Analysis of information sources in references of the Wikipedia article "Monoamine releasing agent" in English language version.
Both d- and l-[amphetamine (AMP)] evoked rapid increases in extraneuronal concentrations of [noradrenaline (NA)] and [dopamine (DA)] that reached a maximum 30 or 60 min after administration. However, the [spontaneously hypertensive rats (SHRs)] were much more responsive to AMP's enantiomers than the [Sprague-Dawleys (SDs)]. Thus, 3 mg/kg d-AMP produced a peak increase in [prefrontal cortex (PFC)] NA of 649 ± 87% (p<0.001) in SHRs compared with 198 ± 39% (p<0.05) in SDs; the corresponding figures for [striatal (STR)] DA were 4898 ± 1912% (p<0.001) versus 1606 ± 391% (p<0.001). At 9 mg/kg, l-AMP maximally increased NA efflux by 1069 ± 105% (p<0.001) in SHRs compared with 157 ± 24% (p<0.01) in SDs; the DA figures were 3294 ± 691% (p<0.001) versus 459 ± 107% (p<0.001).
Both d- and l-[amphetamine (AMP)] evoked rapid increases in extraneuronal concentrations of [noradrenaline (NA)] and [dopamine (DA)] that reached a maximum 30 or 60 min after administration. However, the [spontaneously hypertensive rats (SHRs)] were much more responsive to AMP's enantiomers than the [Sprague-Dawleys (SDs)]. Thus, 3 mg/kg d-AMP produced a peak increase in [prefrontal cortex (PFC)] NA of 649 ± 87% (p<0.001) in SHRs compared with 198 ± 39% (p<0.05) in SDs; the corresponding figures for [striatal (STR)] DA were 4898 ± 1912% (p<0.001) versus 1606 ± 391% (p<0.001). At 9 mg/kg, l-AMP maximally increased NA efflux by 1069 ± 105% (p<0.001) in SHRs compared with 157 ± 24% (p<0.01) in SDs; the DA figures were 3294 ± 691% (p<0.001) versus 459 ± 107% (p<0.001).
The characteristics and mechanisms of efflux are complex and incompletely understood, and have been summarized in many comprehensive reviews including (Reith & Gnegy, 2020; Robertson, Matthies, & Galli, 2009; Sitte & Freissmuth, 2015). [...] Early studies implicating protein phosphorylation in AMPH-evoked DA efflux were performed by Giambalvo, who demonstrated that DAT-mediated uptake of AMPH impacted the activity and subcellular localization of protein kinase C (PKC) with characteristics that correlated with DA efflux (Giambalvo, 1992), and Gnegy and colleagues, whose many studies reinforced the relationship between efflux, kinases, and ionic conditions including PKC, PKCβ, Ca2+, and Ca1+-calmodulin regulated protein kinase (CaMK) (Gnegy et al., 2004; Johnson, Guptaroy, Lund, Shamban, & Gnegy, 2005; Kantor, Hewlett, & Gnegy, 1999). [...] With respect to DAT most studies have focused on PKC, CaMK, and mitogen activated protein kinases (MAPKs), but many other signaling and kinase pathways have been implicated in efflux mechanisms for all of the MATs (Bermingham & Blakely, 2016; Vaughan & Foster, 2013). [...] DAT phosphorylation stimulated by AMPH or METH also occurs on this domain and is blocked by PKC inhibitors (Cervinski et al., 2005; Karam et al., 2017), indicating the capacity of the drugs to involve the PKC pathway. How this occurs is not known, but could potentially follow from drug perturbation of local ion/Ca2+ concentrations that impact PTM enzymes, or alternatively could occur by substrate-driven induction of transporter conformations in which phosphorylation sites become more or less available to enzyme action. [...] There are many other Ser and Thr residues on DAT intracellular loops and domains that may serve as phosphorylation sites, and in vitro studies have demon- strated phosphorylation of N— and C—terminal peptide sequences by many kinases in addition to PKC, ERK, and CaMK that have been implicated in regulation and efflux including protein kinase A (PKA), protein kinase G (PKG), casein kinase 2 (CK2), p38 kinase, JNK2, Cdk5, and Akt1 (Gorentla I et al., 2009; Vaughan & Foster, 2013). [...] The regulatory, efflux, and post-translational characteristics of NET and SERT show many similarities to DAT that indicate conservation of mechanism, but also some distinct properties that reflect requirements of specific neuronal populations. [...] With respect to efflux there are many similarities between SERT, NET, and DAT that indicate conservation of mechanisms.
As expected, MDMA was a potent substrate at SERT (90±7 nM) and DAT (249±19 nM). HHMA was a potent DAT substrate (130±6 nM) but weaker at SERT (1729 ±134). HMMA displayed weak activity at both SERT (607±50 nM) and DAT (3652±252 nM).
Although the pharmacological effect of amphetamine is predominantly mediated by monoamine release, this mechanism is complemented by reuptake inhibition [...] that combine additively or synergistically to augment synaptic monoamine concentrations. The description of amphetamine as a 'monoamine reuptake inhibitor' often causes some confusion, and the difference between the mechanisms of amphetamine, which is a competitive reuptake transport substrate, and classical reuptake inhibitors is illustrated in Figure 3. [...] d-Amphetamine is generally accepted to be a weak dopamine reuptake inhibitor with a Ki value of ~100 nM, a moderately potent inhibitor of noradrenaline reuptake (Ki = 40–50 nM) and a very weak inhibitor of 5-HT reuptake (Ki = 1.4-3.8 µM). [...] the efficacy of amphetamine relative to other indirect monoamine agonists, for example classical reuptake inhibitors, can only be estimated from in vivo experiments. [...] [d- and l-Amphetamine] dose-dependently increased the extracellular concentrations of noradrenaline in the prefrontal cortex (PFC) and dopamine in the striatum. The pharmacodynamics of their effects are typical of those reported for monoamine releasing agents, i.e. a fast onset of action with peak increases of noradrenaline and dopamine efflux occurring at 30–45 min, large effects (400–450% of baseline for noradrenaline and 700–1500% of baseline for dopamine), with a relatively rapid decline after the maximum (Figure 4). [...] the magnitude of the increases produced by amphetamine's isomers are greater than those reported for classical reuptake inhibitors such as atomoxetine or bupropion, and there is no dose-effect ceiling to amphetamine's actions [...] The primary action of amphetamine is to increase synaptic concentrations of monoamine neurotransmitters, thereby indirectly enhancing noradrenergic, dopaminergic neurotransmission in the CNS.
Converging lines of evidence have solidified the notion that DA releasers are substrates of the transporter and once translocated, they reverse the normal direction of transporter flux to evoke release of endogenous neurotransmitters. The nature of this reversal is not well understood, but the entire process is primarily transporter-dependent and requires elevated intracellular sodium concentrations, phosphorylation of DAT, and possible involvement of transporter oligomers (Khoshbouei et al., 2003, 2004; Sitte and Freissmuth, 2010). [...] A library of approximately 1400 phenethylamine compounds (PAL compounds) has been screened using these protocols. Among the active compounds,the smaller DAT ligands were found to be DA releasers while the sterically larger compounds were DAT uptake inhibitors. [...] Generic pharmacophore for biogenic amine transporter ligands. Note that transportable substrate ligands exhibit size constraints defined by the red circle. Functional groups attached to the nitrogen, α-carbon or phenyl ring that extend beyond the "edge" of the pharmacophore will generate partial substrates, transporter blockers or be inactive. [...] phenmetrazine was found to be completely inactive at VMAT2 indicating that a direct interaction of the releaser with VMAT2 is not required for inducing neurotransmitter efflux into the extracellular space (Partilla et al., 2006). Phentermine and benzylpiperazine were also found in the same study to lack VMAT2 activity (Table 5).
Despite the knowledge that amphetamine is a substrate for the DAT and NET, questions still remain as to the physiological mechanism of amphetamine action. [...] At lower doses, amphetamine preferentially releases a newly synthesized pool of DA. [...] DA stores will not be depleted by the AMPT in these short time frames, leading to the conclusion that newly synthesized DA is a principal substrate for amphetamine-stimulated DA efflux. [...] Controversy has surrounded the role of VMAT2 and synaptic vesicles in the mechanism of amphetamine action. [...] Undoubtedly vesicles contribute strongly to the maximal DA released by amphetamine, although VMAT2 is not absolutely required for amphetamine to release DA from nerve terminals (Pifl et al. 1995; Fon et al. 1997; Wang et al. 1997; Patel et al. 2003). [...] Recently, a new model of amphetamine action has been formulated that proposes that amphetamine elevates tonic DA (non-exocytotic) signaling through reverse transport and depleting vesicular stores, but activates phasic DA signaling by enhancing vesicular DA release from the readily releasable pool (Covey et al. 2013). These conclusions were drawn from experiments using fast-scan cyclic voltammetry in either freely moving or anesthetized rats (Avelar et al. 2013; Daberkow et al. 2013). Again, one must strongly consider the dose of amphetamine in interpretation of these actions (Calipari and Ferris 2013).
While the sine qua non property of AMPH at monoamine transporters is the promotion of monoamine release via reverse transport, there are yet profound mysteries in understanding how this works. It is additionally clear that AMPH is an uptake blocker as well as a releaser, and differentiating between elevating extracellular monoamines by reverse transport or uptake blockade can be difficult. Of course, the many AMPH derivatives and different transporters maintain different combinatorial properties, an important topic beyond the range of this article. [...] The mechanism of how reverse transport occurs is unknown, and a very old issue of how reserpine can inhibit uptake but not halt reverse transport remains opaque.
[...] most amphetamines are [monoamine transporter] substrates, which pervert the relay to elicit efflux of monoamines into the synaptic cleft. However, some amphetamines act as transporter inhibitors. [...] Amphetamines also bind to the trace amine receptors TAR1, [...] TAR1 is not activated by all psychoactive amphetamines (p-chloroamphetamine, for instance, is inactive); stimulation of TAR1 actually reduces dopamine release and thus decreases sensitivity to amphetamine [18,19]. [...] amphetamines are taken up by plasma-membrane monoamine transporters as exogenous substrates [31]. Accordingly, they inhibit the physiological monoamine reuptake in a competitive manner [36]. As a consequence of both amphetamine-induced reverse transport and inhibition of reuptake, the synaptic monoamine concentration increases, which in turn activates post- and presynaptic receptors [37]. The activation of postsynaptic receptors propagates the signal and contributes to the biological response. Stimulation of presynaptic autoreceptors decreases the quantal release of monoamines upon excitatory inputs; [...]
An interesting neurochemical issue is the interaction between compounds acting as blockers and substrates. [...] Substrates (e.g. mephedrone) use MAT proteins to release neurotransmitters, while inhibitors (e.g. [MDPV]) prevent the transport of any compounds through MATs. It follows that simultaneous use of a substrate and a blocker should result in an antagonistic mechanism that lowers the mutual potency. Paradoxically, MDPV and mephedrone occur together in mixtures, and in addition they reinforce each other's effects, resulting in very strong stimulation. Simultaneous use of both SCs is reported by users, and studies in rodents involving simultaneous administration of the drugs have shown a significant increase in locomotor activity and additive effect compared to administering these drugs alone (Allen et al., 2019; Benturquia et al., 2019). [...] The explanation for this phenomenon can be found in the action of organic cation transporter (OCT) proteins. OCTs are a family of proteins responsible for endothelial transport of small, organic, hydrophilic, and positively charged molecules, including neurotransmitters and xenobiotics (Couroussé & Gautron, 2015). OCT3 is a protein present in the dopaminergic regions of the central nervous system, where it promotes DA reuptake when it is inhibited for high affinity transporters (DAT) (Couroussé & Gautron, 2015). Monoamines can also be released by OCTs. [...] Ex vivo studies on superior cervical ganglia cells enriched in NET and OCT3 showed that in the presence of MDPV blocking MAT proteins, mephedrone causes neurotransmitter efflux through OCT3, which is insensitive to the inhibitory effects of MDPV. The release of monoamines through OCT3, a low‐affinity transporter, presumably explains the paradoxical synergistic effects of inhibitors and substrates (Mayer et al., 2019). [...] Another feature that distinguishes [synthetic cathinones (SCs)] from amphetamines is their negligible interaction with the trace amine associated receptor 1 (TAAR1). Activation of this receptor reduces the activity of dopaminergic neurones, thereby reducing psychostimulatory effects and addictive potential (Miller, 2011; Simmler et al., 2016). Amphetamines are potent agonists of this receptor, making them likely to self‐inhibit their stimulating effects. In contrast, SCs show negligible activity towards TAAR1 (Kolaczynska et al., 2021; Rickli et al., 2015; Simmler et al., 2014, 2016). [...] It is worth noting, however, that for TAAR1 there is considerable species variability in its interaction with ligands, and it is possible that the in vitro activity of [rodent TAAR1 agonists] may not translate into activity in the human body (Simmler et al., 2016). The lack of self‐regulation by TAAR1 may partly explain the higher addictive potential of SCs compared to amphetamines (Miller, 2011; Simmler et al., 2013).
A number of test drugs displayed no activity in the [3H]dopamine uptake inhibition assay (Table 1). For example, (+)- phenmetrazine and (–)-phenmetrazine, the major metabolites of phendimetrazine (Rothman et al., 2002), were essentially inactive. [...] In contrast, other amphetamine-type agents, such as phentermine, phenmetrazine, and 1-benzylpiperazine, are potent releasers of neuronal dopamine (Baumann et al., 2000, 2005; Rothman et al., 2002), but they are inactive at VMAT2. Agents such as these may prove to be valuable control compounds for determining the importance of vesicular release for the in vivo actions of amphetamine-type agents.
While the determination of drug effects at the isolated target (i.e., DAT, NET, and SERT) can characterize the direct drug action at the target protein, other physiological components can also contribute significantly to the overall effect of the drug. It has been proposed that transporter-mediated, drug-induced efflux of neurotransmitter occurs through effects on the vesicular monoamine transporter 2 (VMAT2), depleting neurotransmitter from the vesicles into the cytosol (Nickell et al. 2014). Accordingly, full assessment of release would require testing the effects of a drug on the membrane transporters (SERT, DAT, and NET) and the effects of a drug at VMAT2. Alternatively, a more physiological system, such as synaptosomes or brain slices, could be used. However, reverse transport can also occur in cell lines that only express the plasma membrane transporters but not VMAT2 (Eshleman et al. 2013; Scholze et al. 2000) and in synaptosomes when VMAT2 is inhibited (Rothman et al. 2001).
In contrast to assay systems involving non-neuronal cells transfected with transporter proteins, synaptosomes possess all of the cellular machinery necessary for neurotransmitter synthesis, release, metabolism, and reuptake. Synaptosomes, however, do not model all of the effects of amphetamine-type agents because the use of reserpine removes any contribution of the vesicular monoamine transporter VMAT2 (SLC18A2) to the release process. In addition to acting as a substrate for plasma membrane monoamine transporters, amphetamine also binds to VMAT, resulting in the redistribution of monoamines from vesicular stores to the cytoplasm (Sulzer et al. 1995; Partilla et al. 2006; Freyberg et al. 2016). Although transporter substrates can induce monoamine release in the absence of VMAT binding (Fon et al. 1997), it is important to recognize that 2-aminoindans may have effects in intact nerve terminals that are not fully replicated in synaptosomes. Follow-up studies will be conducted to evaluate whether 2-aminoindans are capable of interacting with VMAT.
2.2.1. DAT regulation by psychostimulants [...] Considerable evidence supports a role for DAT substrates, like amphetamine, in inducing DAT internalization in vivo and in vitro, thus decreasing DA uptake [390–395]. [...] While several kinases are involved in the regulation of DAT, PKC is by far the most thoroughly investigated [388,403]. PKC activation induces DAT internalization [393,404–406] although the mechanism of PKC activation by DAT substrates remains largely unknown [388,389,403,407,408]
Release by an indirect agonist is termed reverse transport and may require buildup of neurotransmitter in the nerve cytoplasm [...] Stimulants can act to stimulate presynaptic (CNS) or prejunctional (periphery) receptors for the neurotransmitter on nerve terminals to control release from those endings. These receptors are usually inhibitory and are marked as I (either NE, DA, or 5-HT inhibitory receptor) in Figure 1 and as α2 (inhibitory α2- adrenoceptor) in Figure 2, respectively. For instance, α2-adrenoceptors are present as autoreceptors on noradrenergic nerves and mediate an inhibition of NE release both in the periphery and CNS (see Figures 1 and 2).71 [...] Stimulants can act to stimulate, either directly or indirectly via increased release of the monoamine neurotransmitter, receptors for the neurotransmitter on nerves or effector cells situated postsynaptically/postjunctionally to the monoaminergic neuron. [...] Receptor-mediated actions of amphetamine and other amphetamine derivatives [...] may involve trace amine-associated receptors (TAARs) at which amphetamine and MDMA also have significant potency.85–87 Many stimulants have potency at the rat TAAR1 in the micromolar range but tend to be about 5 to 10 times less potent at the human TAAR1, [...] Activation of the TAAR1 receptor causes inhibition of dopaminergic transmission in the mesocorticolimbic system, and TAAR1 agonists attenuated psychostimulant abuse-related behaviors.89 It is likely that TAARs contribute to the actions of specific stimulants to modulate dopaminergic, serotonergic, and glutamate signaling,90 and drugs acting on the TAAR1 may have therapeutic potential.91 In the periphery, stimulants such as MDMA and cathinone produce vasoconstriction, part of which may involve TAARs, although only relatively high concentrations produced vascular contractions resistant to a cocktail of monoamine antagonist drugs.86
The characteristics and mechanisms of efflux are complex and incompletely understood, and have been summarized in many comprehensive reviews including (Reith & Gnegy, 2020; Robertson, Matthies, & Galli, 2009; Sitte & Freissmuth, 2015). [...] Early studies implicating protein phosphorylation in AMPH-evoked DA efflux were performed by Giambalvo, who demonstrated that DAT-mediated uptake of AMPH impacted the activity and subcellular localization of protein kinase C (PKC) with characteristics that correlated with DA efflux (Giambalvo, 1992), and Gnegy and colleagues, whose many studies reinforced the relationship between efflux, kinases, and ionic conditions including PKC, PKCβ, Ca2+, and Ca1+-calmodulin regulated protein kinase (CaMK) (Gnegy et al., 2004; Johnson, Guptaroy, Lund, Shamban, & Gnegy, 2005; Kantor, Hewlett, & Gnegy, 1999). [...] With respect to DAT most studies have focused on PKC, CaMK, and mitogen activated protein kinases (MAPKs), but many other signaling and kinase pathways have been implicated in efflux mechanisms for all of the MATs (Bermingham & Blakely, 2016; Vaughan & Foster, 2013). [...] DAT phosphorylation stimulated by AMPH or METH also occurs on this domain and is blocked by PKC inhibitors (Cervinski et al., 2005; Karam et al., 2017), indicating the capacity of the drugs to involve the PKC pathway. How this occurs is not known, but could potentially follow from drug perturbation of local ion/Ca2+ concentrations that impact PTM enzymes, or alternatively could occur by substrate-driven induction of transporter conformations in which phosphorylation sites become more or less available to enzyme action. [...] There are many other Ser and Thr residues on DAT intracellular loops and domains that may serve as phosphorylation sites, and in vitro studies have demon- strated phosphorylation of N— and C—terminal peptide sequences by many kinases in addition to PKC, ERK, and CaMK that have been implicated in regulation and efflux including protein kinase A (PKA), protein kinase G (PKG), casein kinase 2 (CK2), p38 kinase, JNK2, Cdk5, and Akt1 (Gorentla I et al., 2009; Vaughan & Foster, 2013). [...] The regulatory, efflux, and post-translational characteristics of NET and SERT show many similarities to DAT that indicate conservation of mechanism, but also some distinct properties that reflect requirements of specific neuronal populations. [...] With respect to efflux there are many similarities between SERT, NET, and DAT that indicate conservation of mechanisms.
