Biodistribution.

PET neuroimaging in nonhuman primates has been critical for understanding the mechanism of action of cocaine and related stimulants. An early study focused on the distribution of cocaine binding in the brain of anesthetized baboons using ¹¹C-labeled cocaine (Fowler et al., 1989). Cocaine binding was heterogeneous but showed some selectivity for dopamine transporter (DAT)-rich striatal regions. Striatal cocaine binding was inhibited by pretreatments with pharmacological doses of cocaine and DAT inhibitors but not by norepinephrine transporter or serotonin transporter (SERT) inhibitors. Direct comparisons in human subjects showed a similar distribution of binding with the highest concentration in the striatum. A subsequent study documented significant overlap in the distributions of binding of ¹¹C-labeled cocaine and methylphenidate (Volkow et al., 1995). It is noteworthy that a direct relationship was established between self-reports of “high” induced by cocaine and the time course of striatal uptake (Volkow et al., 1997). More recently, the brain pharmacokinetics of methamphetamine were compared with cocaine in anesthetized baboons using ¹¹C-labeled d-methamphetamine and (−)cocaine (Fowler et al., 2007). The results indicated that the slower clearance of methamphetamine compared with cocaine probably contributed to its longer-lasting stimulant effects. Finally, the reinforcing effects of several cocaine analogs were compared with the time course of uptake of the ¹¹C-labeled drugs in the putamen of awake rhesus monkeys (Kimmel et al., 2008). The cocaine analogs were reliably self-administered, but the rates of responding were lower than those maintained by cocaine. It is noteworthy that there was a clear trend toward an inverse relationship between the time to peak uptake of ¹¹C-labeled drugs in putamen and the peak number of intravenous infusions received, such that the faster-onset drugs produced greater levels of responding relative to the slower-onset drugs. There was also a close correspondence between the time course of drug uptake in brain and drug-induced increases in extracellular dopamine in caudate (Czoty et al., 2002; Ginsburg et al., 2005; Kimmel et al., 2007, 2008). These studies clearly show that PET biodistribution measures inform our understanding of drug mechanism of action and concomitant behavioral effects, such as reinforcing and subjective effects. However, because these studies exclusively examined the brain biodistribution of stimulants, it remains to be determined whether these techniques will prove to be as useful when applied to other drug classes.

Drug Occupancy.

PET neuroimaging in nonhuman primates has documented the relationship between drug occupancy at monoamine transporters and the behavioral effects of stimulants. For example, PET imaging in rhesus monkeys using ¹⁸F-labeled FECNT, a DAT-selective radioligand, showed that FECNT labels a cocaine-sensitive binding site, and high levels of DAT occupancy are associated with behaviorally active cocaine doses (Votaw et al., 2002). More recently, the reinforcing effects of local anesthetics that bind to the DAT in vitro were evaluated for DAT occupancy in vivo in rhesus monkeys (Wilcox et al., 2005). Doses of dimethocaine that maintained maximum response rates produced DAT occupancies between 66 and 82%. These values are highly concordant with results from human PET imaging studies, which found that DAT occupancies were between 60 and 77% for cocaine doses that subjects reported as rewarding (Volkow et al., 1997). They are also concordant with PET imaging data in rhesus monkeys, which revealed that cocaine DAT occupancies between 65 and 76% maintain peak response rates (Wilcox et al., 2002). Unlike dimethocaine, procaine was ineffective in maintaining self-administration and resulted in DAT occupancies between 10 and 41% (Wilcox et al., 2005). However, irrespective of the drug, in vivo microdialysis showed that reinforcing effects and DAT occupancy were closely related to drug-induced increases in extracellular dopamine. These studies illustrate the power of PET imaging to unmask drug mechanisms of action, particularly as it relates to monoamine transporters, and they highlight the utility of the translational nature of PET imaging in nonhuman primates. Consistent with these findings, it has been shown that DAT occupancy by methylphenidate is highly concordant between rhesus monkeys and humans when blood levels of the drug are matched (Wilcox et al., 2008).

