The role of sex and drug use during adolescence in determining the risk for adverse consequences of amphetamines

Abstract

Use of amphetamines during adolescence, a critical period of brain development and reorganization, may lead to particularly adverse outcomes that are long-lasting. Similarly, female users may be uniquely vulnerable to certain aspects of drug use. A recognition of the role of use during adolescence and sex on outcomes of amphetamine and methamphetamine exposure are of critical importance in understanding and treating substance use disorders. This chapter highlights what human research, which has been largely epidemiological, suggests about sex and age differences in drug use patterns and outcomes. We also discuss work in laboratory animals that has typically utilized rats or mice exposed to drugs in a non-contingent manner (i.e., involuntarily) or through volitional self-administration. Lastly, we draw attention to the fact that advancing our understanding of the effects of amphetamine and methamphetamine use, the development of problematic drug taking, and the mechanisms that contribute to relapse will require an emphasis on inclusion of age and sex as moderating factors in future studies.

Introduction

For at least as long as we have had recorded history, the use of mind-altering substances (both natural and manufactured) has been a wide-spread facet of human life. In addition to the purposeful application of these substances for medicinal or spiritual purposes, drugs are often used purely for recreational enjoyment. According to the 2021 National Survey of Drug Use and Health (NSDUH 2020), 22% of Americans aged 12 or older reported having used an illicit substance within the past year, with 3% reporting having misused some sort of central nervous system stimulant. The stimulant class of drugs includes many chemically and pharmacologically distinct compounds, including cocaine, nicotine, and caffeine. The focus here will be on two stimulant drugs with widespread therapeutic and non-therapeutic uses: amphetamine and methamphetamine. These drugs are very similar chemically, with only the addition of a methyl group differentiating the two, and their pharmacological effects are practically indistinguishable (Wilens & Spencer, 1998). Despite the oft-repeated notion that methamphetamine is a more “potent”, “dangerous”, and/or “addictive” form of amphetamine, there is limited empirical evidence to support this belief (Balster & Schuster, 1973; Hall, Stanis, Marquez-Avila & Gulley, 2008; Kirkpatrick, Gunderson, Johanson, Levin, Foltin & Hart, 2012). Both drugs produce a sensation of exhilaration and increased alertness in users, and are often taken to increase mental performance, to extend wakefulness, or simply for the euphoric rush that accompanies the initial onset of the drug’s effects at higher doses.

While the misuse of drugs is undoubtedly problematic at any stage of life, evidence suggests misuse during adolescence may pose unique risks as compared to adult use. In recent studies, 12-17 year old adolescents who initiated misuse of either cannabis or prescription medications were more likely to develop a substance use disorder (SUD) within 12 months compared to those who initiated as young adults (18-25 years old) (Han, Compton, Blanco, & Jones, 2019; Volkow, Han, Einstein, & Compton, 2021). Across the lifetime, the risk of developing an SUD is over four times higher in individuals who initiate use before the age of 15 than for those who do not begin use until after 21 (Grant & Dawson, 1997), and the chances of developing the clinical features of an SUD are far higher in adolescent-onset users compared to those who begin drug taking as adults, even if full diagnosis is not reached (Chen, Storr & Anthony, 2009). It has also been shown that beginning substance use earlier is predictive of greater addiction severity and morbidity, including the use of multiple substances (Taioli & Wynder 1991; Kandel, Yamaguchi & Chen, 1992; Anthony & Petronis, 1995). Furthermore, existing treatments for SUD appear to fail at least half of adolescent-onset users, compared to approximately 30% of adult-onset users, suggesting that current treatments are particularly ineffective for individuals who initiate use during adolescence (Poudel & Gautam 2017). Part of this failure may be due to the dearth of pharmacotherapies available to adolescents with SUDs. Currently, buprenorphine-naloxone is the only pharmacotherapy approved by the FDA for treatment in adolescents (16+ years old) with SUDs and that is for opioid use disorder.