However, another significant question remains: how might amphetaminergic substrates activate PKC in the first place? PKC activity is regulated by Ca2+ and diacylglycerol, a phospholipid metabolite generated in concert with inositol triphosphate by the enzyme phospholipase C (PLC), which is also Ca2+ dependent. [...] Usually, PLC is activated by agonist binding at GPCRs coupled to the Gq/11- type α-subunit; however, compounds, such as amphetamine and methamphetamine, lack significant affinity for GPCRs other than the trace amine-associated receptor 1 (TAAR1), a Gs-coupled receptor.74 [...] Interestingly, inhibition of the Na+/Ca2+ antiporter with amiloride also blocked amphetamine-induced PKC activation, suggesting that the ionic effects of DAT substrate translocation can directly influence activation of intracellular signaling cascades. When substrates are transported across the plasma membrane via the DAT, sodium ions are cotransported, and this increase in intracellular sodium concentration may cause Na+/Ca2+ antiporters at mitochondrial and endoplasmic reticulum membranes to operate in reverse, favoring a net flux of Ca2+ into the cytosolic compartment. [...]
Amphetamine-induced transport reversal at the closely related dopamine transporter (DAT) has been shown previously to be contingent upon modulation by calmodulin kinase IIα (αCaMKII). Here, we show that not only DAT, but also SERT, is regulated by αCaMKII. Inhibition of αCaMKII activity markedly decreased amphetamine-triggered SERT-mediated substrate efflux in both cells coexpressing SERT and αCaMKII and brain tissue preparations. [...] Moreover, we found that genetic deletion of αCaMKII impaired the locomotor response of mice to [MDMA] and blunted d-fenfluramine-induced prolactin release, substantiating the importance of αCaMKII modulation for amphetamine action at SERT in vivo as well. SERT-mediated substrate uptake was neither affected by inhibition of nor genetic deficiency in αCaMKII.
[...] AMPH is a substrate of DAT and causes nonexocytotic efflux of DA by reversing the direction of transport via DAT through a yet not fully understood mechanism (4–7). To improve our insights into AMPH-induced efflux with a focus on the unsettled role of Ca2+, we establish here a novel approach enabling simultaneous assessment of DA efflux and intracellular signaling pathways by live fluorescent imaging of cultured DA midbrain neurons. [...] our data provide strong evidence that AMPH-induced DA efflux via DAT in DArgic neurons does not require Ca2+ but primarily relies on the concerted action of AMPH on VMAT2 and DAT. [...] [...] Summarized, we employ a live imaging approach enabling parallel monitoring of DA efflux and activation of intracellular signaling pathways in cultured DArgic neurons. Our data reveal in the neurons profound AMPH-induced DA efflux that strikingly neither requires Ca2+ signaling nor activation of the kinases PKC and CaMKIIα. However, the efflux depends on both the activity of VMAT2 and DAT, substantiating the unequivocal concerted importance of both transporters for the pharmacological action of AMPH on DArgic neurons. [...]
Although a detailed and rigorous description of the regional distribution of TAAR1 protein and mRNA expression has yet to be accomplished, it is established that TAAR1 protein and mRNA are expressed in monoaminergic brain regions.3,16,26 In our laboratory, we exploited our prowess with assessing changes in kinetic activity of the dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter (SERT) to assess TAAR1 functionality. Because many of the identified ligands that activate TAAR1 are also substrates at the monoamine transporters and TAAR1 localization in transfected cells was shown to remain largely intracellular,4 we had reasoned early on that monoamine transporters could serve as conduits for TAAR1 agonists to enter cells, thereby providing access of the agonist to an intracellular pool of TAAR1 receptors. We speculated that if this were the case there could be a special relationship between TAAR1 and monoamine transporters because they share some of the same ligands. Indeed, we observed substantially enhanced CRE-luciferase signaling in transfected cells that co-expressed both TAAR1 and a monoamine transporter (DAT, NET or SERT).9,16 Because monoamine transporter kinetic regulation and internalization is a consequence of upregulated cellular phosphorylation cascades,27,28,29 we reasoned that the enhanced TAAR1 signaling could in turn trigger changes in the kinetic activity of the monoamine transporters under conditions of co-localization. This too was born out in a series of studies from our laboratory which demonstrated that TAAR1 activation drives the PKA and PKC cellular signaling cascades that result in inhibition of monoamine uptake and transporter reversal (efflux) in DAT/TAAR1, NET/TAAR1 and SERT/TAAR1 co-transfected cells in vitro, as well as in mouse and primate striatal (DAT, SERT) and thalamic (NET) synaptosomes ex vivo.22,23,24,25,30 TAAR1 specificity for mediation of the observed kinetic effects on the monoamine transporters was further confirmed in these studies by demonstrating the absence of noncompetitive effects of the trace amine beta-phenylethylamine (β-PEA),23 the common biogenic amines,24 and methamphetamine25 on uptake inhibition and substrate-induced monoamine efflux in synaptosomes generated from TAAR1 knockout mice. Accordingly, specific drugs that target TAAR1 will likely result in alterations in monoamine kinetic function and brain monoamine levels via this mechanism.
In a later study, Xie and Miller (2009a) resolved this issue by demonstrating that methamphetamine could induce [3 H]dopamine efflux in DAT cells or in TAAR1 knockout mouse striatal synaptosomes when the cells or synaptosomes were loaded with high concentrations of [ 3 H]dopamine (1 μM or higher), indicating that the direct effect of methamphetamine on the dopamine transporter to cause [3 H]dopamine efflux is dependent on the pre-loading concentration of [3 H]dopamine. But the efflux caused by methamphetamine under these conditions in DAT cells was not associated with either the PKA or the PKC phosphorylation pathways whereas it was blocked by methylphenidate. Further studies showed that TAAR1-mediated effects of methamphetamine on [3 H]dopamine efflux occurred at both lower and higher (1 μM) loading concentrations of [ 3 H]dopamine in TAAR1-DAT cells or in wild type mouse striatal synaptosomes, which could be blocked not only by methylphenidate but also by the PKC inhibitor Ro32-0432 (Xie and Miller 2009a). At the higher loading concentration, there was a greater amount of efflux observed in the presence of TAAR1 (in TAAR1-DAT cells or wild-type mouse striatal synaptosomes) than in its absence (in DAT cells or TAAR1 knockout mouse striatal synaptosomes), and it was only this relative greater amount of efflux that was sensitive to PKC inhibition. Accordingly, the TAAR1-mediated effects of methamphetamine on [3 H]dopamine efflux are dependent on PKC phosphorylation, whereas an observed TAAR1-independent, PKC-independent efflux occurs when cells or synaptosomes are loaded with high concentrations of [ 3 H]dopamine. [...] In a different mouse line, Lindemann et al. (2008) reported that TAAR1 knockout mice show an increased locomotive response to d-amphetamine after a single application of either 1 or 2.5 mg/kg. The increased locomotion correlated with a two- to threefold increase in extracellular dopamine, norepinephrine and serotonin levels in the TAAR1 knockout mice as compared with wild-type mice. [...] In further studies, electrophysiological analysis of dopaminergic neurons in the ventral tegmental area of TAAR1 knockout and wild-type mice revealed that the spontaneous firing rate of dopaminergic neurons was 8.6-fold higher in the TAAR1 knockout mice, and that activation of TAAR1 by 10 μM p-tyramine decreased the firing rate of dopaminergic neurons in wild type mice but not in TAAR1 knockout mice (Lindemann et al. 2008).
In both wild-type and Taar 1-/- mice, amphetamine produced a robust increase in striatal release of DA, norepinephrine (NE), and serotonin (5-HT), but this effect was significantly greater in Taar 1-/- mice than in wild type mice (Lindemann et al., 2008; Wolinsky et al., 2007). These findings support the notion that TAAR 1 functionally operates as a negative modulator of monoaminergic neurotransmission. [...] amphetamine had no effect on the release of DA and NA in the NAc of Taar 1 Tg mice, which partially explains the finding that amphetamine-induced a weaker locomotor hyperactivity in Taar 1 Tg mice as compared to their wild type counterparts. [...] There are evidence that TAAR 1 can modulate DAT function in in vitro cell culture experiments. Xie et al reported that β-phenethylamine (β-PEA) inhibited uptake and induced efflux of monoamines in thalamic synaptosomes of rhesus monkeys and wild-type mice, but not in synaptosomes of Taar 1-/- mice. Furthermore, the effect of β-PEA on efflux was blocked by transporter inhibitors in either the transfected cells or wild-type mouse synaptosomes (Xie and Miller, 2008). They also found that methamphetamine inhibited DA uptake, enhanced dopamine efflux, and induced DAT internalization by acting as a TAAR 1 agonist (Xie and Miller, 2009). However, more recent studies show inconsistent results. Leo et al. found that the DA clearance was not changed in Taar 1-/- mice as compared to their wild type counterparts (Leo et al., 2014), suggesting that the DAT function remained intact. In addition, no significant changes of dopamine uptake were observed in slices from Taar 1-/- mice, suggesting that the effect of TAAR 1 activation on DA-related function is independent of DAT (Leo et al., 2014). These apparent discrepancies may be attributable to the assays and tissues used in the studies. Xie et al. used mouse and monkey cellular synaptosome preparations with tissues from putamen and thalamus (Xie and Miller, 2008) while Leo et al. used fast scan cyclic voltammetry to measure DA uptake in mouse striatal slices (Leo et al., 2014). [...] Taar 1-/- mice displayed a behavioral phenotype of supersensitivity to amphetamine and cocaine, as evidenced by drug-induced increases in both locomotor activity and rearing as compared with wild-type littermates (Lindemann et al., 2008; Wolinsky et al., 2007). In contrast, Taar Tg mice responded to amphetamine in an opposite manner and showed a reduced and delayed response to amphetamine as compared with wild-type mice, indicating that Taar Tg mice are hyposensitive to amphetamine (Revel et al., 2012a). Methamphetamine also produced similar behavioral outcome in Taar 1-/- mice (Achat-Mendes et al., 2012).
A significant body of in vitro research has investigated TAAR1 modulation of DAT, primarily by the Miller laboratory. [...] Functionality of DAT is also modulated by TAAR1 as application of DA inhibited [3H]DA uptake and induced [3H]DA release in TAAR1/DAT cells compared to cells only expressing DAT (Xie and Miller, 2007; Xie et al., 2008b). [...] However, it has been argued the conduction of this research in vitro diminishes its validity. Administration of β-PEA or the TAAR1 agonist RO5166017 diminishes hyperlocomotion in DAT-KO mice, indicating activation of TAAR1 functions independently of DAT (Sotnikova et al., 2004; Revel et al., 2011). This theory is bolstered by FSCV experiments. Evoked DA release and uptake, measured by Tau and half-life, are the same between genotypes. DA overflow is greater in the NAc of Taar1-KO than -WT mice, attributed to increased basal DA levels, but DA uptake is still the same between genotypes (Leo et al., 2014). Similarly, the partial TAAR1 agonist RO5203648 diminishes cocaine-induced DA overflow in the NAc without altering DA uptake, also indicating a DAT-independent mechanism (Pei et al., 2014). Further research is needed to better elucidate the interaction between TAAR1 and DAT.
A study using the TAAR1-specific antagonist EPPTB showed that presynaptic blockade of TAAR1 reversed its ability to inhibit the firing rate of VTA dopaminergic neurons, which it normally brings about by activating inwardly rectifying potassium channels [45,48]. An increase in firing rates upon blocking TAAR1 with EPPTB suggests that TAAR1 is either constitutively active or under constant tonic activation from endogenous agonists. [...] A presynaptic interaction between TAAR1 and DAT function has also been suggested because some groups found evidence in cell culture that TAAR1 agonists inhibit DAT function via TAAR1 and D2AR [49,50,65]. Others find, however, that the effects of TAAR1 agonists and antagonist are unaltered in Dat−/− knockout mice, and that dopamine reuptake is unaffected by application of TAAR1 (ant)agonists or in TAAR1-KO animals [46,47]. One hypothesis that could unite the apparently conflicting findings is that TAs exert an inhibitory effect directly on DAT; indeed, PEA administration induces transient hyperlocomotion in mice, similar to the behaviour observed in Dat−/− animals [30]. More recent work suggests that TAAR1 activation mediates internalisation of DAT through regulating RhoA activity [53].
Previous research on TAAR1 modulation of DAT function has produced equivocal findings. In vitro, MA inhibits [3H]DA uptake, and [3H]DA release is increased in striatal tissue from Taar1 WT compared with KO mice (Xie and Miller, 2009). Similar findings were described in cells cotransfected with TAAR1 and DAT, compared with cells transfected only with DAT, in which MA-induced [3H]DA uptake inhibition and release were increased (Xie and Miller, 2007, 2009). However, these findings indicate MA-induced impairment of DAT function is increased when TAAR1 is activated, as opposed to in vivo treatment with AMPH or MDMA, by which striatal extracellular DA levels are increased when TAAR1 is not activated (Wolinsky et al., 2007; Lindemann et al., 2008; Di Cara et al., 2011). We were unable to replicate the results of Xie and Miller (2009) under similar in vitro conditions (Fig. 3). There was no difference in IC50 values for [3H]DA uptake inhibition by MA between synaptosomes from Taar1 WT and KO mice. [...] our results do not support an earlier hypothesis that TAAR1 modulates DAT (Xie and Miller, 2007, 2009; Xie et al., 2008b), as there was no evidence of an interaction under conditions described above. Recent reports support our findings that the DAT is unaffected by TAAR1. Coadministration of MA and the TAAR1 partial agonist RO523648 did not alter [3H]DA uptake and release in striatal synaptosomes in rats (Cotter et al., 2015). Fast-scan cyclic voltammetry showed no difference in DA clearance, as mediated by DAT, in striatal tissue from Taar1 WT compared with KO mice (Leo et al., 2014). [...] Given the lack of interaction, DAT is an improbable mediator of TAAR1 regulation of MA-induced neurotoxicity. [...] activation of TAAR1 did not modulate in vitro MA-impairment of DAT function or DAT expression. As TAAR1 activation did not alter the function or expression of DAT in whole synaptosomes or VMAT2 located on membrane-associated vesicles, these results indicate TAAR1 does not interact with these transporters on the plasma membrane but does affect intracellular VMAT2 function.
Importantly, we have documented that neither Tau nor the half-life of released DA are changed in slices from TAAR1-KO animals, indicating that TAAR1-KO mice exhibit unaltered DA uptake ability and thereby normal dopamine transporter (DAT) functionality. It is believed that Tau and the half-life of released DA are reliable measures for detecting changes in DA uptake because they are strongly correlated with changes in the Km of DAT mediated DA uptake (Yorgason et al., 2011). Thus, these neurochemical in vivo studies, as well as previous demonstrations of the functional activity of TAAR1 ligands in mice lacking the DAT (Sotnikova et al., 2004; Revel et al., 2012a), provide little support for the postulated role of TAAR1 in modulating DAT activity that is based mostly on in vitro cell culture experiments (Miller et al., 2005; Xie et al., 2008; Miller, 2011). [...] Notably, neither [the TAAR1 agonist (RO5166017) nor the TAAR1 antagonist (EPPTB)] changed the kinetics of DA uptake as evidenced by the Tau and DA half-life estimations, indicating that DAT-mediated function is not altered by the action of the drugs on TAAR1.
While our data suggest a role for TAAR1 in eliciting amphetamine-like stimulant effects, it must be borne in mind that the observed in vivo effects are likely to result from interaction with both TAAR1 and monoamine transporters. Thus it has been shown that the selective TAAR1 agonist RO5166017 fully prevented psychostimulant-induced and persistent hyperdopaminergia-related hyperactivity in mice.42 This effect was found to be DAT-independent, since suppression of hyperactivity was observed in DAT-KO mice.42 The collected information leads us to conclude that TAAR1 is a stereoselective binding site for amphetamine and that TAAR1 activation by amphetamine and its congeners may contribute to the stimulant properties of this class of compounds. [...] it has been shown that β-PEA and methamphetamine effects in cells expressing TAAR-DAT significantly exceed those observed in cells expressing DAT only. Consistent with this conclusion is the higher potency of (S)-[amphetamine] in rat synaptosomes relative to cloned human DAT cells (EC50 60 nM vs 240 nM).
