Salsolinol stimulates dopamine neurons in slices of posterior ventral tegmental area indirectly by activating μ-opioid receptors

Abstract

Previous studies in vivo have shown that salsolinol, the condensation product of acetaldehyde and dopamine, has properties that may contribute to alcohol abuse. Although opioid receptors, especially the μ-opioid receptors (MORs), may be involved, the cellular mechanisms mediating the effects of salsolinol have not been fully explored. In the current study, we used whole-cell patch-clamp recordings to examine the effects of salsolinol on dopamine neurons of the ventral tegmental area (VTA) in acute brain slices from Sprague-Dawley rats. Salsolinol (0.01-1 μM) dose-dependently and reversibly increased the ongoing firing of dopamine neurons; this effect was blocked by naltrexone, an antagonist of MORs, and gabazine, an antagonist of GABA(A) receptors. We further showed that salsolinol reduced the frequency without altering the amplitude of spontaneous GABA(A) receptor-mediated inhibitory postsynaptic currents in dopamine neurons. The salsolinol-induced reduction was blocked by both naltrexone and [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin, an agonist of MORs. Thus, salsolinol excites VTA-dopamine neurons indirectly by activating MORs, which inhibit GABA neurons in the VTA. This form of disinhibition seems to be a novel mechanism underlying the effects of salsolinol.

Fig. 1.

Salsolinol (SAL) accelerates spontaneous firing of dopamine neurons in the VTA by an MOR-dependent action. A1, traces illustrate spike discharge at the times indicated in A2. A2, time course of acceleration of pacemaking firing rate of a dopamine neuron by 0.03 μM salsolinol. B1, traces obtained at the times indicated in B2. B2, time course of the effect of salsolinol on the firing rate of a dopamine neuron in the presence of naltrexone, a μ-opioid antagonist (5 μM). C, dose-dependent potentiation of the firing rate of dopamine neurons. NTX, naltrexone. Means and S.E.M. are shown, with numbers of cells in parentheses. *, p < 0.05; #, p < 0.01; by Student's t test, for differences from control.

Fig. 2.

Gabazine prevents SAL-induced excitation of dopamine neurons. A1a, ongoing firing, recorded from a current-clamped dopamine neuron under control condition. A1b, the firing was increased by gabazine. A1c, in the presence of gabazine, salsolinol had minimal effect. These traces were recorded at the times indicated in A2. A2, time course of changes. A3, a scatter plot summarizes all the responses to 10 μM gabazine. B, Data obtained in other tests, where salsolinol was applied in the presence of gabazine (10 μM) and after injecting a small hyperpolarizing current (HPC, −20 pA) to restore the initial firing rate; note: salsolinol-induced excitation was suppressed. B1, traces illustrate spike discharge at the times indicated in B2. B2, time course of the effect of salsolinol on the firing rate of a putative dopamine neuron in the presence of gabazine and HPC. C, Summary (means ± SEM) of increases in firing rate produced by 0.1 μM salsolinol in the absence, or presence of gabazine or gabazine + HPC. In brackets are numbers of recorded neurons. #, p < 0.05, paired t test for salsolinol versus presalsolinol condition. *, p < 0.05, t test for salsolinol versus pre-salsolinol condition or versus without gabazine or gabazine + HPC, as indicated. NS, not significant, t test for salsolinol-induced increase in firing in gabazine versus in gabazine + HPC.

Fig. 3.

Salsolinol depresses eIPSCs in putative dopamine neurons. A, salsolinol (0.1 μM) sharply reduced the peak amplitude of both the first and second IPSC evoked by paired stimulation (at 50-ms interval) within the VTA, but the greater relative reduction of the first response increased the ratio of IPSC2/IPSC (PPR). Labels a-c refer to times in B. B, time course of salsolinol-induced changes in eIPSC amplitude (Amp). C, summary of the effects of salsolinol (0.1 μM) on the amplitude and the PPR of eIPSCs; numbers of recorded neurons are in parentheses. *, p < 0.01, paired t test for salsolinol application versus presalsolinol control. All IPSCs were recorded from putative dopamine neurons at a V_H of −70 mV in the presence of APV (50 μM) and DNQX (20 μM).

Fig. 4.

Salsolinol reduces sIPSC frequency in putative dopamine neurons. A, traces obtained at the times indicated in B. B, time course of salsolinol's effect on sIPSC frequency. C1 and C2, cumulative probability plots of data from the same cell show effects of salsolinol (0.1 μM) on sIPSC frequency (C1) and amplitude (C2). Insets are pooled data (means ± S.E.M.; n = 8). D, salsolinol-induced percentage changes of sIPSC frequency as a function of salsolinol concentration; all data (n = 4–8) were normalized to values obtained in the absence of salsolinol. *, p < 0.05, t test for salsolinol versus presalsolinol condition. E, plot of salsolinol effects on firing of dopamine neurons against the salsolinol effects on sIPSC frequency. Each point corresponds to the effects of one concentration of salsolinol on cell firing rate and sIPSC frequency. The data were fitted by a linear equation with a correlation coefficient of 0.883 (p < 0.0001).

Fig. 5.

Salsolinol-induced depression of sIPSC frequency is prevented by naltrexone (NAL) or DAMGO. A1, current traces obtained at the times indicated in A2. A2, incidence of sIPSCs during application of salsolinol (0.1 μM) in the presence of naltrexone. A3 and A4, pooled data (means ± S.E.M.; n = 6) show the lack of effect of salsolinol in the presence of naltrexone (5 μM) (frequency, A3; amplitude, A4). Numbers of cells are indicated in parentheses. B1, current traces obtained at the times indicated in A2. B2, time course of depression of sIPSC frequency by DAMGO (1 μM), which cancels the effect of salsolinol. B3 and B4, pooled data (means ± S.E.M.; n = 5) summarize these results (frequency, B3; amplitude, B4). *, p < 0.05, paired t test for DAMGO (b) or DAMGO + SAL versus pretreatment condition.

Fig. 6.

Salsolinol does not affect spontaneous miniature IPSCs. mIPSCs were recorded in VTA dopamine neurons in the presence of 0.5 μM TTX. A and B, individual traces (A) and time course (B) are illustrated. C, salsolinol did not change the cumulative probability of interevent intervals (left) and mIPSC amplitudes (right). Insets show mean relative changes (± S.E.M.; n = 5 neurons) induced by 0.1 μM salsolinol.