Adaptor protein-3 produces synaptic vesicles that release phasic dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high-frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a distinct population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Adaptor Protein-3 Produces Synaptic Vesicles that Release Phasic Dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a novel population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Dopamine Dysregulation and Altered Responses to Drugs Affecting Dopaminergic Transmission in a New Dopamine Transporter Knockout (DAT-KO) Rat Model.

Under normal conditions, dopamine (DA) clearance after release largely depends on uptake by the DA transporter (DAT). DAT expression/activity is reduced in some neuropsychiatric and neurological disorders. Our aim was to characterize the behavioral, neurochemical and electrophysiological effects of eliminating DAT in a novel knockout rat model we generated using CRISPR/Cas9. Consistent with existing DAT-KO models, our DAT-KO rats displayed increased locomotion, paradoxical calming by amphetamine, and reduced kinetics of DA clearance after stimulated release. Reduced DA kinetics were demonstrated using fast-scan cyclic voltammetry in brain slices containing the striatum or substantia nigra pars compacta (SNc) and in the dorsal striatum in vivo. Cocaine enhanced DA release in wild-type (WT) but not DAT-KO rats. Basal extracellular DA concentration measured with fast-scan controlled-adsorption voltammetry was higher in DAT-KO rats both in the striatum and SNc and was enhanced by L-DOPA (particularly after pharmacological block of monoamine oxidase), confirming that DA release after L-DOPA is not due to DAT reversal. The baseline firing frequency of SNc neurons was similar in both genotypes. However, D2 receptor-mediated inhibition of firing (by quinpirole or L-DOPA) was blunted in DAT-KO rats, while GABAB-mediated inhibition was preserved. We have also provided new data for the DAT-KO rat regarding the effects of slowing DA diffusion with dextran and blocking organic cation transporter 3 with corticosterone. Together, our results validate our DAT-KO rat and provide new insights into the mechanisms of chronic dysregulation of the DA system by addressing several unresolved issues in previous studies with other DAT-KO models.

Role of homeostatic feedback mechanisms in modulating methylphenidate actions on phasic dopamine signaling in the striatum of awake behaving rats.

Methylphenidate is an established treatment for attention-deficit hyperactivity disorder that also has abuse potential. Both properties may relate to blocking dopamine and norepinephrine reuptake. We measured the effects of methylphenidate on dopamine dynamics in freely moving rats. Methylphenidate alone had no effect on the amplitude of phasic responses to cues or reward. However, when administered with the D2 receptor antagonist raclopride, methylphenidate increased dopamine responses, while raclopride alone had no effect. Using brain slices of substantia nigra or striatum, we confirmed that methylphenidate effects on firing rate of nigral dopamine neurons and dopamine release from terminals are constrained by negative feedback. A computational model using physiologically relevant parameters revealed that actions of methylphenidate on norepinephrine and dopamine transporters, and the effects of changes in tonic dopamine levels on D2 receptors, are necessary and sufficient to account for the experimental findings. In addition, non-linear fitting of the model to the data from freely moving animals revealed that methylphenidate significantly slowed the initial cue response dynamics. These results show that homeostatic regulation of dopamine release in the face of changing tonic levels of extracellular dopamine should be taken into account to understand the therapeutic benefits and abuse potential of methylphenidate.

Action potential and calcium dependence of tonic somatodendritic dopamine release in the Substantia Nigra pars compacta.