The DAT vs SERT ratios of synthetic cathinones are presented in Table 1. [...] Table 1. In vitro dopamine and serotonin uptake transporter inhibition (IC50 values) and release data (EC50 values). [...] The DAT vs SERT ratios of amphetamine derivatives and cocaine are presented in Table 2. [...] Table 2. In vitro dopamine and serotonin uptake transporter inhibition (IC50 values) and release data (EC50 values). [...] * in vitro studies for neurotransmitter reuptake inhibition and release using HEK293 (Human Embryonic Kidney 293) cell line expressing DAT and SERT (IC50 and EC50 values are expressed in μM). [(Note: HEK293 cells lack TAAR1 expression: "HEK-293 cells do not express TAAR1 (Reese et al., 2007; Xie and Miller, 2007)" (Small et al., 2023 [doi:10.1124/jpet.122.001573]).)]
[AMPH and METH] promote DA release from synaptic storage vesicles into the cytoplasm (Partilla et al., 2006) and then into the extracellular space via DAT with an EC50 of 25 nM and a Ki uptake in rat synaptosomes of 34 nM (Rothman et al., 2001). These are drug concentrations approximately 20-30 fold lower than the EC50 values Reese et al. (2007) calculated for eliciting an in vitro functional response from rTAAR1 (0.8 μM). However, experienced METH users can typically consume gram quantities of drug per day (Kramer et al., 1967) and achieve peak blood concentrations of 100 μM (Derlet et al., 1989; Baselt, 2002; Peters et al., 2003). [...] The extracellular free concentration of METH surrounding relevant human dopaminergic synapses in the brain presumably is the relevant pharmacodynamic parameter, at least in part, underlying the desirable effects of the drug. Although this value is not known with certainty for humans METH serum levels typically represent one tenth of what is found in rat brain (Riviere et al., 2000). Consequently, when considered together with the human forensic evidence the in vitro results of Reese et al. (2007) are consistent with the interpretation that in vivo the human TAAR1 is likely to be a mediator of at least some of METH's effects.
The EC50 of (+)-AMPH to release DA via DAT is 25 nM, with Ki uptake values in rat synaptosomes for this neurotransmitter of 34 nM at the DAT, concentrations approximately 20- to 30-fold lower than the EC50 values we calculated for eliciting an in vitro functional response from rTAAR1 (0.8 μM). Chronic METH abusers can typically consume gram quantities of drug per day (Kramer et al., 1967). [...] plasma concentrations of both drugs can reach into the high-micromolar range (Drummer and Odell, 2001; Baselt, 2002; Peters et al., 2003). Although the extracellular free concentration of METH around relevant human dopaminergic synapses presumably involved in producing desirable CNS effects is not known with certainty, in rats METH serum levels are typically 1/10 what is found in brain (Riviere et al., 2000). Forensic evidence indicates that experienced METH users can typically achieve peak blood concentrations of 100 μM (Baselt, 2002; Peters et al., 2003). Both isomers of METH were full agonists of h-rChTAAR1 over a range of EC50 values from 3.5 to ~15 μM, concentrations often exceeded in the blood of human METH addicts (Derlet et al., 1989). If TAAR1s, whether expressed in the CNS or periphery, are exposed to such concentrations, it is possible they could become functionally activated. [...] Given that the EC50 values for METH and AMPH activation of the h-rChTAAR1 in vitro are well below the concentrations frequently found in addicts, we suggest that TAAR1 might be a potential mediator of some of the effects of AMPH and METH in humans, including hyperthermia and stroke.
Interestingly, the concentrations of amphetamine found to be necessary to activate TAAR1 are in line with what was found in drug abusers [3, 51, 52]. Thus, it is likely that some of the effects produced by amphetamines could be mediated by TAAR1. Indeed, in a study in mice, MDMA effects were found to be mediated in part by TAAR1, in a sense that MDMA auto-inhibits its neurochemical and functional actions [46]. Based on this and other studies (see other section), it has been suggested that TAAR1 could play a role in reward mechanisms and that amphetamine activity on TAAR1 counteracts their known behavioral and neurochemical effects mediated via dopamine neurotransmission.
That TAAR1 signaling is coupled to the inhibition of VTA DA neuron firing was a surprising finding. [...] earlier data suggested that TAAR1 activation leads to inhibition of DA uptake by DAT, promotion of DA efflux by DAT and DAT internalization which presumably would augment extracellular DA levels, whereas inhibition of DA neuronal firing rate would likely decrease extracellular DA levels, [...] this mismatch in expectations immediately attracted the attention of those trying to determine whether TAAR1 is a METH/AMPH and DA receptor in vivo as it is in vitro (Bunzow et al., 2001; Wainscott et al., 2007; Xie and Miller, 2009; Panas et al., 2012).
[...] it is unclear if TAAR1 plays any role in the effects of MDMA in humans, as MDMA does not activate human TAAR1 in cellular assays like it does mouse and rat TAAR1.84
Beside the TAAR1-KO mice, animals that overexpress taar1 in the brain were also generated, which was named as taar1 Tg mice (Revel et al. 2012a). Before discussing the behaviors of this line of taar1 Tg mice, it should be kept in mind that taar1 was expressed in all types of neurons in the whole brain of this mice strain, which is in contrast with the specifc expression pattern of that in the wildtype animals (Revel et al. 2012a). The electrophysiological results showed that excitatory and inhibitory inputs into the VTA were altered in the taar1 Tg mice (Revel et al. 2012a). Interestingly, although the basal levels of dopamine and norepinephrine in the nucleus accumbens (NAc) were elevated, amphetamine did not alter dopamine levels in the taar1 Tg mice (Revel et al. 2012a). Consistently, behavioral test showed that AMPH led to hyperactivity in WT but not in taar1 Tg mice (Revel et al. 2012a).
[...] a proposed intracellular target for amphetamine is the [TAAR1], a [GPCR] that is expressed on intracellular membranes in DA neurons (Miller, 2011). Phenethylamine stimulants have been proposed to activate TAAR1, leading to increased cAMP generation and RhoA activation, with subsequent enhancement of DAT reverse transport and endocytosis (Xie and Miller, 2007, 2008, 2009; Underhill et al., 2021). Methamphetamine-induced DAT endocytosis was found to be dependent on TAAR1 expression and PKA activity as suggested by use of the kinase inhibitor H89 (Xie and Miller, 2009). However, evidence indicating that amphetamine-induced endocytosis is independent of TAAR1 includes 1) HEK-293 cells do not express TAAR1 (Reese et al., 2007; Xie and Miller, 2007) but do exhibit amphetamine-induced DAT endocytosis [present studies and (Saunders et al., 2014; Cheng et al., 2015; Wheeler et al., 2015)]; 2) cAMP and PKA activation, which are stimulated by TAAR1, antagonized amphetamine-induced DAT endocytosis in heterologous cells and DA neurons [present studies and (Wheeler et al., 2015)]; and 3) in cell lines and rodent striatal synaptosomes, PKA activation increased DAT Vmax, consistent with increased plasma membrane expression (Pristupa et al., 1998; Page et al., 2004; Batchelor and Schenk, 2018). Additionally, DMAA induced DAT endocytosis (Figs. 3 and 4) despite exhibiting no activity at human TAAR1 in receptor binding studies (Rickli et al., 2019). Therefore, although some evidence does support a role of TAAR1 in modulating amphetamine-induced DAT endocytosis, the present studies suggest that DMAA and amphetamine promote DAT endocytosis through a TAAR1-independent mechanism.
Amphetamine might act on TAAR1 to activate Gαs pathway and phosphorylate monoamine transporter such as dopamine transporter (DAT), leading to its internalization and ceased transport (Bunzow et al. 2001; Miller 2011). Behaviorally, knockout of TAAR1 in mice leads to locomotor supersensitivity induced by amphetamine, although the connection to regulation of monoamine system is undetermined and needs further investigation (Achat-Mendes et al. 2012; Lindemann et al. 2008).
In a recent study it was reported that AMPH induced locomotor function of TAAR1 knockout mice was increased compared to the response of wild type mice (Lindemann et al., 2007). In addition, they reported a 2½ fold increase in dopamine, noradrenaline, and serotonin release, in response to AMPH, in TAAR1 knockout mice compared to wild type mice. These results are similar to the increase in DA and NE release reported in a study of another TAAR1 KO mouse (Wolinsky et al., 2007). These findings of increased DA release and increased locomoter effects in the TAAR1 KO are reconciled with the proposed model as follows. The increased DA release of the TAAR1 KO may be due to reduced DAT internalization. With more DAT (or NET or SERT) remaining at the cell surface, more neurotransmitters can be reverse exchanged upon application of AMPH. The increased DA release would also explain the increased locomotor activity of the KO vs WT. The WT TAAR1 cells would undergo DAT internalization as described in the model. With fewer surface transporters, less DA would be reverse exchanged resulting in less DA release compared to levels in the KO mice.
Moreover, while deletion of TAAR1 led to increased frequency of spontaneous firing frequency of serotonin neurons in brain slices [36], TAAR1 full agonists significantly inhibited the firing frequency in DRN serotonin neurons [28, 38]. [...] In vitro electrophysiological recordings showed a remarkable increase in the spontaneous firing rate of dopamine neurons in the VTA of TAAR1-KO mice [36]. While the endogenous TAAR1 agonist TYR decreased the firing rate of dopamine neurons in slices from WT mice, it had no effect in slices from TAAR1-KO mice [36]. [...] Similar to TYR, [the TAAR1 full agonist] RO5166017 reduced the firing rate of VTA dopamine neurons and DRN serotonin neurons in brain slices from WT but not TAAR1-KO mice, through the activation of K+-mediated outward current, which can be blocked by TAAR1 antagonist EPPTB [38]. [...] [The TAAR1 full agonist] RO5256390 decreased the firing frequency of VTA dopamine and DRN serotonin neurons in brain slices from WT but not TAAR1-KO mice, [...]
β-Keto-analogue cathinones also exhibited approximately 10-fold lower affinity for the TA1 receptor compared with their respective non-β-keto amphetamines. [...] Activation of TA1 receptors negatively modulates dopaminergic neurotransmission. Importantly, methamphetamine decreased DAT surface expression via a TA1 receptor-mediated mechanism and thereby reduced the presence of its own pharmacological target (Xie and Miller, 2009). MDMA and amphetamine have been shown to produce enhanced DA and 5-HT release and locomotor activity in TA1 receptor knockout mice compared with wild-type mice (Lindemann et al., 2008; Di Cara et al., 2011). Because methamphetamine and MDMA auto-inhibit their neurochemical and functional effects via TA1 receptors, low affinity for these receptors may result in stronger effects on monoamine systems by cathinones compared with the classic amphetamines.
Drugs of abuse hijack brain-reward circuitry during the addiction process by augmenting action potential-dependent phasic dopamine release events associated with learning and goal-directed behavior. One prominent exception to this notion would appear to be amphetamine (AMPH) and related analogs, which are proposed instead to disrupt normal patterns of dopamine neurotransmission by depleting vesicular stores and promoting nonexocytotic dopamine efflux via reverse transport. This mechanism of AMPH action, though, is inconsistent with its therapeutic effects and addictive properties, which are thought to be reliant on phasic dopamine signaling. [...] These findings identify upregulation of exocytotic dopamine release as a key AMPH action in behaving animals and support a unified mechanism of abused drugs to activate phasic dopamine signaling. [...] Our results also raise the possibility that enhanced phasic dopamine signaling may underlie important clinical effects of AMPH. Modest activation of dopamine transients at the relatively low dose of 1 mg/kg used here may be an important action of medications that combine amphetamine salts (e.g., Adderall) used clinically (Joyce et al., 2007). Indeed, low doses of AMPH preserve and improve learning and behavioral performance (Mayorga et al., 2000; Wyvell and Berridge, 2000; Taylor and Jentsch, 2001; Zhang et al., 2003; Knutson et al., 2004). In contrast, high doses of AMPH can produce a behavioral profile similar to the positive symptoms associated with schizophrenia (Featherstone et al., 2007; Lisman et al., 2008). [...] Thus, disrupting the tight temporal concurrence between dopamine transients and important external cues due to hyperactive phasic dopamine signaling may contribute to AMPH-induced psychosis (Featherstone et al., 2007).
While there is little debate that behavioral effects of this important psychostimulant are associated with a hyperdopamine state [3–6], the underlying mechanisms by which this condition manifests have been the subject of intense study. Two, what ostensibly appear to be mutually exclusive, views have emerged. On the one hand, AMPH enhances tonic dopamine signaling by reversing dopamine transporter (DAT) direction, leading to a non-exocytotic, action potential-independent type of release or efflux that is driven by vesicular depletion and the redistribution of dopamine to the cytosol [7,8]. On the other hand, AMPH enhances phasic dopamine signaling by promoting burst firing of dopamine neurons [9,10], inhibiting dopamine uptake [11,12], and upregulating vesicular dopamine release [13,14]. How AMPH concurrently activates tonic and phasic dopamine signaling, the two fundamental modes of communication used by dopamine neurons [15], yet elicits opposing actions on vesicular dopamine stores is perplexing and unresolved.
We found that σ1R activation prevents methamphetamine-induced, DAT-mediated increases in firing activity of dopamine neurons. In vitro and in vivo amperometric measurements revealed that σ1R activation decreases methamphetamine-stimulated dopamine efflux without affecting basal dopamine neurotransmission. Consistent with these findings, σ1R activation decreases methamphetamine-induced locomotion, motivated behavior, and enhancement of brain reward function. Notably, we revealed that the σ1R interacts with DAT at or near the plasma membrane and decreases methamphetamine-induced Ca2+ signaling, providing potential mechanisms.
The sampling of extracellular fluids using in vivo microdialysis provides a direct way to study drug effects on synaptic efflux of a neurotransmitter. Comparisons of dexfenfluramine and fluoxetine using this technique have revealed a significant difference between the effects of reuptake inhibition and release on extracellular 5-HT levels.34−36 For example, Tao et al.35 compared the effects of fluoxetine and dexfenfluramine on extracellular 5-HT in the hypothalamus, with the latter increasing basal 5-HT 6−16-fold, compared to 3-fold following fluoxetine (Figure 1A). These differences in magnitude reflect differences in the way that fluoxetine and dexfenfluramine elevate 5-HT. The ability of SSRIs to increase extracellular 5- HT is dependent on neuronal activity (i.e., impulse dependent) and is subject to autoreceptor feedback inhibition. In contrast, (dex)fenfluramine increases extracellular 5-HT by disrupting vesicular storage and promoting nonexocytotic release that is independent of both of these regulatory factors.36 [...] Figure 1. Comparison between a 5-HT releaser (dexfenfluramine) and SSRI (fluoxetine) on (A) extracellular 5-HT release sampled from rat hypothalamus, [...]
Although the precise physiological role of TAAR1/G13-mediated DAT internalization remains to be established, it is likely that this pathway evolved to regulate the actions of endogenous amines including β-PEA, tyramine, 3-methoxytyramine, and DA. DA is a TAAR1 agonist (Fig. 1c) and recently, it was shown that the sustained elevation of extracellular DA seen following burst firing appears to be a consequence of DAT internalization by a Rho-dependent mechanism [51]. These results provide the first evidence that dopamine itself can act as a TAAR1 agonist to trigger transporter internalization in vivo. TAAR1 signaling also has the potential to serve as a sensor of cytoplasmic DA and its metabolites. At high cytosolic concentrations, DA becomes neurotoxic due to the formation of reactive oxygen species and quinones that can cause damage to the neuron [52]. Under normal conditions, DA transported through the DAT from the extracellular space is rapidly repackaged into synaptic vesicles or enzymatically metabolized. However, if cytoplasmic DA concentrations saturate these mechanisms, the reduction in surface levels of DAT mediated by TAAR1/G13 signaling may provide an additional means for maintaining free cytosolic DA below toxic levels.
This flooding of the cytoplasm and synaptic space with the oxidatively labile DA is thought to be a critical first step in the neurotoxic cascade of the amphetamines [73]. These conditions of elevated concentrations of cytosolic monoamines could be further aggravated by inhibition of MAO [91]. Unlike amphetamines, mephedrone and methylone have little if any affinity for VMAT-2 [33]. Therefore, their lack of neurotoxicity could derive from an inability to promote the release of DA from storage vesicles into the cytoplasm.
It has been suggested that the association between PD and ADHD may be explained, in part, by toxic effects of these drugs on DA neurons.241 [...] An important question is whether amphetamines, as they are used clinically to treat ADHD, are toxic to DA neurons. In most of the animal and human studies cited above, stimulant exposure levels are high relative to clinical doses, and dosing regimens (as stimulants) rarely mimic the manner in which these drugs are used clinically. The study by Ricaurte and colleagues248 is an exception. In that study, baboons orally self-administered a racemic (3:1 d/l) amphetamine mixture twice daily in increasing doses ranging from 2.5 to 20 mg/day for four weeks. Plasma amphetamine concentrations, measured at one-week intervals, were comparable to those observed in children taking amphetamine for ADHD. Two to four weeks after cessation of amphetamine treatment, multiple markers of striatal DA function were decreased, including DA and DAT. In another group of animals (squirrel monkeys), d/l amphetamine blood concentration was titrated to clinically comparable levels for four weeks by administering varying doses of amphetamine by orogastric gavage. These animals also had decreased markers of striatal DA function assessed two weeks after cessation of amphetamine.
Amphetamine treatment similar to that used for ADHD has been demonstrated to produce brain dopaminergic neurotoxicity in primates, causing the damage of dopaminergic nerve endings in the striatum that may also occur in other disorders with long-term amphetamine treatment (57).
Though the paradigm used by Ricaurte et al. 53 arguably still incorporates amphetamine exposure at a level above much clinical use,14,55 it raises important unanswered questions. Is there a threshold of amphetamine exposure above which persistent changes in the dopamine system are induced? [...]
Repeated exposure to moderate to high levels of methamphetamine has been related to neurotoxic effects on the dopaminergic and serotonergic systems, leading to potentially irreversible loss of nerve terminals and/or neuron cell bodies (Cho and Melega, 2002). Preclinical evidence suggests that d-amphetamine, even when administered at commonly prescribed therapeutic doses, also results in toxicity to brain dopaminergic axon terminals (Ricaurte et al., 2005).
