Question 1

Like other drugs acting on the dopaminergic system, amphetamine has a high abuse potential, as well
as neurotoxic effects. Its structural similarity to monoamines allows it to act as a competitive substrate
for the reuptake of these neurotransmitters, with relatively weak potency compared with classical
reuptake inhibitors1. Understanding its mechanism of action, and long-term influence on the dopamine-
releasing synapse will help improve clinical approaches and provide a theoretical basis for its effects.

The synaptic availability of dopamine (DA) is increased mainly by action on the DA plasma membrane
transporter (DAT). Amphetamine (AMP) has a moderately high affinity at DAT (Km = 10 μM), in effect
competing with DA reuptake, whose concentration increases in the synaptic cleft2. Within the DA axon,
AMP acts on a second target, namely the vesicular monoamine transporter type-2 (VMAT-2), used for
sequestering DA back in the vesicles. As a result, AMP will be introduced instead of DA. At this level,
AMP impairs the normal process of vesicle acidification, raising the pH from 4 to 72. An acidic
environment helps charge DA, a weak base, thus preventing it from passively diffusing out of the
vesicle. Instead, DA is released in the axoplasm, where it can diffuse in the extracellular space or use
DAT as a reverse transporter. The latter is dependent on sufficient AMP concentration, intracellular
kinases, and L-type Ca2+ channels3. For example, experimentally upregulating CamKIIα in nucleus
accumbens (NAc) improved the AMP behavioural response in mice3. Lastly, AMP inhibits monoamine
oxidase (MAO-A in DA neurons) bound to mitochondria, which would normally limit cytosolic
neurotransmitter by deaminating excess DA2. These three specific targets underlie the immediate
effects of AMP as releasing agent to promote DA availability at the postsynaptic receptor.

While these synaptic effects of AMP are normally limited to 60 minutes in mice3, AMP activity is
changed in several conditions. For instance, acetylcholine (ACh) raises Ca2+ in axoplasm, leading to
prolonged vesicle release, while desensitization lowers the time window. Notably, the combined effect
of the two enantiomers (given a single chiral centre) produces an extended release in the 3:1 d- to l-
isomer mixture3. The authors suggest a competitive action of l-amphetamine at the DAT with the more
efficient d-amphetamine, inducing a prolonged uptake, important in clinical formulations like Adderall.

These mechanisms, illustrated in fig 1, have been modelled differently between in vitro and in vivo
studies, leading to concerns regarding reproducibility of results in animal subjects. As suggested by
Daberkow et al. using electrical stimulation at 2h post-injection, when DA is depleted in vitro, AMP
increased both frequency and amplitude of DA release4. This would not be expected if DA was
depleted from the vesicles following reverse transport via DAT. However, rather than disregarding the
role of DAT in DA efflux, it was argued that prolonged exposure to the drug at the concentration used
in the study (10 mg/kg AMP) is sufficient to produce the effects observed in vitro4. This was suggested
due to the disruption of cue-evoked behaviour in chronic users or at high drug concentrations4.
Concentration, rather than choice of model, may account for the different effect. Indeed, in vitro studies
may bypass limitations encountered in vivo given its neurotoxicity at high drug doses.

Despite its known use in clinical settings, AMP was linked to synaptic degeneration and DA depletion
beyond those seen in the immediate effect at the synapse. Neurotoxicity has been documented in
metabolic interference3, leading to mitochondrial dysfunction in the presynaptic neuron. Repeated use
was seen to induce protein oxidation, as measured by protein carbonyl concentration in NAc and
hippocampus, following reports of oxidative stress conducted in vitro5. While it remains unclear how
AMP acts on metabolic cascades, it was indicated that vesicular dysfunction, leading to high cytosolic
DA, has a pivotal role in the production of reactive oxygen species (ROS)3. Once in the axoplasm, DA
can be oxidized to form reactive quinones. In effect, these can bind to cysteinyl residues and produce
protein-bound cysteinyl catechols, particularly harmful to DA neurons5. By contrast, administration of
antioxidants (e.g., ascorbic acid) reduces the DA depletion linked to neuronal apoptosis, which is
reversed when superoxide dismutase (SOD), normally limiting ROS, is inhibited5. Taken together, the
oxidative stress elicited by AMP is likely mediated presynaptically by DA-induced ROS. However, a
role for excitotoxicity has been proposed beyond the dopaminergic synapse. This involves a
polysynaptic pathway5 enhancing glutamate activity. This initiates abnormal Ca2+ elevations in DA
neurons, a potent signalling molecule raising nitric oxide synthase and superoxide levels. In fact,
experimental perfusion of AMP in vitro does not produce the apoptosis-mediated DA depletion, unless
co-administered with glutamate and in sufficiently warm environments to synchronize their metabolic
processes5. This emphasizes the role of integrated neuronal models in understanding the complete
impact of AMP at the DA synapse.

Unlike neurotoxic effects, other long-term neuroplastic effects have more elusive, but nonetheless
potent influences on dopamine transmission, as seen in substance abuse or ADHD treatments. A
common feature of AMP use is sensitization, linked to increased behavioural activation, which lies in
contrast with an expected tolerance7. Autopsy data of long-term use show decreases in DA, DAT, and
tyrosine hydroxylase, but no effects on VMAT-28. These do suggest a compensatory downregulation,
but studies have not been consistent to explain the contradictory sensitization effects. These are more
persistent than for DA agonists7 and are not likely induced by DAT reduction8. More recent evidence
suggests differential changes in the type 2 DA receptors (D2R) located pre- and post-synaptically9.
Their activity can be reduced by desensitization and eventual internalization in constant stimulation.
However, the opposite was found in neurons lacking DA and DAT, namely an AMP-dependent
increase in D2R. When cells were transfected DAT, they showed a strong reduction of endogenous
D2R9. Authors also found the direct link between AMP levels and D2R function of inhibiting adenylate
cyclase, thus uncovering key neuroplastic events. These changes may be effected by metabolic
actions, as AMP is partially lipophilic3, but remain unexplained. Similarly, different actions at the type 1
DA receptor or variance in effect across brain areas8 point to an insufficient understanding of the role
of AMP in changing synaptic plasticity.

In sum, the complete profile of AMP mechanism of action is incomplete. Following from its short-term
effects of increasing DA synaptic availability by DAT, VMAT-2 substrate competition and selective
MAO-A inhibition, this produces neuroplastic changes at the pre- and post-synaptic level. AMP use is
moreover subject to significant neurotoxic events in high or sustained administration. Elucidating
synaptic effects in conjunction with the availability of DA in key brain areas can help improve drug
treatments and the theoretical framework for its action in substance use disorders.

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Figure 1*. Amphetamine targets at the dopaminergic synapse: 1. Competitive substrate at the
dopamine transporter, inducing dopamine reverse transport. 2. Amphetamine sequestered in synaptic
vesicle increasing axoplasmic dopamine and diffusion. 3. Inhibition of monoamine oxidase (MAO-A).
*Created with BioRender.com

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