Adolescence is a time of considerable emotional, physical, and cognitive changes. This includes significant brain development, with many regions and their associated connections remaining relatively immature through approximately the mid-twenties (Pfefferbaum, Mathalon, Sullivan, Rawles, Zipursky & Lim, 1994; Giedd, 2004). This relatively delayed neurodevelopment occurs along brain region-specific timelines, with sensorimotor areas developing early and higher-order areas such as the prefrontal cortex developing later on in adolescence into young-adulthood. (Sowell, Thompson, Holmes, Jernigan & Toga, 1999, Shaw, Kabani, Lerch, Eckstrand, Lenroot, Gogtay et al, 2008; Gied & Rapoport, 2010; Stiles & Jernigan, 2010). Adolescence is also a time when substance use is initiated, and it is thought that this period of developmental change may represent a time in which the brain is particularly vulnerable to the disruptive effects of stimulant drugs (Dahl, 2004; Casey & Jones, 2010; Gulley & Juraska, 2013). Unfortunately, past-year amphetamine misuse among young adolescents appears to be trending upwards, increasing from 3.8% of eighth graders surveyed in 2016 to 5.3% in 2020 (Schulenberg, Patrick, Johnston, O’Malley, Bachman, & Miech, 2021). This concerning increase in use prevalence further emphasizes the importance of understanding the potential unique impacts of stimulant use on the developing brain.

In addition to adolescent use, gender (in humans) or sex (in laboratory animals) has also been shown to confer specific risks in the context of substance misuse. How gender/sex may interact with adolescent use in drug seeking and taking, and the effects of stimulant use on the brain, is even less fully understood, but key findings in this field suggest that just as normal neurobehavioral development is sex specific (Juraska & Willing, 2017) alterations driven by methamphetamine use may also be different in males versus females (Daiwile, Sullivan, Jayanthi, Goldstein, & Cadet, 2022). This chapter provides an overview of the work that has investigated the effects of amphetamine and methamphetamine use during adolescence, and the role of sex on these outcomes.

Therapeutic use in humans

Attention deficit hyperactivity disorder (ADHD) is one of the most commonly diagnosed psychiatric disorders among adolescents in the United States, with recent studies suggesting prevalence rates around 10% (Danielson, Bitsko, Ghandour, Holbrook, Kogan & Blumberg, 2018). In one sample of children aged 4-18, 62% were on medications to treat their symptoms and less than half (46.7%) were receiving behavioral therapy despite recommendations by the American Academy of Pediatrics stating those 12-17 years old should undergo behavioral interventions in conjunction with medication (Wolraich, Brown, Brown, DuPaul, Earls, Feldman, Ganiats et al., 2011). ADHD is diagnosed 2.1 times more often in boys than in girls in the United States, but this disparity may be due in part to differences in the way symptoms present in boys and girls. Where boys are more often disruptive, girls are more likely to present with primarily inattentive symptoms (Merikangas, He, Brody, Fisher, Bourdon & Koretz, 2010) that can be more challenging to identify and thus lead to fewer girls being diagnosed and treated than boys.

In children, adolescents, and adults diagnosed with ADHD, stimulant drugs are considered the first-line of treatment (Baverstock & Finlay, 2003). These drugs, which typically include methylphenidate, amphetamine, or methamphetamine, are approved by the FDA for treatment given orally, at doses that are generally considered to be safe and with a low likelihood for misuse. Typically, amphetamine is administered either twice daily in formulations designed for immediate release (e.g., Adderall IR tablets, DextroStat) or once daily in formulations designed to release in a slow and sustained manner (Adderall XR). Methamphetamine, which is also FDA-approved for the treatment of ADHD, is far less commonly prescribed. Maximum doses of fast-release amphetamine for children and adolescents are higher than those approved for adults (40 mg and 20 mg, respectively; Berman, Kuczenski, McCracken & London, 2009).