The present findings with MDMA are consistent with our previous data [15,17] and those reported by Simmler et al. [4] and Eshleman et al. [23] who examined the effects of MDMA and related drugs in human embryonic kidney (HEK) cells transfected with human SERT, DAT and NET. Thus, the molecular mechanism of action for MDMA at monoamine transporters is similar in rats and humans. On the other hand, the potency of (±)MDMA for releasing monoamines in rat brain synaptosomes shown here (i.e., 60–70 nM) is greater than its potency in transfected HEK cells (i.e., 1–20 μM). Such discrepancies in absolute potency could be related to species differences in drug responsiveness, differences in release assay methods employed, or the absence of important neuronal membrane proteins in non-neuronal HEK cells.
Table 5. Action of MDMA, MDA, and PMMA as Releasing Agents at the Serotonin (SERT), Dopamine (DAT), and Norepinephrine (NET) Transporters18,59,60 [...] a Data, although from different publications, were obtained from the same laboratory.
PMMA is a 5-HT releasing agent. S(+)PMMA is a potent releaser of 5-HT (EC50 = 41 nM) and NE (EC50 = 147 nM) with reduced activity as a releaser of DA (EC50 = 1,000 nM); the R(−)isomer of PMMA is a releaser of 5-HT (EC50 = 134 nM) with reduced potency for release of NE (EC50 = 1,600 nM) and DA (EC50 > 14,000 nM) (R.B. Rothman, unpublished data).
The most commonly studied DAT substrates are amphetamines, including amphetamine and methamphetamine (Fig. 9). S-(+)-amphetamine releases dopamine with an EC50 of 8.7 nM; the R-(−)-amphetamine is 3-fold weaker, at 27.7 nM (EC50) (Blough, Page et al. 2005). Although weaker, a similar trend is seen for the optical isomers of methamphetamine. S-(+)-methamphetamine releases dopamine with an EC50 of 24.5 nM, while the R-(−)-methamphetamine is 16-fold less active at 416 nM (EC50) (Blough, Page et al. 2005). [...] Blough, B. E., K. M. Page, et al. (2005). "Struture-activity relationship studies of DAT, SERT, and NET releasers." New Perspectives on Neurotransmitter Transporter Pharmacology.
While there is little debate that behavioral effects of this important psychostimulant are associated with a hyperdopamine state [3–6], the underlying mechanisms by which this condition manifests have been the subject of intense study. Two, what ostensibly appear to be mutually exclusive, views have emerged. On the one hand, AMPH enhances tonic dopamine signaling by reversing dopamine transporter (DAT) direction, leading to a non-exocytotic, action potential-independent type of release or efflux that is driven by vesicular depletion and the redistribution of dopamine to the cytosol [7,8]. On the other hand, AMPH enhances phasic dopamine signaling by promoting burst firing of dopamine neurons [9,10], inhibiting dopamine uptake [11,12], and upregulating vesicular dopamine release [13,14]. How AMPH concurrently activates tonic and phasic dopamine signaling, the two fundamental modes of communication used by dopamine neurons [15], yet elicits opposing actions on vesicular dopamine stores is perplexing and unresolved.
We found that σ1R activation prevents methamphetamine-induced, DAT-mediated increases in firing activity of dopamine neurons. In vitro and in vivo amperometric measurements revealed that σ1R activation decreases methamphetamine-stimulated dopamine efflux without affecting basal dopamine neurotransmission. Consistent with these findings, σ1R activation decreases methamphetamine-induced locomotion, motivated behavior, and enhancement of brain reward function. Notably, we revealed that the σ1R interacts with DAT at or near the plasma membrane and decreases methamphetamine-induced Ca2+ signaling, providing potential mechanisms.
Although the pharmacological effect of amphetamine is predominantly mediated by monoamine release, this mechanism is complemented by reuptake inhibition [...] that combine additively or synergistically to augment synaptic monoamine concentrations. The description of amphetamine as a 'monoamine reuptake inhibitor' often causes some confusion, and the difference between the mechanisms of amphetamine, which is a competitive reuptake transport substrate, and classical reuptake inhibitors is illustrated in Figure 3. [...] d-Amphetamine is generally accepted to be a weak dopamine reuptake inhibitor with a Ki value of ~100 nM, a moderately potent inhibitor of noradrenaline reuptake (Ki = 40–50 nM) and a very weak inhibitor of 5-HT reuptake (Ki = 1.4-3.8 µM). [...] the efficacy of amphetamine relative to other indirect monoamine agonists, for example classical reuptake inhibitors, can only be estimated from in vivo experiments. [...] [d- and l-Amphetamine] dose-dependently increased the extracellular concentrations of noradrenaline in the prefrontal cortex (PFC) and dopamine in the striatum. The pharmacodynamics of their effects are typical of those reported for monoamine releasing agents, i.e. a fast onset of action with peak increases of noradrenaline and dopamine efflux occurring at 30–45 min, large effects (400–450% of baseline for noradrenaline and 700–1500% of baseline for dopamine), with a relatively rapid decline after the maximum (Figure 4). [...] the magnitude of the increases produced by amphetamine's isomers are greater than those reported for classical reuptake inhibitors such as atomoxetine or bupropion, and there is no dose-effect ceiling to amphetamine's actions [...] The primary action of amphetamine is to increase synaptic concentrations of monoamine neurotransmitters, thereby indirectly enhancing noradrenergic, dopaminergic neurotransmission in the CNS.
Converging lines of evidence have solidified the notion that DA releasers are substrates of the transporter and once translocated, they reverse the normal direction of transporter flux to evoke release of endogenous neurotransmitters. The nature of this reversal is not well understood, but the entire process is primarily transporter-dependent and requires elevated intracellular sodium concentrations, phosphorylation of DAT, and possible involvement of transporter oligomers (Khoshbouei et al., 2003, 2004; Sitte and Freissmuth, 2010). [...] A library of approximately 1400 phenethylamine compounds (PAL compounds) has been screened using these protocols. Among the active compounds,the smaller DAT ligands were found to be DA releasers while the sterically larger compounds were DAT uptake inhibitors. [...] Generic pharmacophore for biogenic amine transporter ligands. Note that transportable substrate ligands exhibit size constraints defined by the red circle. Functional groups attached to the nitrogen, α-carbon or phenyl ring that extend beyond the "edge" of the pharmacophore will generate partial substrates, transporter blockers or be inactive. [...] phenmetrazine was found to be completely inactive at VMAT2 indicating that a direct interaction of the releaser with VMAT2 is not required for inducing neurotransmitter efflux into the extracellular space (Partilla et al., 2006). Phentermine and benzylpiperazine were also found in the same study to lack VMAT2 activity (Table 5).
Despite the knowledge that amphetamine is a substrate for the DAT and NET, questions still remain as to the physiological mechanism of amphetamine action. [...] At lower doses, amphetamine preferentially releases a newly synthesized pool of DA. [...] DA stores will not be depleted by the AMPT in these short time frames, leading to the conclusion that newly synthesized DA is a principal substrate for amphetamine-stimulated DA efflux. [...] Controversy has surrounded the role of VMAT2 and synaptic vesicles in the mechanism of amphetamine action. [...] Undoubtedly vesicles contribute strongly to the maximal DA released by amphetamine, although VMAT2 is not absolutely required for amphetamine to release DA from nerve terminals (Pifl et al. 1995; Fon et al. 1997; Wang et al. 1997; Patel et al. 2003). [...] Recently, a new model of amphetamine action has been formulated that proposes that amphetamine elevates tonic DA (non-exocytotic) signaling through reverse transport and depleting vesicular stores, but activates phasic DA signaling by enhancing vesicular DA release from the readily releasable pool (Covey et al. 2013). These conclusions were drawn from experiments using fast-scan cyclic voltammetry in either freely moving or anesthetized rats (Avelar et al. 2013; Daberkow et al. 2013). Again, one must strongly consider the dose of amphetamine in interpretation of these actions (Calipari and Ferris 2013).
While the sine qua non property of AMPH at monoamine transporters is the promotion of monoamine release via reverse transport, there are yet profound mysteries in understanding how this works. It is additionally clear that AMPH is an uptake blocker as well as a releaser, and differentiating between elevating extracellular monoamines by reverse transport or uptake blockade can be difficult. Of course, the many AMPH derivatives and different transporters maintain different combinatorial properties, an important topic beyond the range of this article. [...] The mechanism of how reverse transport occurs is unknown, and a very old issue of how reserpine can inhibit uptake but not halt reverse transport remains opaque.
[...] most amphetamines are [monoamine transporter] substrates, which pervert the relay to elicit efflux of monoamines into the synaptic cleft. However, some amphetamines act as transporter inhibitors. [...] Amphetamines also bind to the trace amine receptors TAR1, [...] TAR1 is not activated by all psychoactive amphetamines (p-chloroamphetamine, for instance, is inactive); stimulation of TAR1 actually reduces dopamine release and thus decreases sensitivity to amphetamine [18,19]. [...] amphetamines are taken up by plasma-membrane monoamine transporters as exogenous substrates [31]. Accordingly, they inhibit the physiological monoamine reuptake in a competitive manner [36]. As a consequence of both amphetamine-induced reverse transport and inhibition of reuptake, the synaptic monoamine concentration increases, which in turn activates post- and presynaptic receptors [37]. The activation of postsynaptic receptors propagates the signal and contributes to the biological response. Stimulation of presynaptic autoreceptors decreases the quantal release of monoamines upon excitatory inputs; [...]
An interesting neurochemical issue is the interaction between compounds acting as blockers and substrates. [...] Substrates (e.g. mephedrone) use MAT proteins to release neurotransmitters, while inhibitors (e.g. [MDPV]) prevent the transport of any compounds through MATs. It follows that simultaneous use of a substrate and a blocker should result in an antagonistic mechanism that lowers the mutual potency. Paradoxically, MDPV and mephedrone occur together in mixtures, and in addition they reinforce each other's effects, resulting in very strong stimulation. Simultaneous use of both SCs is reported by users, and studies in rodents involving simultaneous administration of the drugs have shown a significant increase in locomotor activity and additive effect compared to administering these drugs alone (Allen et al., 2019; Benturquia et al., 2019). [...] The explanation for this phenomenon can be found in the action of organic cation transporter (OCT) proteins. OCTs are a family of proteins responsible for endothelial transport of small, organic, hydrophilic, and positively charged molecules, including neurotransmitters and xenobiotics (Couroussé & Gautron, 2015). OCT3 is a protein present in the dopaminergic regions of the central nervous system, where it promotes DA reuptake when it is inhibited for high affinity transporters (DAT) (Couroussé & Gautron, 2015). Monoamines can also be released by OCTs. [...] Ex vivo studies on superior cervical ganglia cells enriched in NET and OCT3 showed that in the presence of MDPV blocking MAT proteins, mephedrone causes neurotransmitter efflux through OCT3, which is insensitive to the inhibitory effects of MDPV. The release of monoamines through OCT3, a low‐affinity transporter, presumably explains the paradoxical synergistic effects of inhibitors and substrates (Mayer et al., 2019). [...] Another feature that distinguishes [synthetic cathinones (SCs)] from amphetamines is their negligible interaction with the trace amine associated receptor 1 (TAAR1). Activation of this receptor reduces the activity of dopaminergic neurones, thereby reducing psychostimulatory effects and addictive potential (Miller, 2011; Simmler et al., 2016). Amphetamines are potent agonists of this receptor, making them likely to self‐inhibit their stimulating effects. In contrast, SCs show negligible activity towards TAAR1 (Kolaczynska et al., 2021; Rickli et al., 2015; Simmler et al., 2014, 2016). [...] It is worth noting, however, that for TAAR1 there is considerable species variability in its interaction with ligands, and it is possible that the in vitro activity of [rodent TAAR1 agonists] may not translate into activity in the human body (Simmler et al., 2016). The lack of self‐regulation by TAAR1 may partly explain the higher addictive potential of SCs compared to amphetamines (Miller, 2011; Simmler et al., 2013).
A number of test drugs displayed no activity in the [3H]dopamine uptake inhibition assay (Table 1). For example, (+)- phenmetrazine and (–)-phenmetrazine, the major metabolites of phendimetrazine (Rothman et al., 2002), were essentially inactive. [...] In contrast, other amphetamine-type agents, such as phentermine, phenmetrazine, and 1-benzylpiperazine, are potent releasers of neuronal dopamine (Baumann et al., 2000, 2005; Rothman et al., 2002), but they are inactive at VMAT2. Agents such as these may prove to be valuable control compounds for determining the importance of vesicular release for the in vivo actions of amphetamine-type agents.
In contrast to assay systems involving non-neuronal cells transfected with transporter proteins, synaptosomes possess all of the cellular machinery necessary for neurotransmitter synthesis, release, metabolism, and reuptake. Synaptosomes, however, do not model all of the effects of amphetamine-type agents because the use of reserpine removes any contribution of the vesicular monoamine transporter VMAT2 (SLC18A2) to the release process. In addition to acting as a substrate for plasma membrane monoamine transporters, amphetamine also binds to VMAT, resulting in the redistribution of monoamines from vesicular stores to the cytoplasm (Sulzer et al. 1995; Partilla et al. 2006; Freyberg et al. 2016). Although transporter substrates can induce monoamine release in the absence of VMAT binding (Fon et al. 1997), it is important to recognize that 2-aminoindans may have effects in intact nerve terminals that are not fully replicated in synaptosomes. Follow-up studies will be conducted to evaluate whether 2-aminoindans are capable of interacting with VMAT.
2.2.1. DAT regulation by psychostimulants [...] Considerable evidence supports a role for DAT substrates, like amphetamine, in inducing DAT internalization in vivo and in vitro, thus decreasing DA uptake [390–395]. [...] While several kinases are involved in the regulation of DAT, PKC is by far the most thoroughly investigated [388,403]. PKC activation induces DAT internalization [393,404–406] although the mechanism of PKC activation by DAT substrates remains largely unknown [388,389,403,407,408]
Release by an indirect agonist is termed reverse transport and may require buildup of neurotransmitter in the nerve cytoplasm [...] Stimulants can act to stimulate presynaptic (CNS) or prejunctional (periphery) receptors for the neurotransmitter on nerve terminals to control release from those endings. These receptors are usually inhibitory and are marked as I (either NE, DA, or 5-HT inhibitory receptor) in Figure 1 and as α2 (inhibitory α2- adrenoceptor) in Figure 2, respectively. For instance, α2-adrenoceptors are present as autoreceptors on noradrenergic nerves and mediate an inhibition of NE release both in the periphery and CNS (see Figures 1 and 2).71 [...] Stimulants can act to stimulate, either directly or indirectly via increased release of the monoamine neurotransmitter, receptors for the neurotransmitter on nerves or effector cells situated postsynaptically/postjunctionally to the monoaminergic neuron. [...] Receptor-mediated actions of amphetamine and other amphetamine derivatives [...] may involve trace amine-associated receptors (TAARs) at which amphetamine and MDMA also have significant potency.85–87 Many stimulants have potency at the rat TAAR1 in the micromolar range but tend to be about 5 to 10 times less potent at the human TAAR1, [...] Activation of the TAAR1 receptor causes inhibition of dopaminergic transmission in the mesocorticolimbic system, and TAAR1 agonists attenuated psychostimulant abuse-related behaviors.89 It is likely that TAARs contribute to the actions of specific stimulants to modulate dopaminergic, serotonergic, and glutamate signaling,90 and drugs acting on the TAAR1 may have therapeutic potential.91 In the periphery, stimulants such as MDMA and cathinone produce vasoconstriction, part of which may involve TAARs, although only relatively high concentrations produced vascular contractions resistant to a cocktail of monoamine antagonist drugs.86
The characteristics and mechanisms of efflux are complex and incompletely understood, and have been summarized in many comprehensive reviews including (Reith & Gnegy, 2020; Robertson, Matthies, & Galli, 2009; Sitte & Freissmuth, 2015). [...] Early studies implicating protein phosphorylation in AMPH-evoked DA efflux were performed by Giambalvo, who demonstrated that DAT-mediated uptake of AMPH impacted the activity and subcellular localization of protein kinase C (PKC) with characteristics that correlated with DA efflux (Giambalvo, 1992), and Gnegy and colleagues, whose many studies reinforced the relationship between efflux, kinases, and ionic conditions including PKC, PKCβ, Ca2+, and Ca1+-calmodulin regulated protein kinase (CaMK) (Gnegy et al., 2004; Johnson, Guptaroy, Lund, Shamban, & Gnegy, 2005; Kantor, Hewlett, & Gnegy, 1999). [...] With respect to DAT most studies have focused on PKC, CaMK, and mitogen activated protein kinases (MAPKs), but many other signaling and kinase pathways have been implicated in efflux mechanisms for all of the MATs (Bermingham & Blakely, 2016; Vaughan & Foster, 2013). [...] DAT phosphorylation stimulated by AMPH or METH also occurs on this domain and is blocked by PKC inhibitors (Cervinski et al., 2005; Karam et al., 2017), indicating the capacity of the drugs to involve the PKC pathway. How this occurs is not known, but could potentially follow from drug perturbation of local ion/Ca2+ concentrations that impact PTM enzymes, or alternatively could occur by substrate-driven induction of transporter conformations in which phosphorylation sites become more or less available to enzyme action. [...] There are many other Ser and Thr residues on DAT intracellular loops and domains that may serve as phosphorylation sites, and in vitro studies have demon- strated phosphorylation of N— and C—terminal peptide sequences by many kinases in addition to PKC, ERK, and CaMK that have been implicated in regulation and efflux including protein kinase A (PKA), protein kinase G (PKG), casein kinase 2 (CK2), p38 kinase, JNK2, Cdk5, and Akt1 (Gorentla I et al., 2009; Vaughan & Foster, 2013). [...] The regulatory, efflux, and post-translational characteristics of NET and SERT show many similarities to DAT that indicate conservation of mechanism, but also some distinct properties that reflect requirements of specific neuronal populations. [...] With respect to efflux there are many similarities between SERT, NET, and DAT that indicate conservation of mechanisms.
