially anesthetized with intramus- cular injections of ketamine (100 mg/kg) and xylazine (6.7 mg/ kg). Additional supplements of ketamine and xylazine were administered approximately every 45 minutes to maintain deep anesthesia through completion of the tracer injections. Prior to injection, the tree shrews were placed in a stereotaxic apparatus and prepared for sterile surgery. A small area of the skull overlying the superior colliculus was removed and the dura reflected, a glass pipette containing either BDA (5% in saline, tip diameter 3 mm) was lowered vertically and the tracer was ejected iontophoretically PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23781