The potential for prescription amphetamines to have long-term, adverse consequences in those who take them for multiple years has long been a matter of concern, and some contention (Diaz Heijtz, Kolb & Forssberg, 2003). Studies have shown that childhood ADHD is positively associated with an increased likelihood of substance use and risk for developing an SUD by the time these children reach adulthood (Molina, Howard, Swanson, Stehli, Mitchell, Kennedy et al, 2018; Elkins, Saunders, Malone, Wilson, McGue & Iacono, 2020). However, a causal link between the use of prescription stimulants and increased prevalence of substance use problems has not been identified. Some researchers argue that use of stimulants to treat ADHD should decrease the propensity to engage in substance misuse, as they are proven to reduce impulsivity in those with ADHD (Cortese, Adamo, Del Giovane, Mohr-Jensen, Hayes, Carucci et al, 2018). Others have suggested that repeated stimulant use during development might sensitize patients to non-prescribed stimulants, increasing their risk of engaging in substance misuse (Ivanov, Bjork, Blair & Newcorn, 2022). Two recent studies support the former view. The first followed 547 children who were prescribed stimulants for ADHD and found they did not have a significant increase in substance use during adolescence or adulthood, nor did they have an increased risk for developing an SUD, when compared to healthy controls (Molina, Kennedy, Howard, Swanson, Arnold & Mitchell, 2023) The second study had responses from 5,034 12^th graders who participated in the Monitoring the Future panel study, and found that adolescents who were prescribed stimulant medication for their ADHD did not differ from healthy controls in their odds of transitioning to later cocaine or methamphetamine use (McCabe, Schulenberg, Wilens, Schepis, McCabe, & Veliz, 2023).

While stimulant use in those with ADHD may not confer an increased risk of later substance use problems, there is evidence that misuse of prescription stimulants is a significant problem for both adults and adolescents. Prevalence rates vary depending on the population being studied, but estimates of lifetime prevalence of prescription stimulant misuse varies between 1.7% and 4.5% in adolescents and between 7.9% and 29% in adults (Novak, Kroutil, Williams & Van Brunt, 2007; Dupont, Coleman, Bucher & Wilford, 2008; Pilkinton & Cannatella, 2012). Nonmedical prescription use of simulants is higher in males than in females, and also varies across factors like SES, race, age, and even sexual identity (Chen, Strain, Alexandre, Alexander, Mojtabai & Martins, 2014; Benson, Flory, Humphreys & Lee, 2015; Dussault & Weyandt, 2013; Philbin, Greene, Martins, LaBossier & Mauro, 2020). Some prescription and even illicit amphetamine misuse, as well as use of other stimulant drugs, may be a form of self-medication in individuals with ADHD, either diagnosed or undiagnosed (for review, see Odell, Reynolds, Fisher, Huckabay, Pedersen, Xandre et al., 2017). How misuse of amphetamines may differentially impact those with ADHD versus those without remains poorly understood since self-medication is difficult to compare to clinician-supervised use of FDA-approved medications.

Non-therapeutic use in humans

Relatively few studies have looked at the effects of amphetamine use in healthy human subjects within a laboratory setting. The work that has been published suggests females experience lower feelings of sedation and higher rates of vigor following a dose of 20 mg methamphetamine compared to males; however, this study was in late adolescents/young adults aged 18-35 years (Mayo, Paul, DeArcangelis, Van Hedger, & de Wit, 2019). Due in part to the ethical concerns surrounding giving these drugs to healthy adolescents, especially young adolescents (12-17 years old), such studies are not conducted and the potential interaction between adolescent use and sex remains unconfirmed in the laboratory setting. Thus, most of what we know about the effects of age and sex on the non-therapeutic use of amphetamines in humans is from epidemiological studies. Over 60% of people who use non-cocaine stimulants report having begun their use prior to age 18, and those that began early are 70% more likely to develop the clinical symptoms of an SUD within 2 years of initial use than those who begin later in life (Chen et al, 2009). Adolescents who engage in methamphetamine use are far more likely than their non-using peers to exhibit risky sexual behavior, polydrug use, and other behavioral problems (Zapata, Hillis, Marchbanks, Curtis. & Lowry, 2008; Embry, Hankins, Biglan & Boles, 2009). They may also exhibit physical and psychiatric health problems, including increased incidence of anxiety, depression, cardiac issues, and even seizures (Sommers, Baskin & Baskin-Sommers, 2006; Degenhardt, Roxburgh & McKetin, 2007; Brecht, Lovinger, Herbeck & Urada, 2013).