Despite the importance of somatodendritic dopamine (DA) release in the Substantia Nigra pars compacta (SNc), its mechanism remains poorly understood. Using a novel approach combining fast-scan controlled-adsorption voltammetry (FSCAV) and single-unit electrophysiology, we have investigated the mechanism of somatodendritic release by directly correlating basal (non-stimulated) extracellular DA concentration ([DA]out ), with pharmacologically-induced changes of firing of nigral dopaminergic neurons in rat brain slices. FSCAV measurements indicated that basal [DA]out in the SNc was 40.7 ± 2.0 nM (at 34 ± 0.5°C), which was enhanced by amphetamine, cocaine, and L-DOPA, and reduced by VMAT2 inhibitor, Ro4-1284. Complete inhibition of firing by TTX decreased basal [DA]out , but this reduction was smaller than the effect of D2 receptor agonist, quinpirole. Despite similar effects on neuronal firing, the larger decrease in [DA]out evoked by quinpirole was attributed to cell membrane hyperpolarization and greater reduction in cytosolic free Ca2+ ([Ca2+ ]in ). Decreasing extracellular Ca2+ also reduced basal [DA]out , despite increasing firing frequency. Furthermore, inhibiting L-type Ca2+ channels decreased basal [DA]out , although specific Cav 1.3 channel inhibition did not affect firing rate. Inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase (SERCA) also decreased [DA]out , demonstrating the importance of intracellular Ca2+ stores for somatodendritic release. Finally, in vivo FSCAV measurements showed that basal [DA]out in the SNc was 79.8 ± 10.9 nM in urethane-anesthetized rats, which was enhanced by amphetamine. Overall, our findings indicate that although tonic somatodendritic DA release is largely independent of action potentials, basal [DA]out is strongly regulated by voltage-dependent Ca2+ influx and release of intracellular Ca2+ . OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Paradoxical lower sensitivity of Locus Coeruleus than Substantia Nigra pars compacta neurons to acute actions of rotenone.

Parkinson's disease (PD) is not only associated with degeneration of dopaminergic (DAergic) neurons in the Substantia Nigra, but also with profound loss of noradrenergic neurons in the Locus Coeruleus (LC). Remarkably, LC degeneration may exceed, or even precede the loss of nigral DAergic neurons, suggesting that LC neurons may be more susceptible to damage by various insults. Using a combination of electrophysiology, fluorescence imaging and electrochemistry, we directly compared the responses of LC, nigral DAergic and nigral non-dopaminergic (non-DAergic) neurons in rat brain slices to acute application of rotenone, a mitochondrial toxin used to create animal and in vitro models of PD. Rotenone (0.01-5.0µM) dose-dependently inhibited the firing of all three groups of neurons, primarily by activating KATP channels. The toxin also depolarised mitochondrial potential (Ψm) and released reactive oxygen species (H2O2). When KATP channels were blocked, rotenone (1µM) increased the firing of LC neurons by activating an inward current associated with dose-dependent increase of cytosolic free Ca2+ ([Ca2+]i). This effect was attenuated by blocking oxidative stress-sensitive TRPM2 channels, and by pre-treatment of slices with anti-oxidants. These results demonstrate that rotenone inhibits the activity of LC neurons mainly by activating KATP channels, and increases [Ca2+]ivia TRPM2 channels. Since the responses of LC neurons were smaller than those of nigral DAergic neurons, our study shows that LC neurons are paradoxically less sensitive to acute effects of this parkinsonian toxin.

Effects of the Parkinsonian toxin MPP+ on electrophysiological properties of nigral dopaminergic neurons.

Although MPP(+) (1-methyl-4-phenylpyridinium) has been widely used to damage dopaminergic neurons of the Substantia Nigra pars compacta (SNc) and produce animal and cellular models of Parkinson's disease, the action of this toxin on ion channels and electrophysiological properties of these neurons remains controversial. Previous work has attributed the early effects of MPP(+) on the membrane potential and firing frequency of SNc neurons either to block of hyperpolarisation-activated (Ih) current, or to activation of ATP-sensitive K(+) (KATP) channels. Using a combination of electrophysiological and pharmacological techniques, we investigated the acute effects of MPP(+) (20 µM) on SNc neurons in rat midbrain slices. Our results show that MPP(+) inhibits the activity of these neurons in distinct stages involving different mechanisms. The early phase of inhibition was dependent on D2 autoreceptors, but [(3)H]raclopride membrane binding and cAMP production assays demonstrated that the toxin (0.001-100 µM) did not directly bind to these receptors nor activated the Gi-linked signalling pathway. Depletion of vesicular dopamine with Ro4-1284 attenuated the early inhibitory effect, indicating that D2 autoreceptors were activated by dopamine released from the somato-dendritic region. After longer exposure (>10-20 min), MPP(+) produced a late phase of inhibition which mainly involved activation of KATP channels, and required uptake of the toxin via dopamine transporter. Although Ih current mediated by hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels was reduced by MPP(+), neither inhibition of firing nor membrane potential hyperpolarisation was significantly attenuated by blocking HCN channels with ZD7288. Our results indicate that the initial cellular events that lead to activation of cell death pathways by MPP(+) are complex and include KATP, and dopamine-dependent components, and show that the inhibitory effect of the toxin is independent of Ih block.