However, another significant question remains: how might amphetaminergic substrates activate PKC in the first place? PKC activity is regulated by Ca2+ and diacylglycerol, a phospholipid metabolite generated in concert with inositol triphosphate by the enzyme phospholipase C (PLC), which is also Ca2+ dependent. [...] Usually, PLC is activated by agonist binding at GPCRs coupled to the Gq/11- type α-subunit; however, compounds, such as amphetamine and methamphetamine, lack significant affinity for GPCRs other than the trace amine-associated receptor 1 (TAAR1), a Gs-coupled receptor.74 [...] Interestingly, inhibition of the Na+/Ca2+ antiporter with amiloride also blocked amphetamine-induced PKC activation, suggesting that the ionic effects of DAT substrate translocation can directly influence activation of intracellular signaling cascades. When substrates are transported across the plasma membrane via the DAT, sodium ions are cotransported, and this increase in intracellular sodium concentration may cause Na+/Ca2+ antiporters at mitochondrial and endoplasmic reticulum membranes to operate in reverse, favoring a net flux of Ca2+ into the cytosolic compartment. [...]
Amphetamine-induced transport reversal at the closely related dopamine transporter (DAT) has been shown previously to be contingent upon modulation by calmodulin kinase IIα (αCaMKII). Here, we show that not only DAT, but also SERT, is regulated by αCaMKII. Inhibition of αCaMKII activity markedly decreased amphetamine-triggered SERT-mediated substrate efflux in both cells coexpressing SERT and αCaMKII and brain tissue preparations. [...] Moreover, we found that genetic deletion of αCaMKII impaired the locomotor response of mice to [MDMA] and blunted d-fenfluramine-induced prolactin release, substantiating the importance of αCaMKII modulation for amphetamine action at SERT in vivo as well. SERT-mediated substrate uptake was neither affected by inhibition of nor genetic deficiency in αCaMKII.
[...] AMPH is a substrate of DAT and causes nonexocytotic efflux of DA by reversing the direction of transport via DAT through a yet not fully understood mechanism (4–7). To improve our insights into AMPH-induced efflux with a focus on the unsettled role of Ca2+, we establish here a novel approach enabling simultaneous assessment of DA efflux and intracellular signaling pathways by live fluorescent imaging of cultured DA midbrain neurons. [...] our data provide strong evidence that AMPH-induced DA efflux via DAT in DArgic neurons does not require Ca2+ but primarily relies on the concerted action of AMPH on VMAT2 and DAT. [...] [...] Summarized, we employ a live imaging approach enabling parallel monitoring of DA efflux and activation of intracellular signaling pathways in cultured DArgic neurons. Our data reveal in the neurons profound AMPH-induced DA efflux that strikingly neither requires Ca2+ signaling nor activation of the kinases PKC and CaMKIIα. However, the efflux depends on both the activity of VMAT2 and DAT, substantiating the unequivocal concerted importance of both transporters for the pharmacological action of AMPH on DArgic neurons. [...]
Although a detailed and rigorous description of the regional distribution of TAAR1 protein and mRNA expression has yet to be accomplished, it is established that TAAR1 protein and mRNA are expressed in monoaminergic brain regions.3,16,26 In our laboratory, we exploited our prowess with assessing changes in kinetic activity of the dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter (SERT) to assess TAAR1 functionality. Because many of the identified ligands that activate TAAR1 are also substrates at the monoamine transporters and TAAR1 localization in transfected cells was shown to remain largely intracellular,4 we had reasoned early on that monoamine transporters could serve as conduits for TAAR1 agonists to enter cells, thereby providing access of the agonist to an intracellular pool of TAAR1 receptors. We speculated that if this were the case there could be a special relationship between TAAR1 and monoamine transporters because they share some of the same ligands. Indeed, we observed substantially enhanced CRE-luciferase signaling in transfected cells that co-expressed both TAAR1 and a monoamine transporter (DAT, NET or SERT).9,16 Because monoamine transporter kinetic regulation and internalization is a consequence of upregulated cellular phosphorylation cascades,27,28,29 we reasoned that the enhanced TAAR1 signaling could in turn trigger changes in the kinetic activity of the monoamine transporters under conditions of co-localization. This too was born out in a series of studies from our laboratory which demonstrated that TAAR1 activation drives the PKA and PKC cellular signaling cascades that result in inhibition of monoamine uptake and transporter reversal (efflux) in DAT/TAAR1, NET/TAAR1 and SERT/TAAR1 co-transfected cells in vitro, as well as in mouse and primate striatal (DAT, SERT) and thalamic (NET) synaptosomes ex vivo.22,23,24,25,30 TAAR1 specificity for mediation of the observed kinetic effects on the monoamine transporters was further confirmed in these studies by demonstrating the absence of noncompetitive effects of the trace amine beta-phenylethylamine (β-PEA),23 the common biogenic amines,24 and methamphetamine25 on uptake inhibition and substrate-induced monoamine efflux in synaptosomes generated from TAAR1 knockout mice. Accordingly, specific drugs that target TAAR1 will likely result in alterations in monoamine kinetic function and brain monoamine levels via this mechanism.
In a later study, Xie and Miller (2009a) resolved this issue by demonstrating that methamphetamine could induce [3 H]dopamine efflux in DAT cells or in TAAR1 knockout mouse striatal synaptosomes when the cells or synaptosomes were loaded with high concentrations of [ 3 H]dopamine (1 μM or higher), indicating that the direct effect of methamphetamine on the dopamine transporter to cause [3 H]dopamine efflux is dependent on the pre-loading concentration of [3 H]dopamine. But the efflux caused by methamphetamine under these conditions in DAT cells was not associated with either the PKA or the PKC phosphorylation pathways whereas it was blocked by methylphenidate. Further studies showed that TAAR1-mediated effects of methamphetamine on [3 H]dopamine efflux occurred at both lower and higher (1 μM) loading concentrations of [ 3 H]dopamine in TAAR1-DAT cells or in wild type mouse striatal synaptosomes, which could be blocked not only by methylphenidate but also by the PKC inhibitor Ro32-0432 (Xie and Miller 2009a). At the higher loading concentration, there was a greater amount of efflux observed in the presence of TAAR1 (in TAAR1-DAT cells or wild-type mouse striatal synaptosomes) than in its absence (in DAT cells or TAAR1 knockout mouse striatal synaptosomes), and it was only this relative greater amount of efflux that was sensitive to PKC inhibition. Accordingly, the TAAR1-mediated effects of methamphetamine on [3 H]dopamine efflux are dependent on PKC phosphorylation, whereas an observed TAAR1-independent, PKC-independent efflux occurs when cells or synaptosomes are loaded with high concentrations of [ 3 H]dopamine. [...] In a different mouse line, Lindemann et al. (2008) reported that TAAR1 knockout mice show an increased locomotive response to d-amphetamine after a single application of either 1 or 2.5 mg/kg. The increased locomotion correlated with a two- to threefold increase in extracellular dopamine, norepinephrine and serotonin levels in the TAAR1 knockout mice as compared with wild-type mice. [...] In further studies, electrophysiological analysis of dopaminergic neurons in the ventral tegmental area of TAAR1 knockout and wild-type mice revealed that the spontaneous firing rate of dopaminergic neurons was 8.6-fold higher in the TAAR1 knockout mice, and that activation of TAAR1 by 10 μM p-tyramine decreased the firing rate of dopaminergic neurons in wild type mice but not in TAAR1 knockout mice (Lindemann et al. 2008).
In both wild-type and Taar 1-/- mice, amphetamine produced a robust increase in striatal release of DA, norepinephrine (NE), and serotonin (5-HT), but this effect was significantly greater in Taar 1-/- mice than in wild type mice (Lindemann et al., 2008; Wolinsky et al., 2007). These findings support the notion that TAAR 1 functionally operates as a negative modulator of monoaminergic neurotransmission. [...] amphetamine had no effect on the release of DA and NA in the NAc of Taar 1 Tg mice, which partially explains the finding that amphetamine-induced a weaker locomotor hyperactivity in Taar 1 Tg mice as compared to their wild type counterparts. [...] There are evidence that TAAR 1 can modulate DAT function in in vitro cell culture experiments. Xie et al reported that β-phenethylamine (β-PEA) inhibited uptake and induced efflux of monoamines in thalamic synaptosomes of rhesus monkeys and wild-type mice, but not in synaptosomes of Taar 1-/- mice. Furthermore, the effect of β-PEA on efflux was blocked by transporter inhibitors in either the transfected cells or wild-type mouse synaptosomes (Xie and Miller, 2008). They also found that methamphetamine inhibited DA uptake, enhanced dopamine efflux, and induced DAT internalization by acting as a TAAR 1 agonist (Xie and Miller, 2009). However, more recent studies show inconsistent results. Leo et al. found that the DA clearance was not changed in Taar 1-/- mice as compared to their wild type counterparts (Leo et al., 2014), suggesting that the DAT function remained intact. In addition, no significant changes of dopamine uptake were observed in slices from Taar 1-/- mice, suggesting that the effect of TAAR 1 activation on DA-related function is independent of DAT (Leo et al., 2014). These apparent discrepancies may be attributable to the assays and tissues used in the studies. Xie et al. used mouse and monkey cellular synaptosome preparations with tissues from putamen and thalamus (Xie and Miller, 2008) while Leo et al. used fast scan cyclic voltammetry to measure DA uptake in mouse striatal slices (Leo et al., 2014). [...] Taar 1-/- mice displayed a behavioral phenotype of supersensitivity to amphetamine and cocaine, as evidenced by drug-induced increases in both locomotor activity and rearing as compared with wild-type littermates (Lindemann et al., 2008; Wolinsky et al., 2007). In contrast, Taar Tg mice responded to amphetamine in an opposite manner and showed a reduced and delayed response to amphetamine as compared with wild-type mice, indicating that Taar Tg mice are hyposensitive to amphetamine (Revel et al., 2012a). Methamphetamine also produced similar behavioral outcome in Taar 1-/- mice (Achat-Mendes et al., 2012).
A study using the TAAR1-specific antagonist EPPTB showed that presynaptic blockade of TAAR1 reversed its ability to inhibit the firing rate of VTA dopaminergic neurons, which it normally brings about by activating inwardly rectifying potassium channels [45,48]. An increase in firing rates upon blocking TAAR1 with EPPTB suggests that TAAR1 is either constitutively active or under constant tonic activation from endogenous agonists. [...] A presynaptic interaction between TAAR1 and DAT function has also been suggested because some groups found evidence in cell culture that TAAR1 agonists inhibit DAT function via TAAR1 and D2AR [49,50,65]. Others find, however, that the effects of TAAR1 agonists and antagonist are unaltered in Dat−/− knockout mice, and that dopamine reuptake is unaffected by application of TAAR1 (ant)agonists or in TAAR1-KO animals [46,47]. One hypothesis that could unite the apparently conflicting findings is that TAs exert an inhibitory effect directly on DAT; indeed, PEA administration induces transient hyperlocomotion in mice, similar to the behaviour observed in Dat−/− animals [30]. More recent work suggests that TAAR1 activation mediates internalisation of DAT through regulating RhoA activity [53].
Previous research on TAAR1 modulation of DAT function has produced equivocal findings. In vitro, MA inhibits [3H]DA uptake, and [3H]DA release is increased in striatal tissue from Taar1 WT compared with KO mice (Xie and Miller, 2009). Similar findings were described in cells cotransfected with TAAR1 and DAT, compared with cells transfected only with DAT, in which MA-induced [3H]DA uptake inhibition and release were increased (Xie and Miller, 2007, 2009). However, these findings indicate MA-induced impairment of DAT function is increased when TAAR1 is activated, as opposed to in vivo treatment with AMPH or MDMA, by which striatal extracellular DA levels are increased when TAAR1 is not activated (Wolinsky et al., 2007; Lindemann et al., 2008; Di Cara et al., 2011). We were unable to replicate the results of Xie and Miller (2009) under similar in vitro conditions (Fig. 3). There was no difference in IC50 values for [3H]DA uptake inhibition by MA between synaptosomes from Taar1 WT and KO mice. [...] our results do not support an earlier hypothesis that TAAR1 modulates DAT (Xie and Miller, 2007, 2009; Xie et al., 2008b), as there was no evidence of an interaction under conditions described above. Recent reports support our findings that the DAT is unaffected by TAAR1. Coadministration of MA and the TAAR1 partial agonist RO523648 did not alter [3H]DA uptake and release in striatal synaptosomes in rats (Cotter et al., 2015). Fast-scan cyclic voltammetry showed no difference in DA clearance, as mediated by DAT, in striatal tissue from Taar1 WT compared with KO mice (Leo et al., 2014). [...] Given the lack of interaction, DAT is an improbable mediator of TAAR1 regulation of MA-induced neurotoxicity. [...] activation of TAAR1 did not modulate in vitro MA-impairment of DAT function or DAT expression. As TAAR1 activation did not alter the function or expression of DAT in whole synaptosomes or VMAT2 located on membrane-associated vesicles, these results indicate TAAR1 does not interact with these transporters on the plasma membrane but does affect intracellular VMAT2 function.
Importantly, we have documented that neither Tau nor the half-life of released DA are changed in slices from TAAR1-KO animals, indicating that TAAR1-KO mice exhibit unaltered DA uptake ability and thereby normal dopamine transporter (DAT) functionality. It is believed that Tau and the half-life of released DA are reliable measures for detecting changes in DA uptake because they are strongly correlated with changes in the Km of DAT mediated DA uptake (Yorgason et al., 2011). Thus, these neurochemical in vivo studies, as well as previous demonstrations of the functional activity of TAAR1 ligands in mice lacking the DAT (Sotnikova et al., 2004; Revel et al., 2012a), provide little support for the postulated role of TAAR1 in modulating DAT activity that is based mostly on in vitro cell culture experiments (Miller et al., 2005; Xie et al., 2008; Miller, 2011). [...] Notably, neither [the TAAR1 agonist (RO5166017) nor the TAAR1 antagonist (EPPTB)] changed the kinetics of DA uptake as evidenced by the Tau and DA half-life estimations, indicating that DAT-mediated function is not altered by the action of the drugs on TAAR1.
While our data suggest a role for TAAR1 in eliciting amphetamine-like stimulant effects, it must be borne in mind that the observed in vivo effects are likely to result from interaction with both TAAR1 and monoamine transporters. Thus it has been shown that the selective TAAR1 agonist RO5166017 fully prevented psychostimulant-induced and persistent hyperdopaminergia-related hyperactivity in mice.42 This effect was found to be DAT-independent, since suppression of hyperactivity was observed in DAT-KO mice.42 The collected information leads us to conclude that TAAR1 is a stereoselective binding site for amphetamine and that TAAR1 activation by amphetamine and its congeners may contribute to the stimulant properties of this class of compounds. [...] it has been shown that β-PEA and methamphetamine effects in cells expressing TAAR-DAT significantly exceed those observed in cells expressing DAT only. Consistent with this conclusion is the higher potency of (S)-[amphetamine] in rat synaptosomes relative to cloned human DAT cells (EC50 60 nM vs 240 nM).
The DAT vs SERT ratios of synthetic cathinones are presented in Table 1. [...] Table 1. In vitro dopamine and serotonin uptake transporter inhibition (IC50 values) and release data (EC50 values). [...] The DAT vs SERT ratios of amphetamine derivatives and cocaine are presented in Table 2. [...] Table 2. In vitro dopamine and serotonin uptake transporter inhibition (IC50 values) and release data (EC50 values). [...] * in vitro studies for neurotransmitter reuptake inhibition and release using HEK293 (Human Embryonic Kidney 293) cell line expressing DAT and SERT (IC50 and EC50 values are expressed in μM). [(Note: HEK293 cells lack TAAR1 expression: "HEK-293 cells do not express TAAR1 (Reese et al., 2007; Xie and Miller, 2007)" (Small et al., 2023 [doi:10.1124/jpet.122.001573]).)]
[AMPH and METH] promote DA release from synaptic storage vesicles into the cytoplasm (Partilla et al., 2006) and then into the extracellular space via DAT with an EC50 of 25 nM and a Ki uptake in rat synaptosomes of 34 nM (Rothman et al., 2001). These are drug concentrations approximately 20-30 fold lower than the EC50 values Reese et al. (2007) calculated for eliciting an in vitro functional response from rTAAR1 (0.8 μM). However, experienced METH users can typically consume gram quantities of drug per day (Kramer et al., 1967) and achieve peak blood concentrations of 100 μM (Derlet et al., 1989; Baselt, 2002; Peters et al., 2003). [...] The extracellular free concentration of METH surrounding relevant human dopaminergic synapses in the brain presumably is the relevant pharmacodynamic parameter, at least in part, underlying the desirable effects of the drug. Although this value is not known with certainty for humans METH serum levels typically represent one tenth of what is found in rat brain (Riviere et al., 2000). Consequently, when considered together with the human forensic evidence the in vitro results of Reese et al. (2007) are consistent with the interpretation that in vivo the human TAAR1 is likely to be a mediator of at least some of METH's effects.
The EC50 of (+)-AMPH to release DA via DAT is 25 nM, with Ki uptake values in rat synaptosomes for this neurotransmitter of 34 nM at the DAT, concentrations approximately 20- to 30-fold lower than the EC50 values we calculated for eliciting an in vitro functional response from rTAAR1 (0.8 μM). Chronic METH abusers can typically consume gram quantities of drug per day (Kramer et al., 1967). [...] plasma concentrations of both drugs can reach into the high-micromolar range (Drummer and Odell, 2001; Baselt, 2002; Peters et al., 2003). Although the extracellular free concentration of METH around relevant human dopaminergic synapses presumably involved in producing desirable CNS effects is not known with certainty, in rats METH serum levels are typically 1/10 what is found in brain (Riviere et al., 2000). Forensic evidence indicates that experienced METH users can typically achieve peak blood concentrations of 100 μM (Baselt, 2002; Peters et al., 2003). Both isomers of METH were full agonists of h-rChTAAR1 over a range of EC50 values from 3.5 to ~15 μM, concentrations often exceeded in the blood of human METH addicts (Derlet et al., 1989). If TAAR1s, whether expressed in the CNS or periphery, are exposed to such concentrations, it is possible they could become functionally activated. [...] Given that the EC50 values for METH and AMPH activation of the h-rChTAAR1 in vitro are well below the concentrations frequently found in addicts, we suggest that TAAR1 might be a potential mediator of some of the effects of AMPH and METH in humans, including hyperthermia and stroke.