It must be acknowledged that substance use at any stage of life does not occur in the absence of a myriad of influential factors. Thus, it can be difficult to separate the outcomes of an individual’s substance misuse from the antecedents that lead them to misuse drugs in the first place. The biological changes occurring in the body and brain during adolescence underlie shifts in behavioral tendencies as well as functional roles, as the individual assumes more adult responsibilities and establishes their identity (Blakemore & Choudhury, 2006). Adolescents in general may be more sensation-seeking, as well as more susceptible to influence by their peers (Forbes & Dahl, 2010). Among the adolescent population, there are a number of predictors of amphetamine use and developing dysfunctions related to that use, including a history of aggression, having parents who use drugs, smoking cigarettes at/before 14 years of age, and experiencing childhood sexual abuse (Hayatbakhsh, Najman, Bor, and Williams, 2009; Boden, Foulds, Cantal, Jones, Dent, Mora et al., 2023). To what extent these predictors, whether they be environmental or genetic in nature, underlie or perhaps interact with the effects of drug use during the adolescent period is challenging to study. These factors cannot be easily teased apart in humans. Further research, especially longitudinal studies which begin prior to any drug use and include genetically related participants (i.e., siblings, identical twins) would be required to further elucidate the role of behavioral and genetic pre-dispositions to stimulant use, and whether they may play a role in the outcomes associated with adolescent drug use.

Gender (in human work) or sex (in laboratory animal work) has also been implicated as a factor in the adverse outcomes associated with substance use, especially with stimulants. Despite males having a higher rate of drug use and diagnosis of SUD (NASDUH, 2020), research in both humans and laboratory animal models suggests that females are specifically vulnerable to certain elements of drug misuse. One frequently identified phenomenon that has been repeatedly described in research on stimulants like cocaine and amphetamines (Griffin, Weiss, Mirin & Lange, 1989; Brecht et al, 2004; Haas & Peters, 2000), as well as other drugs like alcohol (Piazza et al, 1989; Diehl, Croissant, Batra, Mundle, Nakovics & Mann, 2007), is the “telescoping effect” whereby drug users progress rapidly from initial use to problematic drug taking behavior. Telescoping has been reported to occur more frequently in females compared to males (Towers et al, 2023). Pre-clinical work in animal models has also demonstrated this phenomenon, and this is discussed later in this chapter. The role of both adolescent use and sex in vulnerability to developing methamphetamine use disorder has been investigated, albeit infrequently. Adolescent female methamphetamine users report initiating use earlier than males (Hser, Evans & Huang, 2005; He, Xie, Tao, Su, Wu, Zou, et al, 2013) and are more likely to report methamphetamine as their “drug of choice” (Rawson, Gonzales, Obert, Mccann & Brethen, 2005). Because users of amphetamines rarely, if indeed ever, use these drugs in isolation (i.e., without also using alcohol, cannabis, or other drugs), the specific way in which age and sex may interact to confer increased vulnerability onto adolescent female users is difficult to study in humans.

Laboratory animals: non-contingent exposure effects

Due to the challenges of human studies, many scientists have utilized laboratory animal models that can be controlled much more easily. Most studies on the effects of exposure to amphetamines have used laboratory rodents, though other animals including non-human primates have been studied as well (Soto, Wilcox, Zhou, Kumar, Ator, Riddle, et al, 2012). Unfortunately, the persistent bias in laboratory animal research towards the use of male subjects only has impeded our understanding of the role of sex in the consequences of exposure to amphetamines. As late as 2011, males were six times more likely to be studied in preclinical neuroscience research compared to females (Beery & Zucker, 2011). An often-cited reason for this bias is the erroneous belief that female animals have “messier data” due to cycling ovarian hormones (Wald and Wu, 2010), even though meta-analyses have shown that female rats are not more variable than males (Becker, Prendergast, & Liang, 2016). More recent work has begun to include females, and we highlight this growing literature whenever possible.