PET imaging has also been used to study protein occupancy by other stimulants. The interoceptive and reinforcing effects of the substituted phenethylamine MDMA share some similarity to other stimulants (Fantegrossi et al., 2002; Murnane et al., 2009), but the role of the DAT in the behavioral effects of MDMA is not well documented, especially in primates. Accordingly, a study assessed the role of the DAT in the behavioral effects of racemic MDMA in nonhuman primates (Fantegrossi et al., 2009). A dose of MDMA that suppressed operant behavior had negligible effects on extracellular dopamine in the caudate, and the percentage of DAT occupancy of MDMA was marginal. This work was subsequently corroborated by findings that higher doses of MDMA were required to elicit significant release of dopamine (Murnane et al., 2010). Collectively, these results indicate that the rate-suppressant effects of MDMA are not mediated by the DAT and that MDMA has lower potency at the DAT compared with other stimulants, such as cocaine or amphetamine. Similar to MDMA, the role of the DAT in the behavioral effects of the wake-promoting drug modafinil is not well documented. It is noteworthy that a number of clinical studies suggest that modafinil may improve clinical outcomes for the treatment of cocaine dependence by reducing self-reports of craving and cocaine-induced euphoria (Dackis et al., 2003, 2005; Hart et al., 2008; Anderson et al., 2009) through a possible DAT-mediated mechanism (Volkow et al., 2009; Zolkowska et al., 2009). To this end, a study in rhesus monkeys demonstrated that the in vivo effects of modafinil at the DAT are similar to other stimulants, such as cocaine (Andersen et al., 2010). Modafinil induced nocturnal locomotor-stimulant effects and reinstated previously extinguished cocaine self-administration. An effective dose of modafinil resulted in approximately 60% DAT occupancy in the striatum and significantly increased extracellular dopamine levels, comparable with effects observed after cocaine doses that reliably maintain self-administration (Ito et al., 2002; Votaw et al., 2002; Wilcox et al., 2002, 2005). The results obtained provide important information about the mechanism of action of modafinil and show low-potency DAT-related effects in nonhuman primates. Although these studies collectively demonstrate the power of PET imaging to characterize the transporter-related effects of stimulants, the relationship between presynaptic transporter effects and postsynaptic dopamine receptor effects requires further elucidation.

Neurotransmitter Release.

Competition between radiolabeled ligands and endogenous neurotransmitters provides an effective means of evaluating drug-induced changes in extracellular neurotransmitter concentrations in vivo (see Laruelle, 2000). Similar to other PET measures, findings in nonhuman primates and human subjects exhibit considerable overlap (Table 2). PET neuroimaging with ¹⁸F-labeled fluoroclebopride (FCP) as a reversible D2 receptor ligand characterized stimulant-induced dopamine release in rhesus monkeys (Mach et al., 1997). Intravenous administration of cocaine, amphetamine, methylphenidate, and methamphetamine increased rates of FCP washout from the basal ganglia, consistent with the capacity of each drug to elevate extracellular dopamine. ¹¹C-labeled raclopride studies in baboons (Dewey et al., 1992; Villemagne et al., 1998; Volkow et al., 1999) and ¹⁸F-labeled fallypride studies in rhesus monkeys (Mukherjee et al., 1997) documented that these effects can be demonstrated with several radioligands and in several primate species. Other studies in baboons and rhesus monkeys have begun to document the usefulness of drug-induced displacement of ¹⁸F-labeled fallypride to characterize dopamine release in extrastriatal brain regions (Mukherjee et al., 1997; Slifstein et al., 2004). Taken together, these results validate the use of PET imaging as an in vivo measure of neurotransmitter release in nonhuman primates and provide a solid foundation for human studies (Martinez et al., 2007; Volkow et al., 2008). However, most studies have been limited to dopamine displacement of D2 binding in the striatum. In apparent contrast to the effects of dopamine releasers, the serotonin releaser fenfluramine significantly increased extracellular serotonin in rhesus monkeys without displacing the ¹⁸F-labeled serotonin 1A receptor ligand MPPF [4-fluoro-N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)benzamide] (Udo de Haes et al., 2006). Moreover, dopamine depletion did not affect the binding potential of the ¹¹C-labeled D2 receptor ligand FLB 457 [(S)-5-bromo-N-[(1-cyclopropylmethyl-2-pyrrolidinyl)methyl]-2,3-dimethoxybenzamide] in human cortex (Frankle et al., 2010). The relative affinity of the radioligand and the endogenous neurotransmitter, protein density in specific brain regions, and direct interactions between the drug and its metabolites with the protein target all are important considerations that may influence the outcome and interpretation of in vivo displacement studies.