That TAAR1 signaling is coupled to the inhibition of VTA DA neuron firing was a surprising finding. [...] earlier data suggested that TAAR1 activation leads to inhibition of DA uptake by DAT, promotion of DA efflux by DAT and DAT internalization which presumably would augment extracellular DA levels, whereas inhibition of DA neuronal firing rate would likely decrease extracellular DA levels, [...] this mismatch in expectations immediately attracted the attention of those trying to determine whether TAAR1 is a METH/AMPH and DA receptor in vivo as it is in vitro (Bunzow et al., 2001; Wainscott et al., 2007; Xie and Miller, 2009; Panas et al., 2012).
[...] it is unclear if TAAR1 plays any role in the effects of MDMA in humans, as MDMA does not activate human TAAR1 in cellular assays like it does mouse and rat TAAR1.84
Beside the TAAR1-KO mice, animals that overexpress taar1 in the brain were also generated, which was named as taar1 Tg mice (Revel et al. 2012a). Before discussing the behaviors of this line of taar1 Tg mice, it should be kept in mind that taar1 was expressed in all types of neurons in the whole brain of this mice strain, which is in contrast with the specifc expression pattern of that in the wildtype animals (Revel et al. 2012a). The electrophysiological results showed that excitatory and inhibitory inputs into the VTA were altered in the taar1 Tg mice (Revel et al. 2012a). Interestingly, although the basal levels of dopamine and norepinephrine in the nucleus accumbens (NAc) were elevated, amphetamine did not alter dopamine levels in the taar1 Tg mice (Revel et al. 2012a). Consistently, behavioral test showed that AMPH led to hyperactivity in WT but not in taar1 Tg mice (Revel et al. 2012a).
[...] a proposed intracellular target for amphetamine is the [TAAR1], a [GPCR] that is expressed on intracellular membranes in DA neurons (Miller, 2011). Phenethylamine stimulants have been proposed to activate TAAR1, leading to increased cAMP generation and RhoA activation, with subsequent enhancement of DAT reverse transport and endocytosis (Xie and Miller, 2007, 2008, 2009; Underhill et al., 2021). Methamphetamine-induced DAT endocytosis was found to be dependent on TAAR1 expression and PKA activity as suggested by use of the kinase inhibitor H89 (Xie and Miller, 2009). However, evidence indicating that amphetamine-induced endocytosis is independent of TAAR1 includes 1) HEK-293 cells do not express TAAR1 (Reese et al., 2007; Xie and Miller, 2007) but do exhibit amphetamine-induced DAT endocytosis [present studies and (Saunders et al., 2014; Cheng et al., 2015; Wheeler et al., 2015)]; 2) cAMP and PKA activation, which are stimulated by TAAR1, antagonized amphetamine-induced DAT endocytosis in heterologous cells and DA neurons [present studies and (Wheeler et al., 2015)]; and 3) in cell lines and rodent striatal synaptosomes, PKA activation increased DAT Vmax, consistent with increased plasma membrane expression (Pristupa et al., 1998; Page et al., 2004; Batchelor and Schenk, 2018). Additionally, DMAA induced DAT endocytosis (Figs. 3 and 4) despite exhibiting no activity at human TAAR1 in receptor binding studies (Rickli et al., 2019). Therefore, although some evidence does support a role of TAAR1 in modulating amphetamine-induced DAT endocytosis, the present studies suggest that DMAA and amphetamine promote DAT endocytosis through a TAAR1-independent mechanism.
Amphetamine might act on TAAR1 to activate Gαs pathway and phosphorylate monoamine transporter such as dopamine transporter (DAT), leading to its internalization and ceased transport (Bunzow et al. 2001; Miller 2011). Behaviorally, knockout of TAAR1 in mice leads to locomotor supersensitivity induced by amphetamine, although the connection to regulation of monoamine system is undetermined and needs further investigation (Achat-Mendes et al. 2012; Lindemann et al. 2008).
Moreover, while deletion of TAAR1 led to increased frequency of spontaneous firing frequency of serotonin neurons in brain slices [36], TAAR1 full agonists significantly inhibited the firing frequency in DRN serotonin neurons [28, 38]. [...] In vitro electrophysiological recordings showed a remarkable increase in the spontaneous firing rate of dopamine neurons in the VTA of TAAR1-KO mice [36]. While the endogenous TAAR1 agonist TYR decreased the firing rate of dopamine neurons in slices from WT mice, it had no effect in slices from TAAR1-KO mice [36]. [...] Similar to TYR, [the TAAR1 full agonist] RO5166017 reduced the firing rate of VTA dopamine neurons and DRN serotonin neurons in brain slices from WT but not TAAR1-KO mice, through the activation of K+-mediated outward current, which can be blocked by TAAR1 antagonist EPPTB [38]. [...] [The TAAR1 full agonist] RO5256390 decreased the firing frequency of VTA dopamine and DRN serotonin neurons in brain slices from WT but not TAAR1-KO mice, [...]
β-Keto-analogue cathinones also exhibited approximately 10-fold lower affinity for the TA1 receptor compared with their respective non-β-keto amphetamines. [...] Activation of TA1 receptors negatively modulates dopaminergic neurotransmission. Importantly, methamphetamine decreased DAT surface expression via a TA1 receptor-mediated mechanism and thereby reduced the presence of its own pharmacological target (Xie and Miller, 2009). MDMA and amphetamine have been shown to produce enhanced DA and 5-HT release and locomotor activity in TA1 receptor knockout mice compared with wild-type mice (Lindemann et al., 2008; Di Cara et al., 2011). Because methamphetamine and MDMA auto-inhibit their neurochemical and functional effects via TA1 receptors, low affinity for these receptors may result in stronger effects on monoamine systems by cathinones compared with the classic amphetamines.
Drugs of abuse hijack brain-reward circuitry during the addiction process by augmenting action potential-dependent phasic dopamine release events associated with learning and goal-directed behavior. One prominent exception to this notion would appear to be amphetamine (AMPH) and related analogs, which are proposed instead to disrupt normal patterns of dopamine neurotransmission by depleting vesicular stores and promoting nonexocytotic dopamine efflux via reverse transport. This mechanism of AMPH action, though, is inconsistent with its therapeutic effects and addictive properties, which are thought to be reliant on phasic dopamine signaling. [...] These findings identify upregulation of exocytotic dopamine release as a key AMPH action in behaving animals and support a unified mechanism of abused drugs to activate phasic dopamine signaling. [...] Our results also raise the possibility that enhanced phasic dopamine signaling may underlie important clinical effects of AMPH. Modest activation of dopamine transients at the relatively low dose of 1 mg/kg used here may be an important action of medications that combine amphetamine salts (e.g., Adderall) used clinically (Joyce et al., 2007). Indeed, low doses of AMPH preserve and improve learning and behavioral performance (Mayorga et al., 2000; Wyvell and Berridge, 2000; Taylor and Jentsch, 2001; Zhang et al., 2003; Knutson et al., 2004). In contrast, high doses of AMPH can produce a behavioral profile similar to the positive symptoms associated with schizophrenia (Featherstone et al., 2007; Lisman et al., 2008). [...] Thus, disrupting the tight temporal concurrence between dopamine transients and important external cues due to hyperactive phasic dopamine signaling may contribute to AMPH-induced psychosis (Featherstone et al., 2007).
While there is little debate that behavioral effects of this important psychostimulant are associated with a hyperdopamine state [3–6], the underlying mechanisms by which this condition manifests have been the subject of intense study. Two, what ostensibly appear to be mutually exclusive, views have emerged. On the one hand, AMPH enhances tonic dopamine signaling by reversing dopamine transporter (DAT) direction, leading to a non-exocytotic, action potential-independent type of release or efflux that is driven by vesicular depletion and the redistribution of dopamine to the cytosol [7,8]. On the other hand, AMPH enhances phasic dopamine signaling by promoting burst firing of dopamine neurons [9,10], inhibiting dopamine uptake [11,12], and upregulating vesicular dopamine release [13,14]. How AMPH concurrently activates tonic and phasic dopamine signaling, the two fundamental modes of communication used by dopamine neurons [15], yet elicits opposing actions on vesicular dopamine stores is perplexing and unresolved.
We found that σ1R activation prevents methamphetamine-induced, DAT-mediated increases in firing activity of dopamine neurons. In vitro and in vivo amperometric measurements revealed that σ1R activation decreases methamphetamine-stimulated dopamine efflux without affecting basal dopamine neurotransmission. Consistent with these findings, σ1R activation decreases methamphetamine-induced locomotion, motivated behavior, and enhancement of brain reward function. Notably, we revealed that the σ1R interacts with DAT at or near the plasma membrane and decreases methamphetamine-induced Ca2+ signaling, providing potential mechanisms.
The sampling of extracellular fluids using in vivo microdialysis provides a direct way to study drug effects on synaptic efflux of a neurotransmitter. Comparisons of dexfenfluramine and fluoxetine using this technique have revealed a significant difference between the effects of reuptake inhibition and release on extracellular 5-HT levels.34−36 For example, Tao et al.35 compared the effects of fluoxetine and dexfenfluramine on extracellular 5-HT in the hypothalamus, with the latter increasing basal 5-HT 6−16-fold, compared to 3-fold following fluoxetine (Figure 1A). These differences in magnitude reflect differences in the way that fluoxetine and dexfenfluramine elevate 5-HT. The ability of SSRIs to increase extracellular 5- HT is dependent on neuronal activity (i.e., impulse dependent) and is subject to autoreceptor feedback inhibition. In contrast, (dex)fenfluramine increases extracellular 5-HT by disrupting vesicular storage and promoting nonexocytotic release that is independent of both of these regulatory factors.36 [...] Figure 1. Comparison between a 5-HT releaser (dexfenfluramine) and SSRI (fluoxetine) on (A) extracellular 5-HT release sampled from rat hypothalamus, [...]
Although the precise physiological role of TAAR1/G13-mediated DAT internalization remains to be established, it is likely that this pathway evolved to regulate the actions of endogenous amines including β-PEA, tyramine, 3-methoxytyramine, and DA. DA is a TAAR1 agonist (Fig. 1c) and recently, it was shown that the sustained elevation of extracellular DA seen following burst firing appears to be a consequence of DAT internalization by a Rho-dependent mechanism [51]. These results provide the first evidence that dopamine itself can act as a TAAR1 agonist to trigger transporter internalization in vivo. TAAR1 signaling also has the potential to serve as a sensor of cytoplasmic DA and its metabolites. At high cytosolic concentrations, DA becomes neurotoxic due to the formation of reactive oxygen species and quinones that can cause damage to the neuron [52]. Under normal conditions, DA transported through the DAT from the extracellular space is rapidly repackaged into synaptic vesicles or enzymatically metabolized. However, if cytoplasmic DA concentrations saturate these mechanisms, the reduction in surface levels of DAT mediated by TAAR1/G13 signaling may provide an additional means for maintaining free cytosolic DA below toxic levels.
This flooding of the cytoplasm and synaptic space with the oxidatively labile DA is thought to be a critical first step in the neurotoxic cascade of the amphetamines [73]. These conditions of elevated concentrations of cytosolic monoamines could be further aggravated by inhibition of MAO [91]. Unlike amphetamines, mephedrone and methylone have little if any affinity for VMAT-2 [33]. Therefore, their lack of neurotoxicity could derive from an inability to promote the release of DA from storage vesicles into the cytoplasm.
It has been suggested that the association between PD and ADHD may be explained, in part, by toxic effects of these drugs on DA neurons.241 [...] An important question is whether amphetamines, as they are used clinically to treat ADHD, are toxic to DA neurons. In most of the animal and human studies cited above, stimulant exposure levels are high relative to clinical doses, and dosing regimens (as stimulants) rarely mimic the manner in which these drugs are used clinically. The study by Ricaurte and colleagues248 is an exception. In that study, baboons orally self-administered a racemic (3:1 d/l) amphetamine mixture twice daily in increasing doses ranging from 2.5 to 20 mg/day for four weeks. Plasma amphetamine concentrations, measured at one-week intervals, were comparable to those observed in children taking amphetamine for ADHD. Two to four weeks after cessation of amphetamine treatment, multiple markers of striatal DA function were decreased, including DA and DAT. In another group of animals (squirrel monkeys), d/l amphetamine blood concentration was titrated to clinically comparable levels for four weeks by administering varying doses of amphetamine by orogastric gavage. These animals also had decreased markers of striatal DA function assessed two weeks after cessation of amphetamine.
Amphetamine treatment similar to that used for ADHD has been demonstrated to produce brain dopaminergic neurotoxicity in primates, causing the damage of dopaminergic nerve endings in the striatum that may also occur in other disorders with long-term amphetamine treatment (57).
Though the paradigm used by Ricaurte et al. 53 arguably still incorporates amphetamine exposure at a level above much clinical use,14,55 it raises important unanswered questions. Is there a threshold of amphetamine exposure above which persistent changes in the dopamine system are induced? [...]
Repeated exposure to moderate to high levels of methamphetamine has been related to neurotoxic effects on the dopaminergic and serotonergic systems, leading to potentially irreversible loss of nerve terminals and/or neuron cell bodies (Cho and Melega, 2002). Preclinical evidence suggests that d-amphetamine, even when administered at commonly prescribed therapeutic doses, also results in toxicity to brain dopaminergic axon terminals (Ricaurte et al., 2005).
The present findings with MDMA are consistent with our previous data [15,17] and those reported by Simmler et al. [4] and Eshleman et al. [23] who examined the effects of MDMA and related drugs in human embryonic kidney (HEK) cells transfected with human SERT, DAT and NET. Thus, the molecular mechanism of action for MDMA at monoamine transporters is similar in rats and humans. On the other hand, the potency of (±)MDMA for releasing monoamines in rat brain synaptosomes shown here (i.e., 60–70 nM) is greater than its potency in transfected HEK cells (i.e., 1–20 μM). Such discrepancies in absolute potency could be related to species differences in drug responsiveness, differences in release assay methods employed, or the absence of important neuronal membrane proteins in non-neuronal HEK cells.
The major goal of this study was to establish a high-throughput assay to detect [monoamine transporter substrates (SBSTs)] for use in a project to develop new drugs with dual activity as SBSTs of DAT and SERT. METHODS. Using minor modifications of published procedures, rat brain synaptosomes were preloaded with either [3H]DA, [3H]NE or [3H]5-HT. Test drugs were added and the reaction terminated by rapid filtration over Whatman GF/B filters. Release was quantified by counting how much tritium was retained on the filters. RESULTS. Using optimized conditions known SBSTs potently decreased retained tritium in a dose-dependent manner whereas known [uptake inhibitors (UIs)] were weak or ineffective. UIs shifted SBST inhibition curves to the right, consistent with antagonist-like activity. CONCLUSION. We have developed high throughput assays which detect SBSTs for the DA, 5-HT and NE transporters.
RESULTS. Methamphetamine and amphetamine potently released NE (IC50s = 14.3 and 7.0 nM) and DA (IC50s = 40.4 nM and 24.8 nM), and were much less potent releasers of 5-HT (IC50s = 740 nM and 1765 nM). Phentermine released all three biogenic amines with an order of potency NE (IC50 = 28.8 nM)> DA (IC50 = 262 nM)> 5-HT (IC50 = 2575 nM). Aminorex released NE (IC50 = 26.4 nM), DA (IC50 = 44.8 nM) and 5-HT (IC50 = 193 nM). Chlorphentermine was a very potent 5-HT releaser (IC50 = 18.2 nM), a weaker DA releaser (IC50 = 935 nM) and inactive in the NE release assay. Chlorphentermine was a moderate potency inhibitor of [3H]NE uptake (Ki = 451 nM). Diethylpropion, which is self-administered, was a weak DA uptake inhibitor (Ki = 15 µM) and NE uptake inhibitor (Ki = 18.1 µM) and essentially inactive in the other assays. Phendimetrazine, which is self-administered, was a weak DA uptake inhibitor (IC50 = 19 µM), a weak NE uptake inhibitor (8.3 µM) and essentially inactive in the other assays.
Table 5. Action of MDMA, MDA, and PMMA as Releasing Agents at the Serotonin (SERT), Dopamine (DAT), and Norepinephrine (NET) Transporters18,59,60 [...] a Data, although from different publications, were obtained from the same laboratory.
Although the pharmacological effect of amphetamine is predominantly mediated by monoamine release, this mechanism is complemented by reuptake inhibition [...] that combine additively or synergistically to augment synaptic monoamine concentrations. The description of amphetamine as a 'monoamine reuptake inhibitor' often causes some confusion, and the difference between the mechanisms of amphetamine, which is a competitive reuptake transport substrate, and classical reuptake inhibitors is illustrated in Figure 3. [...] d-Amphetamine is generally accepted to be a weak dopamine reuptake inhibitor with a Ki value of ~100 nM, a moderately potent inhibitor of noradrenaline reuptake (Ki = 40–50 nM) and a very weak inhibitor of 5-HT reuptake (Ki = 1.4-3.8 µM). [...] the efficacy of amphetamine relative to other indirect monoamine agonists, for example classical reuptake inhibitors, can only be estimated from in vivo experiments. [...] [d- and l-Amphetamine] dose-dependently increased the extracellular concentrations of noradrenaline in the prefrontal cortex (PFC) and dopamine in the striatum. The pharmacodynamics of their effects are typical of those reported for monoamine releasing agents, i.e. a fast onset of action with peak increases of noradrenaline and dopamine efflux occurring at 30–45 min, large effects (400–450% of baseline for noradrenaline and 700–1500% of baseline for dopamine), with a relatively rapid decline after the maximum (Figure 4). [...] the magnitude of the increases produced by amphetamine's isomers are greater than those reported for classical reuptake inhibitors such as atomoxetine or bupropion, and there is no dose-effect ceiling to amphetamine's actions [...] The primary action of amphetamine is to increase synaptic concentrations of monoamine neurotransmitters, thereby indirectly enhancing noradrenergic, dopaminergic neurotransmission in the CNS.
