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Amphetamine has well-established actions on pre-synaptic dopamine signaling, such as inhibiting uptake and degradation, activating synthesis, depleting vesicular stores, and promoting dopamine-transporter reversal and non-exocytotic release. Recent in vivo studies have identified an additional mechanism: augmenting vesicular release. In this study, we investigated how amphetamine elicits this effect. Our hypothesis was that amphetamine enhances vesicular dopamine release in dorsal and ventral striata by differentially targeting dopamine synthesis and degradation. In urethane-anesthetized rats, we employed voltammetry to monitor dopamine, electrical stimulation to deplete stores or assess vesicular release and uptake, and pharmacology to isolate degradation and synthesis. While amphetamine increased electrically evoked dopamine levels, inhibited uptake, and up-regulated vesicular release in both striatal sub-regions in controls, this psychostimulant elicited region-specific effects on evoked levels and vesicular release but not uptake in drug treatments. Evoked levels better correlated with vesicular release compared with uptake, supporting enhanced vesicular release as an important amphetamine mechanism. Taken together, these results suggested that amphetamine enhances vesicular release in the dorsal striatum by activating dopamine synthesis and inhibiting dopamine degradation, but targeting an alternative mechanism in the ventral striatum. Region-distinct activation of vesicular dopamine release highlights complex cellular actions of amphetamine and may have implications for its behavioral effects.
Substantive evidence indicates that amphetamine (AMPH), a highly addictive drug of abuse exhibiting clinical efficacy for treating narcolepsy and attention deficit hyperactivity disorder (Heal et al. 2009; Peacock and Benca 2010), targets pre-synaptic dopamine (DA) signaling. Effects include inhibiting the DA transporter (DAT) and monoamine oxidase and activating tyrosine hydroxylase, but depleting vesicular DA stores and promoting non-exocytotic DA release via DAT reversal are considered primary (Fleckenstein et al. 2007; Sulzer 2011). More recently, AMPH has been shown to augment vesicular DA release in both dorsal and ventral striata in vivo (Ramsson et al. 2011b; Daberkow et al. 2013). While the significance of this unexpected finding to overall drug effect remains to be determined, enhanced vesicular DA release may drive AMPH-induced increases in phasic DA signaling (Ramsson et al. 2011b; Daberkow et al. 2013), which is important for reinforcement-learning in goal-directed behavior and addiction (Hyman 2005; Wanat et al. 2009). Several other DAT inhibitors have also been shown to increase vesicular DA release (Ewing et al. 1983; Kuhr et al. 1986; Jones et al. 1995; Lee et al. 2001; Venton et al. 2006; Oleson et al. 2009; Kile et al. 2010; Chadchankar et al. 2012), suggesting a common action for a major psychostimulant class.
How AMPH augments vesicular DA release is unknown, but potential mechanisms are suggested by other DAT inhibitors. Cocaine and methylphenidate act on DA storage pools associated with synapsin (Venton et al. 2006; Kile et al. 2010) and α-synuclein (Chadchankar et al. 2012), respectively. Several DAT inhibitors re-distribute cytosolic and membrane-bound vesicles (Riddle et al. 2002, 2007; Volz et al. 2007) and increase vesicular DA uptake (Brown et al. 2001; Volz et al. 2008). As a drug with complex actions, AMPH could exert additional, unique effects, including the inhibition of DA degradation (Scorza et al. 1997) and activation of DA synthesis (Kuczenski 1975) leading to elevated cytosolic DA levels and vesicular packaging, promoting exocytosis by liberating intracellular Ca2+ stores (Mundorf et al. 1999), and increasing membrane excitability as a DAT substrate (Ingram et al. 2002).
This study used voltammetry and electrical stimulation to investigate the mechanism by which AMPH augments vesicular DA release in dorsal and ventral striata in vivo. We employed an experimental design previously used to demonstrate that amfonelic acid and cocaine reinstate vesicular DA release after its near-complete depletion (Ewing et al. 1983; Kuhr et al. 1986; Venton et al. 2006). These results were interpreted as the two psychostimulants mobilizing the reserve DA pool to replenish the readily releasable DA pool independently of an action on DA synthesis, because tyrosine hydroxylase was pharmacologically blocked. However, vesicular mobilization was not directly assessed and thus not proven. We selected this design, because the robust response serves as a gauge of AMPH's effectiveness and because amfonelic acid and cocaine are perhaps the best-established DAT inhibitors for up-regulating vesicular DA release. Indeed, amfonelic acid has been recognized for decades as an archetypal enhancer of vesicular release (Aceto et al. 1970; Shore 1976), and this cocaine effect manifests across brain-slice (Jones et al. 1995; Lee et al. 2001; Kile et al. 2010), anesthetized (Ramsson et al. 2011b), and awake (Oleson et al. 2009) preparations. Because AMPH could conceivably act by inhibiting DA degradation, in addition to activating DA synthesis, we modified the design to also incorporate pharmacological blockade of monoamine oxidase, in order to assess the respective contributions of both pre-synaptic mechanisms. The experimental design also permitted resolving the respective contributions of vesicular DA release and DA uptake to observed AMPH-induced changes in electrically evoked DA levels. The hypothesis tested was that AMPH distinctly up-regulates vesicular DA release in striatal sub-regions by differentially targeting DA synthesis and degradation. Our results are consistent with a mechanism of AMPH action characterized by generalized uptake inhibition and up-regulation of vesicular release across striatal sub-regions, but a specific degradation- and synthesis-sensitive enhancement of vesicular release in the dorsal striatum only.