PET imaging has been used in nonhuman primates to examine the receptor pharmacology influencing dopamine release. In one study, pretreatment with the metabotropic glutamate receptor 1 antagonist 2-methyl-6-(phenylethynyl)pyridine attenuated dopamine release by methamphetamine, as measured by ¹¹C-labeled MNPA (Tokunaga et al., 2009). Likewise, the metabotropic glutamate receptor 2 agonist (2S,4S)-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-dioic acid (LY354740) potentiated amphetamine-elicited dopamine release, as measured by ¹¹C-labeled raclopride (van Berckel et al., 2006). Similar to biodistribution studies, displacement of radiotracers by drug-induced increases in neurotransmitter levels can be used to study the time course of drug action (Narendran et al., 2007). In addition, the importance of the intrinsic efficacy of PET radioligands has become recognized. For example, the D2 receptor agonist radioligand MNPA is more sensitive than the D2 antagonist radioligand raclopride to amphetamine-elicited increases in dopamine levels (Seneca et al., 2006). This is consistent with prior in vitro competition binding work showing that agonists have a higher apparent affinity for receptors labeled with agonist radioligands than antagonist radioligands (Sleight et al., 1996). Moving forward, these pharmacodynamic and pharmacokinetic studies are likely to yield new insights into mechanisms that modulate the neurochemical and behavioral effects of drugs of abuse.

Cerebral Blood Flow and Metabolism.

The noninvasive measurement of cerebral blood flow with PET neuroimaging and [¹⁵O]water provides a useful means to characterize acute drug-induced changes in brain activity. Early studies in human cocaine abusers using single-photon emission computed tomography imaging largely showed regional decreases in cerebral blood flow after acute administration of cocaine (Pearlson et al., 1993; Wallace et al., 1996; Johnson et al., 1998). Functional changes in cerebral blood flow using PET imaging have also been determined in awake, drug-naive rhesus monkeys after acute intravenous cocaine administration (Howell et al., 2001, 2002). In contrast to the deactivations found in humans with a history of cocaine abuse, brain activation maps normalized to global flow showed prominent cocaine-induced activation of prefrontal cortex, especially dorsolaterally. These differences in the direction of the change in blood flow induced by cocaine may have been caused by differences in the methodology implemented, the dose of cocaine tested, the drug history of the subject, or other factors. Nevertheless, importantly, the selective SERT inhibitor alaproclate attenuated the brain activational effects of cocaine, cocaine-induced increases in striatal dopamine, and self-administration of cocaine in rhesus monkeys (Czoty et al., 2002; Howell et al., 2002). Hence, there was close concordance between in vivo measures of behavior, neurochemistry, and functional imaging. Another study was the first to use PET imaging with [¹⁵O]water to document acute cocaine-induced changes in brain activity during cocaine self-administration in nonhuman primates (Howell et al., 2010). The area of major activation included the anterior cingulate cortex, a region associated with the extended limbic system. Furthermore, drug-associated stimuli elicited increases in regional cerebral blood flow in the dorsomedial prefrontal cortex, indicating robust cortical activation. Consistent with these findings, a study in cocaine-addicted humans showed that a dose of methylphenidate that increased dopamine levels, as indexed by displacement of ¹¹C-labeled raclopride binding, only elicited drug “craving” when presented in combination with drug-associated stimuli (Volkow et al., 2008). In another study in nonhuman primates, functional changes in glucose metabolism were characterized in rhesus monkeys with ¹⁸F-labeled flurodeoxyglucose (FDG) after acute administration of cocaine (Henry et al., 2010). Similar to the results obtained using [¹⁵O]water to map cerebral blood flow, metabolic mapping demonstrated acute cocaine-induced activation of extended limbic regions in cocaine-naive rhesus monkeys. Collectively, these studies enhance our understanding of the neurobiological effects of drugs of abuse. However, although extensive PET imaging work has examined dopaminergic mechanisms (Fig. 1) of drugs of abuse in nonhuman primates, a relative paucity of work regarding other mechanisms of action, such as serotonergic effects, has been published.

