Salsolinol Facilitates Glutamate Transmission Figure 2. Salsolinol increases frequency of spontaneous EPSCs (sEPSCs) recorded from p-VTA DA neurons. EPSCs were recorded in the presence of bicuculline (10 mM) at a holding potential of 270 mV. A1, Traces obtained at the times indicated in A2. A2, Time course of 0.1 mM salsolinol-induced enhancement of sEPSC frequency in one experiment. B, C, Cumulative probability plots of data from the same cell show effects of salsolinol (0.1 mM) on sEPSC frequency (B) and amplitudes (C). Insets: mean data (6 S.E.M., from seven neurons). *P,0.05, **P,0.01, paired t-test for salsolinol vs. pre-salsolinol control. doi:10.1371/journal.pone.0036716.g002 Salsolinol enhances evoked EPSCs (eEPSCs) in DA neurons The above data indicate that salsolinol increases Gluergic transmission via a presynaptic mechanism. To confirm this, we tested the effect of salsolinol on EPSCs evoked by pairs of stimuli (at 50 ms interval) applied by a local stimulating electrode in the presence of 10 mM bicuculline and at a Vh of –70 mV. These EPSCs were completely blocked by 20 mM DNQX (not shown), indicating that they were mediated by AMPA receptors. Salsolinol (0.1 mM) enhanced the amplitude of the first eEPSC (EPSC1, by 34.862.5%, n = 11; P,0.01, by paired t-test, Fig. 4A–C), but not the second eEPSC (EPSC2) and thus decreased the paired-pulse ratio (PPR = EPSC2/EPSC1, by 23.263.4% from 1.0860.05 to 0.8760.03; n = 11, P,0.05, paired t-test, Fig. 4A and C). It’s well documented that changes in transmitter release generally affect the PPR [29,30]. Salsolinol-induced increase in EPSCs is abolished by a D1Rs antagonist, SKF 83566 In the VTA, D1Rs are expressed on Gluergic axons [31] but not on the soma of DA neurons [31,32]. The activation of D1Rs increases glutamate levels in the VTA [20] as well as Gluergic transmission in the globus pallidus [33]. As was recently shown by our lab that ethanol-induced enhancement of Gluergic transmission onto VTA DA neurons involves D1Rs localized on the Gluergic Figure 3. Tetrodotoxin (TTX) abolishes Salsolinol’s increase of sEPSCs. The miniature EPSCs (mEPSCs) were recorded in p-VTA DA neurons at a holding potential of 270 mV, in the presence of bicuculline (10 mM) and TTX (0.5 mM). A1, Traces obtained at the times indicated in A2. (A2) Time course of salsolinol-induced changes of mEPSC frequency in one experiment. B, C, Cumulative probability plots of inter-event intervals between mEPSCs (B) and their amplitudes (C). Insets: mean data (6 S.E.M., from six neurons) confirm the absence of significant effect of salsolinol (0.1 mM) on the frequency and the amplitude of mEPSCs. *P,0.05, paired t-test for salsolinol vs. pre- salsolinol control. doi:10.1371/journal.pone.0036716.g003 terminals [21,22], we wonder if salsolinol-induced enhancement of Gluergic transmission onto VTA DA neurons also involves D1Rs. We compared the effects of salsolinol on eEPSCs in the absence and presence of SKF83566 (10 mM). As depicted in Fig. 5A–C, in the presence of SKF83566, salsolinol (0.1 mM) failed to change either eEPSC1 amplitude (by 26.7611.3%, n = 9, P = 0.6, paired t-test), or the PPR (by 18.462.8% of control, from 1.0760.04 to 1.2560.08; n = 9, P = 0.2, paired t-test). These results indicated that D1Rs are essential for salsolinol’s enhancement of Gluergic transmission. Salsolinol-induced excitation of VTA DA neurons is attenuated by SKF 83566 To further assess the contribution of D1Rs to salsolinol’s stimulation of DA neurons, we compared the effect of salsolinol on the ongoing discharges of p-VTA DA neurons in the absence and presence of SKF83566. In the presence of 10 mM SKF83566, salsolinol (0.1 mM) significantly increased the firing rate, but only PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36716