Tectopulvinar Synapses and Synaptic Potentials Figure 5. In vitro recording methods. A) A parasaggital section of the tree shrew brain stained for the type 2 vesicular glutamate transporter (vGLUT2) illustrates the location of the whole cell recordings in the caudal pulvinar nucleus and the location of the 8 electrode stimulus array in the stratum griseum superficial (SGS) and stratum opticum (SO) of the superior colliculus. B, C) High magnification views of the Pc (B) and Pd (C) illustrated in panel A. Immunohistochemical staining for vGLUT2 is a marker for tectopulvinar terminals [9] and reveals the distinct arrangements of tectal terminals in the dorsal (Pd) central (Pc) pulvinar nucleus. The Pd contains dense clusters of tectal terminals and tubular clusters line long lengths of dendrites (C). In contrast, the Pc contains smaller more sparsely distributed clusters of tectal terminals (B). D) Voltage fluctuations recorded in response to the injection of depolarizing or hyperpolarizing current steps of varying size revealed that all recorded cells fired with both tonic action potentials, and low threshold calcium bursts. E) Drawing of a biocytin-labeled cell recorded in the juvenile pulvinar, F) Drawing of a biocytin-labeled cell recorded in the adult pulvinar. Scale in A = 1 mm. Scale in C = 30 mm and also applies to B. Scales in E and F = 10 mm. dLGN, dorsal lateral geniculate nucleus. doi:10.1371/journal.pone.0023781.g005 addition, the rise time (10-90% of maximum amplitude) of short latency, large amplitude EPSPs (2.9060.25 ms n = 17) was significantly faster than that of the slow latency, small amplitude EPSPs (3.826 0.27 ms, n = 8, p,0.05). The two types of tectopulvinar EPSPs display distinct short term plasticity The short-term plasticity of these two types of EPSPs was tested with trains of stimuli varying in frequency from 1Hz to 10 Hz (Figure 7), as well as paired-pulse stimuli with interstimulus intervals of 0.1s to 2s (Figure 8). For each type of EPSP we used stimulation currents sufficient to evoke EPSPs at 50% of maximum. The short latency/large amplitude EPSPs were depressed by high frequency stimulation (frequencies .1 Hz, n = 10). With train stimuli, relative to the first EPSP of the train, the second EPSP decreased by 16.6762.35% at 2.5 Hz, 23.2963.88% at 5 Hz, and 29.4263.42% at 10Hz (Figure 7A and B). Similar values were observed using paired-pulse stimuli (a 9.1964.99% reduction in the amplitude of the second pulse relative to the first pulse at interstimulus intervals of 0.6s; 17.1767.51% at 0.2s and 37.1868.27% at 0.1 s; Fig. 8A and C, n = 5). In contrast, for the slower latency/smaller amplitude EPSPs, there was no correlation between mean amplitudes and stimulation frequencies or paired-pulse interstimulus intervals (Fig. 7C and D, n = 6; Fig. 8B and D, n = 4). The two types of tectopulvinar EPSP display different degrees of convergence All recorded tectopulvinar EPSP amplitudes (n = 28) increased as the stimulation intensity was increased, suggesting that each postsynaptic cell receives input from multiple convergent tectal axons. However, the degree of convergence was found to be greatest for the slower/smaller/nondepressing EPSPs. The peak amplitudes of these EPSPs increased up to 3.34 fold above threshold amplitude values, while peak amplitudes of the faster/ larger/depressing EPSPs increased to 2.11 fold above threshold values. As illustrated in Figure 6 (B and C), the rate of amplitude increase as a function of stimulation current was greater for nondepressing EPSPs compared to the depressing EPSPs. ‘‘Mixed’’ EPSPs For the majority of tectopulvinar EPSPs (25 of 28) the latencies did not change as the stimulation intensity was increased (Figure 6B, C). However, in some cases (n = 3) EPSPs appeared to contain a mixture of the two EPSP types described above. The amplitude of these ‘‘mixed’’ EPSPs initially increased gradually as the stimulation current was increased, but then exhibited a sudden large increase in amplitude, suggesting that the threshold was reached to recruit a different type of axon. Supporting this conclusion, there was a corresponding decrease in EPSP latency with the jump in EPSP amplitude (Fig. 9, A and B). Furthermore, PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23781