Converging lines of evidence have solidified the notion that DA releasers are substrates of the transporter and once translocated, they reverse the normal direction of transporter flux to evoke release of endogenous neurotransmitters. The nature of this reversal is not well understood, but the entire process is primarily transporter-dependent and requires elevated intracellular sodium concentrations, phosphorylation of DAT, and possible involvement of transporter oligomers (Khoshbouei et al., 2003, 2004; Sitte and Freissmuth, 2010). [...] A library of approximately 1400 phenethylamine compounds (PAL compounds) has been screened using these protocols. Among the active compounds,the smaller DAT ligands were found to be DA releasers while the sterically larger compounds were DAT uptake inhibitors. [...] Generic pharmacophore for biogenic amine transporter ligands. Note that transportable substrate ligands exhibit size constraints defined by the red circle. Functional groups attached to the nitrogen, α-carbon or phenyl ring that extend beyond the "edge" of the pharmacophore will generate partial substrates, transporter blockers or be inactive. [...] phenmetrazine was found to be completely inactive at VMAT2 indicating that a direct interaction of the releaser with VMAT2 is not required for inducing neurotransmitter efflux into the extracellular space (Partilla et al., 2006). Phentermine and benzylpiperazine were also found in the same study to lack VMAT2 activity (Table 5).
[...] most amphetamines are [monoamine transporter] substrates, which pervert the relay to elicit efflux of monoamines into the synaptic cleft. However, some amphetamines act as transporter inhibitors. [...] Amphetamines also bind to the trace amine receptors TAR1, [...] TAR1 is not activated by all psychoactive amphetamines (p-chloroamphetamine, for instance, is inactive); stimulation of TAR1 actually reduces dopamine release and thus decreases sensitivity to amphetamine [18,19]. [...] amphetamines are taken up by plasma-membrane monoamine transporters as exogenous substrates [31]. Accordingly, they inhibit the physiological monoamine reuptake in a competitive manner [36]. As a consequence of both amphetamine-induced reverse transport and inhibition of reuptake, the synaptic monoamine concentration increases, which in turn activates post- and presynaptic receptors [37]. The activation of postsynaptic receptors propagates the signal and contributes to the biological response. Stimulation of presynaptic autoreceptors decreases the quantal release of monoamines upon excitatory inputs; [...]
In contrast to assay systems involving non-neuronal cells transfected with transporter proteins, synaptosomes possess all of the cellular machinery necessary for neurotransmitter synthesis, release, metabolism, and reuptake. Synaptosomes, however, do not model all of the effects of amphetamine-type agents because the use of reserpine removes any contribution of the vesicular monoamine transporter VMAT2 (SLC18A2) to the release process. In addition to acting as a substrate for plasma membrane monoamine transporters, amphetamine also binds to VMAT, resulting in the redistribution of monoamines from vesicular stores to the cytoplasm (Sulzer et al. 1995; Partilla et al. 2006; Freyberg et al. 2016). Although transporter substrates can induce monoamine release in the absence of VMAT binding (Fon et al. 1997), it is important to recognize that 2-aminoindans may have effects in intact nerve terminals that are not fully replicated in synaptosomes. Follow-up studies will be conducted to evaluate whether 2-aminoindans are capable of interacting with VMAT.
2.2.1. DAT regulation by psychostimulants [...] Considerable evidence supports a role for DAT substrates, like amphetamine, in inducing DAT internalization in vivo and in vitro, thus decreasing DA uptake [390–395]. [...] While several kinases are involved in the regulation of DAT, PKC is by far the most thoroughly investigated [388,403]. PKC activation induces DAT internalization [393,404–406] although the mechanism of PKC activation by DAT substrates remains largely unknown [388,389,403,407,408]
Amphetamine-induced transport reversal at the closely related dopamine transporter (DAT) has been shown previously to be contingent upon modulation by calmodulin kinase IIα (αCaMKII). Here, we show that not only DAT, but also SERT, is regulated by αCaMKII. Inhibition of αCaMKII activity markedly decreased amphetamine-triggered SERT-mediated substrate efflux in both cells coexpressing SERT and αCaMKII and brain tissue preparations. [...] Moreover, we found that genetic deletion of αCaMKII impaired the locomotor response of mice to [MDMA] and blunted d-fenfluramine-induced prolactin release, substantiating the importance of αCaMKII modulation for amphetamine action at SERT in vivo as well. SERT-mediated substrate uptake was neither affected by inhibition of nor genetic deficiency in αCaMKII.
[...] AMPH is a substrate of DAT and causes nonexocytotic efflux of DA by reversing the direction of transport via DAT through a yet not fully understood mechanism (4–7). To improve our insights into AMPH-induced efflux with a focus on the unsettled role of Ca2+, we establish here a novel approach enabling simultaneous assessment of DA efflux and intracellular signaling pathways by live fluorescent imaging of cultured DA midbrain neurons. [...] our data provide strong evidence that AMPH-induced DA efflux via DAT in DArgic neurons does not require Ca2+ but primarily relies on the concerted action of AMPH on VMAT2 and DAT. [...] [...] Summarized, we employ a live imaging approach enabling parallel monitoring of DA efflux and activation of intracellular signaling pathways in cultured DArgic neurons. Our data reveal in the neurons profound AMPH-induced DA efflux that strikingly neither requires Ca2+ signaling nor activation of the kinases PKC and CaMKIIα. However, the efflux depends on both the activity of VMAT2 and DAT, substantiating the unequivocal concerted importance of both transporters for the pharmacological action of AMPH on DArgic neurons. [...]
Although a detailed and rigorous description of the regional distribution of TAAR1 protein and mRNA expression has yet to be accomplished, it is established that TAAR1 protein and mRNA are expressed in monoaminergic brain regions.3,16,26 In our laboratory, we exploited our prowess with assessing changes in kinetic activity of the dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter (SERT) to assess TAAR1 functionality. Because many of the identified ligands that activate TAAR1 are also substrates at the monoamine transporters and TAAR1 localization in transfected cells was shown to remain largely intracellular,4 we had reasoned early on that monoamine transporters could serve as conduits for TAAR1 agonists to enter cells, thereby providing access of the agonist to an intracellular pool of TAAR1 receptors. We speculated that if this were the case there could be a special relationship between TAAR1 and monoamine transporters because they share some of the same ligands. Indeed, we observed substantially enhanced CRE-luciferase signaling in transfected cells that co-expressed both TAAR1 and a monoamine transporter (DAT, NET or SERT).9,16 Because monoamine transporter kinetic regulation and internalization is a consequence of upregulated cellular phosphorylation cascades,27,28,29 we reasoned that the enhanced TAAR1 signaling could in turn trigger changes in the kinetic activity of the monoamine transporters under conditions of co-localization. This too was born out in a series of studies from our laboratory which demonstrated that TAAR1 activation drives the PKA and PKC cellular signaling cascades that result in inhibition of monoamine uptake and transporter reversal (efflux) in DAT/TAAR1, NET/TAAR1 and SERT/TAAR1 co-transfected cells in vitro, as well as in mouse and primate striatal (DAT, SERT) and thalamic (NET) synaptosomes ex vivo.22,23,24,25,30 TAAR1 specificity for mediation of the observed kinetic effects on the monoamine transporters was further confirmed in these studies by demonstrating the absence of noncompetitive effects of the trace amine beta-phenylethylamine (β-PEA),23 the common biogenic amines,24 and methamphetamine25 on uptake inhibition and substrate-induced monoamine efflux in synaptosomes generated from TAAR1 knockout mice. Accordingly, specific drugs that target TAAR1 will likely result in alterations in monoamine kinetic function and brain monoamine levels via this mechanism.
In a later study, Xie and Miller (2009a) resolved this issue by demonstrating that methamphetamine could induce [3 H]dopamine efflux in DAT cells or in TAAR1 knockout mouse striatal synaptosomes when the cells or synaptosomes were loaded with high concentrations of [ 3 H]dopamine (1 μM or higher), indicating that the direct effect of methamphetamine on the dopamine transporter to cause [3 H]dopamine efflux is dependent on the pre-loading concentration of [3 H]dopamine. But the efflux caused by methamphetamine under these conditions in DAT cells was not associated with either the PKA or the PKC phosphorylation pathways whereas it was blocked by methylphenidate. Further studies showed that TAAR1-mediated effects of methamphetamine on [3 H]dopamine efflux occurred at both lower and higher (1 μM) loading concentrations of [ 3 H]dopamine in TAAR1-DAT cells or in wild type mouse striatal synaptosomes, which could be blocked not only by methylphenidate but also by the PKC inhibitor Ro32-0432 (Xie and Miller 2009a). At the higher loading concentration, there was a greater amount of efflux observed in the presence of TAAR1 (in TAAR1-DAT cells or wild-type mouse striatal synaptosomes) than in its absence (in DAT cells or TAAR1 knockout mouse striatal synaptosomes), and it was only this relative greater amount of efflux that was sensitive to PKC inhibition. Accordingly, the TAAR1-mediated effects of methamphetamine on [3 H]dopamine efflux are dependent on PKC phosphorylation, whereas an observed TAAR1-independent, PKC-independent efflux occurs when cells or synaptosomes are loaded with high concentrations of [ 3 H]dopamine. [...] In a different mouse line, Lindemann et al. (2008) reported that TAAR1 knockout mice show an increased locomotive response to d-amphetamine after a single application of either 1 or 2.5 mg/kg. The increased locomotion correlated with a two- to threefold increase in extracellular dopamine, norepinephrine and serotonin levels in the TAAR1 knockout mice as compared with wild-type mice. [...] In further studies, electrophysiological analysis of dopaminergic neurons in the ventral tegmental area of TAAR1 knockout and wild-type mice revealed that the spontaneous firing rate of dopaminergic neurons was 8.6-fold higher in the TAAR1 knockout mice, and that activation of TAAR1 by 10 μM p-tyramine decreased the firing rate of dopaminergic neurons in wild type mice but not in TAAR1 knockout mice (Lindemann et al. 2008).
In both wild-type and Taar 1-/- mice, amphetamine produced a robust increase in striatal release of DA, norepinephrine (NE), and serotonin (5-HT), but this effect was significantly greater in Taar 1-/- mice than in wild type mice (Lindemann et al., 2008; Wolinsky et al., 2007). These findings support the notion that TAAR 1 functionally operates as a negative modulator of monoaminergic neurotransmission. [...] amphetamine had no effect on the release of DA and NA in the NAc of Taar 1 Tg mice, which partially explains the finding that amphetamine-induced a weaker locomotor hyperactivity in Taar 1 Tg mice as compared to their wild type counterparts. [...] There are evidence that TAAR 1 can modulate DAT function in in vitro cell culture experiments. Xie et al reported that β-phenethylamine (β-PEA) inhibited uptake and induced efflux of monoamines in thalamic synaptosomes of rhesus monkeys and wild-type mice, but not in synaptosomes of Taar 1-/- mice. Furthermore, the effect of β-PEA on efflux was blocked by transporter inhibitors in either the transfected cells or wild-type mouse synaptosomes (Xie and Miller, 2008). They also found that methamphetamine inhibited DA uptake, enhanced dopamine efflux, and induced DAT internalization by acting as a TAAR 1 agonist (Xie and Miller, 2009). However, more recent studies show inconsistent results. Leo et al. found that the DA clearance was not changed in Taar 1-/- mice as compared to their wild type counterparts (Leo et al., 2014), suggesting that the DAT function remained intact. In addition, no significant changes of dopamine uptake were observed in slices from Taar 1-/- mice, suggesting that the effect of TAAR 1 activation on DA-related function is independent of DAT (Leo et al., 2014). These apparent discrepancies may be attributable to the assays and tissues used in the studies. Xie et al. used mouse and monkey cellular synaptosome preparations with tissues from putamen and thalamus (Xie and Miller, 2008) while Leo et al. used fast scan cyclic voltammetry to measure DA uptake in mouse striatal slices (Leo et al., 2014). [...] Taar 1-/- mice displayed a behavioral phenotype of supersensitivity to amphetamine and cocaine, as evidenced by drug-induced increases in both locomotor activity and rearing as compared with wild-type littermates (Lindemann et al., 2008; Wolinsky et al., 2007). In contrast, Taar Tg mice responded to amphetamine in an opposite manner and showed a reduced and delayed response to amphetamine as compared with wild-type mice, indicating that Taar Tg mice are hyposensitive to amphetamine (Revel et al., 2012a). Methamphetamine also produced similar behavioral outcome in Taar 1-/- mice (Achat-Mendes et al., 2012).
Previous research on TAAR1 modulation of DAT function has produced equivocal findings. In vitro, MA inhibits [3H]DA uptake, and [3H]DA release is increased in striatal tissue from Taar1 WT compared with KO mice (Xie and Miller, 2009). Similar findings were described in cells cotransfected with TAAR1 and DAT, compared with cells transfected only with DAT, in which MA-induced [3H]DA uptake inhibition and release were increased (Xie and Miller, 2007, 2009). However, these findings indicate MA-induced impairment of DAT function is increased when TAAR1 is activated, as opposed to in vivo treatment with AMPH or MDMA, by which striatal extracellular DA levels are increased when TAAR1 is not activated (Wolinsky et al., 2007; Lindemann et al., 2008; Di Cara et al., 2011). We were unable to replicate the results of Xie and Miller (2009) under similar in vitro conditions (Fig. 3). There was no difference in IC50 values for [3H]DA uptake inhibition by MA between synaptosomes from Taar1 WT and KO mice. [...] our results do not support an earlier hypothesis that TAAR1 modulates DAT (Xie and Miller, 2007, 2009; Xie et al., 2008b), as there was no evidence of an interaction under conditions described above. Recent reports support our findings that the DAT is unaffected by TAAR1. Coadministration of MA and the TAAR1 partial agonist RO523648 did not alter [3H]DA uptake and release in striatal synaptosomes in rats (Cotter et al., 2015). Fast-scan cyclic voltammetry showed no difference in DA clearance, as mediated by DAT, in striatal tissue from Taar1 WT compared with KO mice (Leo et al., 2014). [...] Given the lack of interaction, DAT is an improbable mediator of TAAR1 regulation of MA-induced neurotoxicity. [...] activation of TAAR1 did not modulate in vitro MA-impairment of DAT function or DAT expression. As TAAR1 activation did not alter the function or expression of DAT in whole synaptosomes or VMAT2 located on membrane-associated vesicles, these results indicate TAAR1 does not interact with these transporters on the plasma membrane but does affect intracellular VMAT2 function.
While our data suggest a role for TAAR1 in eliciting amphetamine-like stimulant effects, it must be borne in mind that the observed in vivo effects are likely to result from interaction with both TAAR1 and monoamine transporters. Thus it has been shown that the selective TAAR1 agonist RO5166017 fully prevented psychostimulant-induced and persistent hyperdopaminergia-related hyperactivity in mice.42 This effect was found to be DAT-independent, since suppression of hyperactivity was observed in DAT-KO mice.42 The collected information leads us to conclude that TAAR1 is a stereoselective binding site for amphetamine and that TAAR1 activation by amphetamine and its congeners may contribute to the stimulant properties of this class of compounds. [...] it has been shown that β-PEA and methamphetamine effects in cells expressing TAAR-DAT significantly exceed those observed in cells expressing DAT only. Consistent with this conclusion is the higher potency of (S)-[amphetamine] in rat synaptosomes relative to cloned human DAT cells (EC50 60 nM vs 240 nM).
The DAT vs SERT ratios of synthetic cathinones are presented in Table 1. [...] Table 1. In vitro dopamine and serotonin uptake transporter inhibition (IC50 values) and release data (EC50 values). [...] The DAT vs SERT ratios of amphetamine derivatives and cocaine are presented in Table 2. [...] Table 2. In vitro dopamine and serotonin uptake transporter inhibition (IC50 values) and release data (EC50 values). [...] * in vitro studies for neurotransmitter reuptake inhibition and release using HEK293 (Human Embryonic Kidney 293) cell line expressing DAT and SERT (IC50 and EC50 values are expressed in μM). [(Note: HEK293 cells lack TAAR1 expression: "HEK-293 cells do not express TAAR1 (Reese et al., 2007; Xie and Miller, 2007)" (Small et al., 2023 [doi:10.1124/jpet.122.001573]).)]
[AMPH and METH] promote DA release from synaptic storage vesicles into the cytoplasm (Partilla et al., 2006) and then into the extracellular space via DAT with an EC50 of 25 nM and a Ki uptake in rat synaptosomes of 34 nM (Rothman et al., 2001). These are drug concentrations approximately 20-30 fold lower than the EC50 values Reese et al. (2007) calculated for eliciting an in vitro functional response from rTAAR1 (0.8 μM). However, experienced METH users can typically consume gram quantities of drug per day (Kramer et al., 1967) and achieve peak blood concentrations of 100 μM (Derlet et al., 1989; Baselt, 2002; Peters et al., 2003). [...] The extracellular free concentration of METH surrounding relevant human dopaminergic synapses in the brain presumably is the relevant pharmacodynamic parameter, at least in part, underlying the desirable effects of the drug. Although this value is not known with certainty for humans METH serum levels typically represent one tenth of what is found in rat brain (Riviere et al., 2000). Consequently, when considered together with the human forensic evidence the in vitro results of Reese et al. (2007) are consistent with the interpretation that in vivo the human TAAR1 is likely to be a mediator of at least some of METH's effects.
That TAAR1 signaling is coupled to the inhibition of VTA DA neuron firing was a surprising finding. [...] earlier data suggested that TAAR1 activation leads to inhibition of DA uptake by DAT, promotion of DA efflux by DAT and DAT internalization which presumably would augment extracellular DA levels, whereas inhibition of DA neuronal firing rate would likely decrease extracellular DA levels, [...] this mismatch in expectations immediately attracted the attention of those trying to determine whether TAAR1 is a METH/AMPH and DA receptor in vivo as it is in vitro (Bunzow et al., 2001; Wainscott et al., 2007; Xie and Miller, 2009; Panas et al., 2012).