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Fig. 1.

Elements of the dopamine neurovascular unit that have been previously imaged using PET imaging in nonhuman primate drug abuse research. As can be seen, numerous radiotracers have been developed and validated for studying a broad range of dopaminergic elements. Inset, the key for the elements.

Neurobiological Changes.

A major advantage of PET imaging is the ability to use longitudinal designs that involve repeated measures over extended periods of time. This approach has been used effectively in nonhuman primates to characterize enduring changes in brain chemistry associated with chronic drug exposure (Table 3). For example, PET imaging studies conducted in socially housed cynomolgus monkeys characterized the effects of chronic cocaine exposure in dominant and subordinate subjects. Although dominant monkeys initially exhibited higher D2 receptor availability and lower rates of cocaine self-administration (Morgan et al., 2002), chronic exposure to self-administered cocaine resulted in reductions in D2 receptor availability to levels comparable with subordinate monkeys (Czoty et al., 2004). A subsequent study examined D2 receptor availability during extended cocaine abstinence (Nader et al., 2006). In subjects with short-term exposure, D2 receptor availability returned to predrug levels within 3 weeks. In subjects with long-term exposure, some showed complete recovery within 3 months, whereas others did not recover after 1 year of abstinence. However, the rate of recovery did not correlate with total drug intake. Nevertheless, individual differences in D2 receptor availability recovery rates have also been observed after drug-induced increases by D2 receptor antagonists (Czoty et al., 2005). An early study of cocaine self-administration effects on DAT levels largely showed increases in DAT levels, but in a dose-, brain region-, and exposure duration-dependent fashion (Letchworth et al., 2001). A study of DAT availability using ¹⁸F-labeled (+)-N-(4-fluorobenzyl)-2β-propanoyl-3β-(4-chlorophenyl)tropane examined the effects of cocaine self-administration in rhesus monkeys under contingencies that resulted in low drug intake (Czoty et al., 2007). Self-administration of a low cocaine dose over 9 weeks did not significantly affect DAT availability in any brain region. After more prolonged histories of cocaine self-administration, significant increases in striatal SERT were observed in experiments using ¹¹C-labeled DASB (Banks et al., 2008). In other work, a noncontingent dosing regimen of morphine decreased DAT availability in rhesus monkeys (Xiao et al., 2006). Collectively, these studies demonstrate substantial, but yet to be fully elucidated, plasticity of the dopaminergic system in response to exposure to drugs of abuse.

Evaluations of drug-induced changes in protein binding in vivo are complemented by a study that documented cocaine-induced changes in brain metabolic activity as a function of cocaine self-administration history (Henry et al., 2010). Experimentally naive rhesus monkeys were given increasing access to cocaine self-administration. PET neuroimaging with ¹⁸F-labeled FDG was used to measure acute cocaine-induced changes in brain metabolism in the cocaine-naive state and in limited- and extended-access conditions. In the cocaine-naive state, cocaine-induced increases in brain metabolism were restricted to the anterior cingulate and medial prefrontal cortex. Increased cocaine exposure from limited through extended access recruited cocaine-induced metabolic effects in additional frontal cortical areas and within the striatum. In apparent contrast, tolerance to cocaine- and amphetamine-induced synaptic release of dopamine in the striatum was observed in these same animals under both access conditions (Kirkland Henry et al., 2009). It is noteworthy that blunting of dopamine release has also been recorded in cocaine-dependent humans, in experiments using ¹¹C-labeled raclopride (Martinez et al., 2007). Furthermore, this blunting of dopamine release was associated with whether the subject would choose cocaine over money. Accordingly, further combined study of the relationship between drug self-administration and both tolerance to the dopaminergic effects of cocaine and its recruitment of cortical activation may be highly relevant toward efforts to develop treatments for cocaine addiction. Indeed, a treatment that reverses these effects may have significant clinical value.

