Figure 6. SKF83566 attenuates salsolinol-induced excitation of p-VTA DA neurons. A, Ongoing firing in the presence of SKF83566 (10 mM), recorded from a current-clamped p-VTA DA neuron, was increased by salsolinol (0.1 mM). B, Pooled data show that in the presence of SKF83566, salsolinol induced an increase in firing rate of p- VTA DA neurons from thirteen cells. C, Summary (means 6 S.E.M.) of the effects of salsolinol (0.1 mM) on DA neuron firing rate in the absence (ACSF) and presence of SKF 83566 (SKF 63566). Numbers of cells are indicated in the brackets. *P,0.05 by un-paired t-test, salsolinol vs. SKF 83566+salsolinol. doi:10.1371/journal.pone.0036716.g006 demonstrated that ethanol enhanced Gluergic transmission to VTA DA neurons [21,22]. In this study, we found salsolinol enhanced Gluergic transmission onto DA neurons. The applica- tion of APV+DNQX substantially attenuated salsolinol-induced increase in the firing rate of DA neurons, indicating that Gluergic activity contributes considerably to salsolinol-induced excitation of DA neurons. Moreover, TTX abolished the increase in the frequency of spontaneous EPSC induced by salsolinol, indicating that salsolinol’s effect was dependent on TTX-sensitive Na+ channels. Salsolinol did not alter the amplitude of both sEPSCs and mEPSCs, indicating that salsolinol acted on the presynaptic site. The presynaptic loci were further supported by salsolinol’s effects on evoked EPSCs: it enhanced the amplitude of the first EPSCs in response to the first stimuli of the paired stimuli but decreased their paired-pulse ratio. Role of D1Rs in salsolinol-induced excitation of VTA DA neurons DA receptors consist of D1-like (D1 and D5 receptors) and D2- like (D2, D3 and D4 receptors) families. Both D1R and the D2R family (D2R in particular) [41] have been implicated in the mechanisms of drug dependence and abuse. Disruption of D1R gene expression [42] or administration of D1R antagonist [43] Salsolinol Facilitates Glutamate Transmission attenuates or prevents alcohol-seeking behavior. D1Rs are expressed on Gluergic axons [31] but not on the soma of VTA 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]. Previous works in our lab showed that ethanol may enhance glutamate release by potentiation of D1R function [21,22]. In this study, we found that SKF83566 abolished salsolinol’s enhancement of the eEPSCs, indicating that this enhancement is mediated by D1Rs. Salsolinol may increase the somatodendritic release of dopamine, which may retrogradely activate the D1Rs on the glutamate-releasing terminals, which in turn increases glutamate release and the excitability of DA neurons. Somatodendritic release of dopamine in the midbrain dopamine neurons is Na+ channel-dependent (see [44] for review). In keeping with this, we found that the increase in the frequency of spontaneous EPSCs was abolished by TTX. Interestingly, salsolinol is more potent than ethanol in enhancing Gluergic transmission as well as in excitation of DA neurons. The effective concentration of salsolinol in enhancing Gluergic transmission is much smaller than that of ethanol (peak effect: salsolinol: 0.1 mM vs ethanol: 40 mM [21,22]. This is consistent with both in vivo and in vitro findings that DA system is highly sensitive to salsolinol [8,9,10]. We previously showed that ethanol-induced increase in the release of glutamate (EPSCs) in the VTA was eliminated when dopamine was depleted by the pretreatment of reserpine. Since salsolinol is the condensation product of dopamine and acetalde- hyde, the metabolic product of ethanol, depletion of dopamine may prevent the formation of salsolinol. On the basis of the results of this study, we propose that the observation that ethanol failed to increase Gluergic transmission when dopamine was depleted may well be explained by the lack of salsolinol formation. VTA DA neurons receive numerous inputs which in turn can modulate their eventual outputs. Integration of synaptic inputs and intrinsic properties sets the frequency and pattern of firing [45,46,47]. In this study, APV and DNQX, as well as SKF 83566, significantly attenuated but not totally abolished salsolinol- induced increase in the firing rate of VTA DA neurons. This is in line with our recent finding that salsolinol stimulates DA neurons partly through activation of m opioid receptors, which leads to the inhibition of GABAergic transmission onto VTA DA neurons. It is worth noting that even in the presence of a mixture containing APV+DNQX+gabazine, salsolinol could still significantly increase the firing rate of p-VTA DA neurons (by 27.365.6%, n = 10, P = 0.03) (G Xie and JH Ye, unpublished observation). Further- more, since the sum of the reductions induced by gabazine and by APV+DNQX in salsolinol-induced increase in firing rate of DA neurons is larger than one, the effects of salsolinol on GABAergic and Gluergic pathways may not be paralleled. This observation indicates the complex nature of salsolinol’s effects. Other mechanisms underlying salsolinol-induced excitation of VTA DA neurons remain to be fully unveiled. On the bases of the findings of this study and our recent report [10], we propose that both Gluergic and GABAergic transmissions play essential roles in salsolinol-induced stimulation of VTA DA neurons. Our present work offers new insights into salsolinol’s central mechanism under its rewarding properties. Author Contributions Conceived and designed the experiments: JHY GX. Performed the experiments: GX. Analyzed the data: GX. Wrote the paper: JHY GX. PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e36716