Salsolinol Facilitates Glutamate Transmission Figure 4. Salsolinol increases amplitude and decreases paired- pulse ratio (PPR) of evoked EPSCs (eEPSCs) recorded from p- VTA DA neurons. eEPSCs were recorded in the presence of bicuculline (10 mM) at a holding potential of 270 mV. A, Salsolinol (0.1 mM) sharply increased the peak amplitude of eEPSC1 and had minimal effect on that of eEPSC2 evoked by paired stimulation (at 50- ms interval) within the VTA, and hence reduced the PPR (EPSC2/EPSC1). B, Time course of salsolinol-induced enhancement of eEPSC1 amplitude in one experiment. C, Summary of salsolinol-induced changes (%) in eEPSC1 amplitude and PPR (Mean 6 SEM, from eleven neurons). *P,0.05, **P,0.01, paired t-test for salsolinol vs. pre-salsolinol control. doi:10.1371/journal.pone.0036716.g004 by 34.9614.8% (from 1.360.4 to 1.660.5 Hz, n = 13, P = 0.036, Fig. 6A–B), which is .50% smaller (Fig. 6C, P,0.05, by unpaired t test) than 100.3%, the increase induced by 0.1 mM salsolinol alone. Discussion We show here the first electrophysiological evidence of salsolinol facilitation of Gluergic transmission onto p-VTA DA neurons in acute midbrain slices. Salsolinol (0.1 mM) robustly increased the spontaneous firing rate of DA neurons, which was substantially attenuated by APV and DNQX. Furthermore, salsolinol signifi- cantly increased the frequency of sEPSCs and the amplitude of the first eEPSCs, but reduced the paired-pulse ratio of eEPSCs. The effects of salsolinol on EPSCs were eliminated by the D1R antagonist SKF83566. Finally, SKF83566 attenuated salsolinol- induced increase in firing rate of p-VTA DA neurons. Taken together, these data indicate that salsolinol, via the activation of Figure 5. The D1R antagonist SKF 83566 eliminates salsolinol’s augmentation of eEPSCs. A, Current traces show that salsolinol did not change the amplitude of eEPSC1 and PPR in the presence of 10 mM SKF 83566. B, Time course of salsolinol-induced changes of eEPSC1 amplitude in nine experiments. C, Summary of salsolinol-induced changes (%) in eEPSC1 amplitude and PPR in the presence of SKF 83566 (data from nine cells). *P,0.05, **P,0.01, paired t-test for salsolinol application vs. pre-salsolinol control. doi:10.1371/journal.pone.0036716.g005 presynaptic D1Rs and consequently facilitation of Gluergic transmission contributes substantially to salsolinol-induced excita- tion of p-VTA DA neurons. Hence, salsolinol’s effect on DA neurons involves activation of Gluergic transmission, in addition to the suppression of GABAergic transmission [10]. Gluergic transmission is involved in Salsolinol-induced excitation of VTA DA neurons Recently we have shown that salsolinol (0.01–1 mM) stimulated p-VTA DA neurons in brain slices of rats [10]. In this study, we showed that salsolinol’s stimulation was substantially attenuated by APV and DNQX. This finding clearly points to the involvement of Gluergic transmission in salsolinol’s action. It is well accepted that VTA DA neurons are important for the response to reinforcement and reward signals. Accumulating evidence suggests that the input and output elements of DA neurons are essential components in the reward pathways. Gluergic transmission plays an important role in the effects of ethanol [34,35]. In several brain regions, ethanol inhibits NMDA and non-NMDA glutamate receptors, as well as glutamate release [36]. However, there are also reports that ethanol increases glutamate release under some circumstances. Systemic adminis- tration of ethanol increases glutamate release in the nucleus accumbens of low-alcohol sensitive rats [37] and addiction-prone Lewis rats [38]. Studies on the central nucleus of the amygdale reveal that acute ethanol increases glutamate release only in rats receiving chronic ethanol treatment [39,40]. We have previously PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e36716