Neuron, Vol. 5, 797-808, December, 1990, Copyright 0 1990 by Cell Press Amphetamine and Other Psychostimulants Reduce pH Gradients in Midbrain Dopaminergic Neurons and Chromaffin Granules: A Mechanism of Action David Sulzer and Stephen Rayport Department of Psychiatry and Center for Neurobiology and Behavior Columbia University New York, New York 10032 Department of Neuropathology New York State Psychiatric Institute New York, New York 10032 Summary Rewarding properties of psychostimulants result from reduced uptake and/or increased release of dopamine at mesolimbic synapses. As exemplified by cocaine, many psychostimulants act by binding to the dopamine uptake transporter. However, this does not explain the action of other psychostimulants, including amphetamine. As most psychostimulants are weak bases and dopamine uptake into synaptic vesicles uses an interior-acidic pH gradient, we examined the possibility that psychostimu- lants might inhibit acidification. Pharmacologically rele- vant concentrations of amphetamine as well as cocaine and phencyclidine rapidly reduced pH gradients in cul- tured midbrain dopaminergic neurons. To examine di- rect effects on vesicles, we used chromaffin granules. The three psychostimulants, as well as fenfluramine, imipramine, and tyramine, reduced the pH gradient, re- sulting in reduced uptake and increased release of neuro- transmitter. Inhibition of acidification by psychoactive amines contributes to their pharmacology and may pro- vide a principal molecular mechanism of action of am- phetamine. introduction Ventral tegmental area (VTA) dopaminergic neurons projecting to the nucleus accumbens are thought to mediate the rewarding effects of psychostimulants (Koob and Bloom, 1988). Amphetamine, cocaine, phencyclidine, and other major drugs of abuse act by increasing extraneuronal dopamine in this projec- tion, as measured by in vivo microdialysis and voltam- metry (Carboni et al., 1989; Di Chiara and Imperato, 1988; Hernandez et al., 1988; Mifsud et al., 1989). Ani- mals self-administer cocaine and amphetamine di- rectly into the nucleus accumbens, whereas lesioning or pharmacological blockade of dopaminergic trans- mission at this site blocks rewarding effects (Goeders and Smith, 1986; Hoebel et al., 1983). Anti-psychotics, which are dopamine receptor antagonists, abolish self-administration in animals and euphoria in man (Wise and Bozarth, 1987). Indeed, chronic or high doses of amphetamine produce symptoms behavior- ally indistinguishable from schizophreniform psycho- sis (Ellinwood et al., 1973). The increased synaptic concentration of dopamine produced by psychostimulants results from a reduc- tion in presynaptic dopamine reuptake and/or in- creased release (Wise and Bozarth, 1987). While some psychostimulants, including cocaine, appear to in- crease extracellular dopamine by inhibiting dopa- mine reuptake at the plasma membrane dopamine transporter, this does not adequately explain the ef- fects of other psychostimulants, particularly amphet- amine. Amphetamine induces alterations in behavior and releases dopamine far in excess of what is pre- dicted by its relatively weak binding to the dopamine transporter (Andersen, 1987; Ritz et al., 1987). The ac- tion of amphetamine is apparently neither dependent on activity nor due to dopamine receptor binding (Kuczenski et al., 1990; Shalaby et al., 1983). Indeed, amphetamine reduces accumulation of monoamines in synaptic vesicle preparations that lack the plasma membrane transporter (Knepper et al., 1988). Rather, amphetamine is thought to displace dopamine in syn- aptic vesicles, leading to increased synaptic levels; however, the molecular mechanism of amphetamine action has remained obscure (Carboni et al., 1989; Knepper et al., 1988; Liang and Rutledge, 1982; Zaczek et al., 1990). Catecholaminergic vesicles use an interior-acidic proton gradient for transmitter uptake. Amphetamine and several other psychostimulants are lipophilic weak bases (see Beckett and Moffat, 1969; Mack and Bonisch, 1979). We show here that such drugs perturb proton gradients in intracellular compartments in VTA dopaminergic neurons. The drugs work directly at thevesicular level, as shown in chromaffin granules used as a model vesicle preparation. This inhibition of proton gradients strongly affects neurotransmitter accumulation. We suggest that a weak base mecha- nism may in part explain how psychostimulants in- crease synaptic levels of dopamine, thereby mediat- ing their euphoriant and psychotogenic effects. Results intracellular Acidification in Dopaminergic Cells VTA dopaminergic neurons give rise to the meso- limbic projection and are thus the cellular substrate for psychostimulant action. Cells from the VTA were isolated from postnatal rats and grown for 7-10 days in vitro on a glial monolayer. In culture, these neu- rons show most of the distinctive characteristics of dopaminergic cells in vivo or in krain slice prepara- tions (Rayport et al., Sot. Neurosci., abstract, 1988, 1990).Asdopaminergicneuronsaretheonlycatechol- aminergic cells in the ventral midbrain, we used 5-hy- droxydopamine, which is specifically taken up by catecholaminergic synaptic vesicles and forms an electron-dense product (Tranzer and Thoenen, 1967), to examine them at the electron microscopic level. Two sizes of labeled vesicles were observed, chiefly