There has been significant interest in the potential neurotoxic effects of amphetamine derivatives, such as methamphetamine and MDMA. Under a variety of conditions, MDMA has selective and enduring effects on markers of brain serotonin systems, which some investigators interpret as neurotoxicity. However, early studies were limited by biochemical and histological analyses that required between-subject comparisons. An early PET imaging study in a baboon characterized the effects of MDMA on in vivo SERT availability using ¹¹C-labeled McN5652 (Scheffel et al., 1998). After treatment with MDMA twice daily for 4 consecutive days, PET scans showed reductions in SERT availability in all brain regions analyzed at 13 to 40 days after drug treatment but regional differences in its apparent recovery at 9 and 13 months. Similar results have been reported for methamphetamine-induced reductions in DAT availability in baboons (Villemagne et al., 1998) and rhesus monkeys (Hashimoto et al., 2007). However, other studies provided more ambiguous results (Melega et al., 2008), including small and transient changes in D1 receptor availability, using ¹¹C-labeled SCH23390 (Hashimoto et al., 2007). Furthermore, behavioral decrements resulting from neurochemical changes induced by exposure to amphetamine derivatives have been much more difficult to establish (Winsauer et al., 2002; Saadat et al., 2006). It is critical to note that studies reporting neurotoxic effects of amphetamine derivatives have relied on noncontingent drug administration and have typically administered large and/or repeated doses. In one of the first studies to characterize the neurochemical effects of self-administered MDMA in nonhuman primates, rhesus monkeys self-administered MDMA for approximately 18 months. PET neuroimaging with ¹¹C-labeled DTBZ was used to quantify vesicular monoamine transporter availability after at least 2 months of drug abstinence (Fantegrossi et al., 2004). The reinforcing effects of MDMA were selectively attenuated by chronic MDMA self-administration but there was no significant change in vesicular monoamine transporter binding potential and no significant changes in serotonin or dopamine levels in postmortem brains. Another study found a similar lack of significant SERT availability changes after MDMA self-administration in rhesus monkeys using ¹¹C-labeled DASB (Banks et al., 2008). Hence, noncontingent drug administration has yielded neurochemical changes in the absence of behavioral correlates, whereas drug self-administration has yielded behavioral alterations in the absence of any significant neurochemical correlates. As such, given the important public health implications of drug-induced neurotoxicity, further study is clearly warranted. In this regard, PET imaging in rhesus monkeys has shown that before or after exposure treatment with the antibiotic minocycline prevents methamphetamine-elicited reductions in DAT availability (Hashimoto et al., 2007). Such an approach may be highly beneficial in the prevention or treatment of any neurotoxic effects of amphetamine derivatives.

Vulnerability Factors.

It has become well accepted that behavior, brain chemistry, and neuronal function can be readily influenced by pharmacological challenges and environmental conditions. Differences in the dominance rank among socially housed nonhuman primates have been associated with differential levels of dopamine D2 receptors as measured with ¹⁸F-labeled FCP (see Nader and Czoty, 2005). Social housing of male cynomolgus monkeys increased the availability of D2 receptors in dominant animals without producing any changes in subordinate group members, and these changes seemed to exert significant effects on cocaine self-administration (Morgan et al., 2002). Cocaine reliably functioned as a reinforcer in subordinate subjects but failed to maintain self-administration in dominant monkeys. Likewise, subordinate animals were more sensitive to the reinforcing effects of cocaine evaluated with a choice procedure, such that they would choose a lower dose of cocaine over food compared with dominant animals (Czoty et al., 2005). In addition, the protective effects associated with high D2 receptor density in dominant animals can be attenuated with prolonged exposure to cocaine (Czoty et al., 2004). Hence, a trait variable that is associated with low vulnerability to stimulant abuse may become less important with continued exposure to stimulants. Furthermore, the observation that female cynomolgus monkeys show significant changes in D2 binding potential associated with menstrual cycle phase may have direct relevance to understanding the neurobiological basis of human sex differences in sensitivity to stimulants (Czoty et al., 2009). The success of these studies suggests that the identification of additional vulnerability factors other than D2 levels would be of great benefit to drug abuse research.