their physiological and pharmacological properties as described by our many published documents [21,23,28]. Most DA neurons fired spontaneously in a regular pacemaker mode. As the basal firing rate varied, each neuron served as its own control. We recorded spontaneous firing for 5–10 min in the absence of salsolinol and then switched the perfusion to a solution containing salsolinol for 5–10 min before returning to control solution. Recently, we have shown that salsolinol (0.01–1 mM) dose- dependently increases the firing of DA neurons with a peak effect at 0.1 mM [10]. In keeping with this, salsolinol (0.1 mM) robustly increased the ongoing discharges recorded from current clamped DA neurons (by 100.3615.6%, from 0.8860.17 to 1.8760.50 Hz, n = 7, P,0.001, by paired t test; Fig. 1A). This effect was reversible upon washout of salsolinol. An identical effect of salsolinol was observed using the loose-patch cell-attached configuration (data not shown). Excitatory synaptic input mediated by glutamate is a key component in the regulation of DA cell excitability [15]. To assess whether salsolinol-induced excitation of DA neurons involves activation of Gluergic ionotropic receptors, we compared salsolinol’s effects in the absence and presence of the mixture of APV (50 mM) and DNQX (20 mM), the antagonists of NMDA and AMPA receptors. APV+DNQX slightly but significantly reduced the firing rate of DA neurons to 80.066.4% of control (from 1.960.4 to 1.660.5 Hz, n = 11, P = 0.011; Fig. 1C). This indicates a significant Gluergic influence which tonically facilitates DA cells’ activity. After the response to APV+DNQX stabilized, the addition of salsolinol (0.1 mM) increased the firing by 33.567.4 % (n = 6) (Fig. 1B). This increase was significant (P,0.05. vs. in the absence of salsolinol), but was substantially smaller than 100.3615.6% that induced by salsolinol (0.1 mM) alone (Fig. 1D, P,0.05, student’s unpaired t test). These data indicate that salsolinol-induced stimulation of DA neurons is mediated, at least partly, by Gluergic activity. Salsolinol increases the frequency but not the amplitude of spontaneous EPSCs (sEPSCs) in putative DA Neurons We then examined if salsolinol would affect Gluergic transmis- sion onto DA neurons. We recorded sEPSCs in the presence of 10 mM bicuculline at a holding potential (Vh) of 270 mV. Their suppression by 20 mM DNQX (not shown) indicated that they were indeed Gluergic events mediated by AMPA receptors. Salsolinol (0.1 mM) increased their frequency by 50.9612.4% (from 3.761.3 to 5.661.8 per 10 s, n = 7, P = 0.006, by paired t- test, Fig. 2A). This is further shown in Fig. 2B by the sharp increase in the probability of shorter intervals between successive sEPSCs (Kolmogorov-Smirnov, K–S test, P,0.01). After washout of salsolinol, sEPSC frequency returned to control levels (Fig. 2A). Conversely, salsolinol (0.1 mM) did not alter sEPSC amplitudes (control, 23.662.3 pA; salsolinol, 24.860.7 pA, n = 7, p = 0.53, paired t-test). The cumulative probability plot of amplitudes between successive sEPSCs also confirms this finding (K–S test, P.0.5, Fig. 2C). It is generally believed that a significant effect on sEPSC frequency without a concomitant effect on their amplitude is good evidence for an interference with the release mechanisms. Tetrodotoxin (TTX) abolishes Salsolinol’s increases of spontaneous EPSCs in DA Neurons To further characterize the effects of salsolinol on the spontaneous EPSCs, we recorded miniature EPSCs (mEPSCs) in the presence of bicuculline (10 mM) and TTX (0.5 mM), which blocks action potential-generated events. TTX (0.5 mM) lowered the frequency of spontaneous EPSCs to 33.466.0% of control Salsolinol Facilitates Glutamate Transmission Figure 1. Salsolinol-induced stimulation of dopaminergic (DA) neurons is attenuated by APV and DNQX. A1, Traces illustrate spike discharge at the times indicated in A2. A2, Time course of the increase in the ongoing pacemaking firing rate, recorded from a current-clamped DA neuron in the posterior ventral tegmental area (p- VTA) of a rat, by 0.1 mM salsolinol. B1, Traces obtained at the times indicated in B2. B2, Time course of the effect of salsolinol on the firing rate of a DA neuron in the presence of APV (50 mM) and DNQX (20 mM), the antagonists of NMDA and AMPA receptors. C, Summary plot of decease in firing rate by APV + DNQX. D, Summary plot (means 6 S.E.M.) of increase in firing rate of p-VTA DA neurons induced by salsolinol (0.1 mM) in ACSF is larger than that in the APV+DNQX. Numbers in bars indicate numbers of neurons tested. *P,0.05, **P,0.01, paired t-test for salsolinol vs. pre-salsolinol control. Unpaired t-test for salsolinol vs. APV+DNQX+salsolinol. doi:10.1371/journal.pone.0036716.g001 (from 2.760.7 to 0.860.4 per 10 s, n = 3, P = 0.008, paired t-test) without altering their amplitude (97.061.3% of control, from 15.363.1 to 14.963.1 pA, n = 3, p = 0.14, paired t-test). In the presence of TTX (0.5 mM), salsolinol (0.1 mM) did not significantly altered either the mEPSC frequency (by 16.7615.5%, from 0.860.1 to 1.060.2 per 10 s, n = 6, P = 0.33, paired t-test), or their amplitude (by 1.264.0%, control, 20.761.3 pA; salsolinol, 20.961.5 pA, n = 6, P = 0.9, paired t-test; Fig.3A–C). Thus, salsolinol’s enhancement of Gluergic transmission is dependent on TTX-sensitive Na+ channels. PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e36716