[...] it is unclear if TAAR1 plays any role in the effects of MDMA in humans, as MDMA does not activate human TAAR1 in cellular assays like it does mouse and rat TAAR1.84
Beside the TAAR1-KO mice, animals that overexpress taar1 in the brain were also generated, which was named as taar1 Tg mice (Revel et al. 2012a). Before discussing the behaviors of this line of taar1 Tg mice, it should be kept in mind that taar1 was expressed in all types of neurons in the whole brain of this mice strain, which is in contrast with the specifc expression pattern of that in the wildtype animals (Revel et al. 2012a). The electrophysiological results showed that excitatory and inhibitory inputs into the VTA were altered in the taar1 Tg mice (Revel et al. 2012a). Interestingly, although the basal levels of dopamine and norepinephrine in the nucleus accumbens (NAc) were elevated, amphetamine did not alter dopamine levels in the taar1 Tg mice (Revel et al. 2012a). Consistently, behavioral test showed that AMPH led to hyperactivity in WT but not in taar1 Tg mice (Revel et al. 2012a).
[...] a proposed intracellular target for amphetamine is the [TAAR1], a [GPCR] that is expressed on intracellular membranes in DA neurons (Miller, 2011). Phenethylamine stimulants have been proposed to activate TAAR1, leading to increased cAMP generation and RhoA activation, with subsequent enhancement of DAT reverse transport and endocytosis (Xie and Miller, 2007, 2008, 2009; Underhill et al., 2021). Methamphetamine-induced DAT endocytosis was found to be dependent on TAAR1 expression and PKA activity as suggested by use of the kinase inhibitor H89 (Xie and Miller, 2009). However, evidence indicating that amphetamine-induced endocytosis is independent of TAAR1 includes 1) HEK-293 cells do not express TAAR1 (Reese et al., 2007; Xie and Miller, 2007) but do exhibit amphetamine-induced DAT endocytosis [present studies and (Saunders et al., 2014; Cheng et al., 2015; Wheeler et al., 2015)]; 2) cAMP and PKA activation, which are stimulated by TAAR1, antagonized amphetamine-induced DAT endocytosis in heterologous cells and DA neurons [present studies and (Wheeler et al., 2015)]; and 3) in cell lines and rodent striatal synaptosomes, PKA activation increased DAT Vmax, consistent with increased plasma membrane expression (Pristupa et al., 1998; Page et al., 2004; Batchelor and Schenk, 2018). Additionally, DMAA induced DAT endocytosis (Figs. 3 and 4) despite exhibiting no activity at human TAAR1 in receptor binding studies (Rickli et al., 2019). Therefore, although some evidence does support a role of TAAR1 in modulating amphetamine-induced DAT endocytosis, the present studies suggest that DMAA and amphetamine promote DAT endocytosis through a TAAR1-independent mechanism.
Amphetamine might act on TAAR1 to activate Gαs pathway and phosphorylate monoamine transporter such as dopamine transporter (DAT), leading to its internalization and ceased transport (Bunzow et al. 2001; Miller 2011). Behaviorally, knockout of TAAR1 in mice leads to locomotor supersensitivity induced by amphetamine, although the connection to regulation of monoamine system is undetermined and needs further investigation (Achat-Mendes et al. 2012; Lindemann et al. 2008).
Moreover, while deletion of TAAR1 led to increased frequency of spontaneous firing frequency of serotonin neurons in brain slices [36], TAAR1 full agonists significantly inhibited the firing frequency in DRN serotonin neurons [28, 38]. [...] In vitro electrophysiological recordings showed a remarkable increase in the spontaneous firing rate of dopamine neurons in the VTA of TAAR1-KO mice [36]. While the endogenous TAAR1 agonist TYR decreased the firing rate of dopamine neurons in slices from WT mice, it had no effect in slices from TAAR1-KO mice [36]. [...] Similar to TYR, [the TAAR1 full agonist] RO5166017 reduced the firing rate of VTA dopamine neurons and DRN serotonin neurons in brain slices from WT but not TAAR1-KO mice, through the activation of K+-mediated outward current, which can be blocked by TAAR1 antagonist EPPTB [38]. [...] [The TAAR1 full agonist] RO5256390 decreased the firing frequency of VTA dopamine and DRN serotonin neurons in brain slices from WT but not TAAR1-KO mice, [...]
β-Keto-analogue cathinones also exhibited approximately 10-fold lower affinity for the TA1 receptor compared with their respective non-β-keto amphetamines. [...] Activation of TA1 receptors negatively modulates dopaminergic neurotransmission. Importantly, methamphetamine decreased DAT surface expression via a TA1 receptor-mediated mechanism and thereby reduced the presence of its own pharmacological target (Xie and Miller, 2009). MDMA and amphetamine have been shown to produce enhanced DA and 5-HT release and locomotor activity in TA1 receptor knockout mice compared with wild-type mice (Lindemann et al., 2008; Di Cara et al., 2011). Because methamphetamine and MDMA auto-inhibit their neurochemical and functional effects via TA1 receptors, low affinity for these receptors may result in stronger effects on monoamine systems by cathinones compared with the classic amphetamines.
Drugs of abuse hijack brain-reward circuitry during the addiction process by augmenting action potential-dependent phasic dopamine release events associated with learning and goal-directed behavior. One prominent exception to this notion would appear to be amphetamine (AMPH) and related analogs, which are proposed instead to disrupt normal patterns of dopamine neurotransmission by depleting vesicular stores and promoting nonexocytotic dopamine efflux via reverse transport. This mechanism of AMPH action, though, is inconsistent with its therapeutic effects and addictive properties, which are thought to be reliant on phasic dopamine signaling. [...] These findings identify upregulation of exocytotic dopamine release as a key AMPH action in behaving animals and support a unified mechanism of abused drugs to activate phasic dopamine signaling. [...] Our results also raise the possibility that enhanced phasic dopamine signaling may underlie important clinical effects of AMPH. Modest activation of dopamine transients at the relatively low dose of 1 mg/kg used here may be an important action of medications that combine amphetamine salts (e.g., Adderall) used clinically (Joyce et al., 2007). Indeed, low doses of AMPH preserve and improve learning and behavioral performance (Mayorga et al., 2000; Wyvell and Berridge, 2000; Taylor and Jentsch, 2001; Zhang et al., 2003; Knutson et al., 2004). In contrast, high doses of AMPH can produce a behavioral profile similar to the positive symptoms associated with schizophrenia (Featherstone et al., 2007; Lisman et al., 2008). [...] Thus, disrupting the tight temporal concurrence between dopamine transients and important external cues due to hyperactive phasic dopamine signaling may contribute to AMPH-induced psychosis (Featherstone et al., 2007).
While there is little debate that behavioral effects of this important psychostimulant are associated with a hyperdopamine state [3–6], the underlying mechanisms by which this condition manifests have been the subject of intense study. Two, what ostensibly appear to be mutually exclusive, views have emerged. On the one hand, AMPH enhances tonic dopamine signaling by reversing dopamine transporter (DAT) direction, leading to a non-exocytotic, action potential-independent type of release or efflux that is driven by vesicular depletion and the redistribution of dopamine to the cytosol [7,8]. On the other hand, AMPH enhances phasic dopamine signaling by promoting burst firing of dopamine neurons [9,10], inhibiting dopamine uptake [11,12], and upregulating vesicular dopamine release [13,14]. How AMPH concurrently activates tonic and phasic dopamine signaling, the two fundamental modes of communication used by dopamine neurons [15], yet elicits opposing actions on vesicular dopamine stores is perplexing and unresolved.
We found that σ1R activation prevents methamphetamine-induced, DAT-mediated increases in firing activity of dopamine neurons. In vitro and in vivo amperometric measurements revealed that σ1R activation decreases methamphetamine-stimulated dopamine efflux without affecting basal dopamine neurotransmission. Consistent with these findings, σ1R activation decreases methamphetamine-induced locomotion, motivated behavior, and enhancement of brain reward function. Notably, we revealed that the σ1R interacts with DAT at or near the plasma membrane and decreases methamphetamine-induced Ca2+ signaling, providing potential mechanisms.
Although the precise physiological role of TAAR1/G13-mediated DAT internalization remains to be established, it is likely that this pathway evolved to regulate the actions of endogenous amines including β-PEA, tyramine, 3-methoxytyramine, and DA. DA is a TAAR1 agonist (Fig. 1c) and recently, it was shown that the sustained elevation of extracellular DA seen following burst firing appears to be a consequence of DAT internalization by a Rho-dependent mechanism [51]. These results provide the first evidence that dopamine itself can act as a TAAR1 agonist to trigger transporter internalization in vivo. TAAR1 signaling also has the potential to serve as a sensor of cytoplasmic DA and its metabolites. At high cytosolic concentrations, DA becomes neurotoxic due to the formation of reactive oxygen species and quinones that can cause damage to the neuron [52]. Under normal conditions, DA transported through the DAT from the extracellular space is rapidly repackaged into synaptic vesicles or enzymatically metabolized. However, if cytoplasmic DA concentrations saturate these mechanisms, the reduction in surface levels of DAT mediated by TAAR1/G13 signaling may provide an additional means for maintaining free cytosolic DA below toxic levels.
This flooding of the cytoplasm and synaptic space with the oxidatively labile DA is thought to be a critical first step in the neurotoxic cascade of the amphetamines [73]. These conditions of elevated concentrations of cytosolic monoamines could be further aggravated by inhibition of MAO [91]. Unlike amphetamines, mephedrone and methylone have little if any affinity for VMAT-2 [33]. Therefore, their lack of neurotoxicity could derive from an inability to promote the release of DA from storage vesicles into the cytoplasm.
Amphetamine treatment similar to that used for ADHD has been demonstrated to produce brain dopaminergic neurotoxicity in primates, causing the damage of dopaminergic nerve endings in the striatum that may also occur in other disorders with long-term amphetamine treatment (57).
Though the paradigm used by Ricaurte et al. 53 arguably still incorporates amphetamine exposure at a level above much clinical use,14,55 it raises important unanswered questions. Is there a threshold of amphetamine exposure above which persistent changes in the dopamine system are induced? [...]
The present findings with MDMA are consistent with our previous data [15,17] and those reported by Simmler et al. [4] and Eshleman et al. [23] who examined the effects of MDMA and related drugs in human embryonic kidney (HEK) cells transfected with human SERT, DAT and NET. Thus, the molecular mechanism of action for MDMA at monoamine transporters is similar in rats and humans. On the other hand, the potency of (±)MDMA for releasing monoamines in rat brain synaptosomes shown here (i.e., 60–70 nM) is greater than its potency in transfected HEK cells (i.e., 1–20 μM). Such discrepancies in absolute potency could be related to species differences in drug responsiveness, differences in release assay methods employed, or the absence of important neuronal membrane proteins in non-neuronal HEK cells.
Table 5. Action of MDMA, MDA, and PMMA as Releasing Agents at the Serotonin (SERT), Dopamine (DAT), and Norepinephrine (NET) Transporters18,59,60 [...] a Data, although from different publications, were obtained from the same laboratory.
The major goal of this study was to establish a high-throughput assay to detect [monoamine transporter substrates (SBSTs)] for use in a project to develop new drugs with dual activity as SBSTs of DAT and SERT. METHODS. Using minor modifications of published procedures, rat brain synaptosomes were preloaded with either [3H]DA, [3H]NE or [3H]5-HT. Test drugs were added and the reaction terminated by rapid filtration over Whatman GF/B filters. Release was quantified by counting how much tritium was retained on the filters. RESULTS. Using optimized conditions known SBSTs potently decreased retained tritium in a dose-dependent manner whereas known [uptake inhibitors (UIs)] were weak or ineffective. UIs shifted SBST inhibition curves to the right, consistent with antagonist-like activity. CONCLUSION. We have developed high throughput assays which detect SBSTs for the DA, 5-HT and NE transporters.
RESULTS. Methamphetamine and amphetamine potently released NE (IC50s = 14.3 and 7.0 nM) and DA (IC50s = 40.4 nM and 24.8 nM), and were much less potent releasers of 5-HT (IC50s = 740 nM and 1765 nM). Phentermine released all three biogenic amines with an order of potency NE (IC50 = 28.8 nM)> DA (IC50 = 262 nM)> 5-HT (IC50 = 2575 nM). Aminorex released NE (IC50 = 26.4 nM), DA (IC50 = 44.8 nM) and 5-HT (IC50 = 193 nM). Chlorphentermine was a very potent 5-HT releaser (IC50 = 18.2 nM), a weaker DA releaser (IC50 = 935 nM) and inactive in the NE release assay. Chlorphentermine was a moderate potency inhibitor of [3H]NE uptake (Ki = 451 nM). Diethylpropion, which is self-administered, was a weak DA uptake inhibitor (Ki = 15 µM) and NE uptake inhibitor (Ki = 18.1 µM) and essentially inactive in the other assays. Phendimetrazine, which is self-administered, was a weak DA uptake inhibitor (IC50 = 19 µM), a weak NE uptake inhibitor (8.3 µM) and essentially inactive in the other assays.
FIGURE 2-6: Release: Effects of the specified test drug on monoamine release by DAT (red circles), NET (blue squares), and SERT (black traingles) in rat brain tissue. [...] EC50 values determined for the drug indicated within the panel. [...]
It has been suggested that the association between PD and ADHD may be explained, in part, by toxic effects of these drugs on DA neurons.241 [...] An important question is whether amphetamines, as they are used clinically to treat ADHD, are toxic to DA neurons. In most of the animal and human studies cited above, stimulant exposure levels are high relative to clinical doses, and dosing regimens (as stimulants) rarely mimic the manner in which these drugs are used clinically. The study by Ricaurte and colleagues248 is an exception. In that study, baboons orally self-administered a racemic (3:1 d/l) amphetamine mixture twice daily in increasing doses ranging from 2.5 to 20 mg/day for four weeks. Plasma amphetamine concentrations, measured at one-week intervals, were comparable to those observed in children taking amphetamine for ADHD. Two to four weeks after cessation of amphetamine treatment, multiple markers of striatal DA function were decreased, including DA and DAT. In another group of animals (squirrel monkeys), d/l amphetamine blood concentration was titrated to clinically comparable levels for four weeks by administering varying doses of amphetamine by orogastric gavage. These animals also had decreased markers of striatal DA function assessed two weeks after cessation of amphetamine.
While no in vivo studies have been done with selective sigma 2 receptor ligands measuring amphetamine-mediated locomotor activity, it was found that sigma 2 receptor agonists potentiate amphetamine-mediated dopamine efflux in PC12 cells in vitro (Izenwasser et al., 1998; Weatherspoon and Werling, 1999). These in vitro results indicate that sigma 2 receptor agonists would potentiate amphetamine-, and potentially cocaine-mediated locomotor activity in vivo. Due to the lack of selective sigma 2 ligands, the precise mechanism of sigma 2 enhancement of dopamine release is not known. However, it has been postulated that sigma 2 activation leads to an increase in intracellular calcium, either through an interaction with the plasma membrane bound L-type calcium channels, or via the ER-bound IP3 ligand gated calcium channels (Zhang and Cuevas, 2002; Cassano et al., 2006, 2009). This increase in intracellular calcium is hypothesized to promote the activation of PKC, and therefore the phosphorylation of DAT, leading to an increase in extracellular dopamine (Derbez et al., 2002).
[...] it is unclear if TAAR1 plays any role in the effects of MDMA in humans, as MDMA does not activate human TAAR1 in cellular assays like it does mouse and rat TAAR1.84
The characteristics and mechanisms of efflux are complex and incompletely understood, and have been summarized in many comprehensive reviews including (Reith & Gnegy, 2020; Robertson, Matthies, & Galli, 2009; Sitte & Freissmuth, 2015). [...] Early studies implicating protein phosphorylation in AMPH-evoked DA efflux were performed by Giambalvo, who demonstrated that DAT-mediated uptake of AMPH impacted the activity and subcellular localization of protein kinase C (PKC) with characteristics that correlated with DA efflux (Giambalvo, 1992), and Gnegy and colleagues, whose many studies reinforced the relationship between efflux, kinases, and ionic conditions including PKC, PKCβ, Ca2+, and Ca1+-calmodulin regulated protein kinase (CaMK) (Gnegy et al., 2004; Johnson, Guptaroy, Lund, Shamban, & Gnegy, 2005; Kantor, Hewlett, & Gnegy, 1999). [...] With respect to DAT most studies have focused on PKC, CaMK, and mitogen activated protein kinases (MAPKs), but many other signaling and kinase pathways have been implicated in efflux mechanisms for all of the MATs (Bermingham & Blakely, 2016; Vaughan & Foster, 2013). [...] DAT phosphorylation stimulated by AMPH or METH also occurs on this domain and is blocked by PKC inhibitors (Cervinski et al., 2005; Karam et al., 2017), indicating the capacity of the drugs to involve the PKC pathway. How this occurs is not known, but could potentially follow from drug perturbation of local ion/Ca2+ concentrations that impact PTM enzymes, or alternatively could occur by substrate-driven induction of transporter conformations in which phosphorylation sites become more or less available to enzyme action. [...] There are many other Ser and Thr residues on DAT intracellular loops and domains that may serve as phosphorylation sites, and in vitro studies have demon- strated phosphorylation of N— and C—terminal peptide sequences by many kinases in addition to PKC, ERK, and CaMK that have been implicated in regulation and efflux including protein kinase A (PKA), protein kinase G (PKG), casein kinase 2 (CK2), p38 kinase, JNK2, Cdk5, and Akt1 (Gorentla I et al., 2009; Vaughan & Foster, 2013). [...] The regulatory, efflux, and post-translational characteristics of NET and SERT show many similarities to DAT that indicate conservation of mechanism, but also some distinct properties that reflect requirements of specific neuronal populations. [...] With respect to efflux there are many similarities between SERT, NET, and DAT that indicate conservation of mechanisms.
