Tectopulvinar Synapses and Synaptic Potentials Figure 4. Distribution of synapsins and vGLUTs in the pulvinar nucleus. Tree shrew pulvinar 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 of the Pd (single 0.2 mm scan with a 100x objective) are illustrated. Corticopulvinar terminals (RS profiles labeled with the vGLUT1 antibody) contain synapsin I (A), but not synapsin II (C). Tectopulvinar terminals (large clusters of RM profiles labeled with the vGLUT2 antibody) contain both synapsin I (B) and synapsin II (D). Scale = 10 mm. doi:10.1371/journal.pone.0023781.g004 subdivisions originates exclusively from layer VI [6] while cortical input to the SC originates exclusively from layer V [42]. Therefore SC stimulation cannot activate corticopulvinar inputs. In addition, the pretectum does not project to the Pd or Pc [9], so current spread to this region does not activate any pulvinar inputs. Finally, cholinergic inputs from the parabigeminal nucleus that travel through the superficial SC and optic tract to the dLGN [43] do not innervate the pulvinar nucleus [9]. Figure 5A illustrates the approximate location of the recording pipette in the pulvinar nucleus and the location of the 8 electrode stimulation array in the SC (spanning the superficial layers, stratum griseum superficiale and stratum opticum). The array spanned a distance of 1 mm and stimulation could be produced between any two electrodes in the array; the anode and cathode positions were varied to obtain the best response. With this configuration, tectopulvinar EPSPs could be elicited with a high rate of success (28 of 46 cells; 15 of 29 cells in juvenile tissue, and 10 of 17 cells in adult tissue) in the caudal pulvinar, which corresponds to the Pd (as described by Lyon et al., 2003[44]). In sections stained for vGLUT2, the Pd is identified as a region with densely distributed large clusters of terminals (Figure 5C) while the Pc contains smaller clusters of terminals that are more sparsely distributed (Figure 5D). We were unable to elicit tectopulvinar responses from more rostral regions of the pulvinar nucleus; in the parasagittal slice preparation, tectal axons traveling to the rostral pulvinar were most likely severed before they reached their synaptic targets. As illustrated in Figure 5B, in response to the injection of depolarizing or hyperpolarizing current steps, all recorded neurons fired with either tonic action potentials or low threshold calcium spikes respectively, firing properties exhibited by thalamic relay cells [45]. Biocytin was included in the recording pipette and all recovered cells (n = 20) displayed morphologies consistent with their identification as relay cells (Figure 5E, F). Tectopulvinar EPSPs can be divided into two groups based on latency, and amplitude As illustrated in Figure 6, tectopulvinar EPSPs fell into two groups based on differences in their average latencies, as well as their threshold and peak amplitudes, suggesting that two different types of axon arbors were activated. EPSPs with the shortest latencies (2.1160.10 ms, n = 17; 10 in juvenile tissue and 7 in adult tissue) exhibited the largest threshold EPSP amplitudes (4.8260.57 mV, n = 17; Figure 6B) and EPSPs with longer latencies (3.2060.15 ms, n = 8; 5 in juvenile tissue and 3 in adult tissue) exhibited smaller threshold amplitudes (1.5860.27 mV, n = 8; Figure 6C). These parameters were found to be significantly different (latencies p,0.05; threshold amplitudes p,0.05), sup- porting the division of the tectopulvinar EPSPs into two groups. In PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23781