In rats, much like in humans, there is no clear age at which adolescence begins and ends. A common hallmark of adolescent onset in humans is the beginning of puberty, and in rats puberty is associated with vaginal opening (females), preputial separation (males), and a surge in gonadal hormones that occurs around postnatal day (P) 35 in females and P42 in males (Juraska & Willing, 2017). Most adult-like characteristics, including sexual maturity and parental independence, are observed in rats by P60. For these reasons, as well as the highly influential work in this field by Linda Spear, a generally accepted time frame for adolescence in rats is P27-P60 (Spear, 2000). Note however, this may be different in mice where adolescence is suggested to begin earlier and pubertal onset occurs in some strains at nearly the same age for females and males (Hoops & Flores, 2017).

There have been numerous studies of the effects of non-contingent (i.e., involuntary) exposure to amphetamines, but most have been done in male rodents that were either adolescents or adults at the time of drug exposure. We and others have reviewed this literature previously (Gulley & Juraska, 2013; Hankosky & Gulley, 2016; Westbrook, Carrica, Banks & Gulley, 2020a; Phillips & Aldrich, 2022), so the focus here is on studies that addressed the role of sex and age of exposure by using groups of adolescent and adult subjects from both sexes. To date, there have been a relatively small number of publications that meet this criterion, perhaps in part due to the complexity, cost, and labor-intensive nature of experiments that require so many groups of subjects.

In one of the earliest studies to address the role of both sex and age, Mathews and McCormick (2007) used male and female Long-Evans rats to investigate amphetamine-induced locomotor activation and reward using a conditioned place preference paradigm. They found that across a range of doses (0.5-1.0 mg/kg), female adolescents (P46-P51) were less sensitive to the acute, stimulant effects of the first drug injection compared to female adults (P70-P75); there were no age differences in males. By the last drug injection, female adolescents exhibited greater locomotor sensitization (i.e., reverse tolerance) than female adults, as well as compared to males of both age groups. Interestingly, there were no significant sex or age differences in amphetamine reward as measured by place preference, but females in diestrus had a greater preference for amphetamine than those in proestrus/estrus. These results were interpreted by the authors as evidence for delayed development of the mesocorticolimbic dopamine system, which is critically involved in the behavioral response to stimulant drugs like amphetamine.

Many subsequent studies have elucidated some of the key developmental changes in dopamine circuitry that likely contribute to age-dependent differences in responses to these drugs (for review, see Reynolds & Flores, 2021). Very recent work in mice suggests the intriguing possibility that during early adolescence (P22-P31), males may be more sensitive to the effects of repeated amphetamine exposure on axonal guidance cues (Netrin-1/DCC) that are critical for the normal structural and functional organization of mesocorticolimbic circuitry during adolescent development (Reynolds, Hernandez, MacGowan, Popescu, Nouel, Cuesta et al, 2023). Drug exposure later in adolescence (P35-P44) led to more significant effects on guidance cues in females, but they appear to have more robust compensatory mechanisms: amphetamine-induced deficits in dopaminergic innervation of the prefrontal cortex and deficits in impulse control during adulthood were only observed in males.

Previous work from our laboratory also suggests the timing of drug exposure during the adolescent period likely plays an important role in the differential consequences observed in females compared to males (Hammerslag, Waldman & Gulley, 2014). In rats exposed to 3 mg/kg amphetamine every other day from P27-P45 (adolescent group) or P85-P103 (young adult group), and who were tested in an inhibitory control task at least 60 days after their last drug injection, we found that drug exposure induced impulsivity in adolescent males compared to adult males. This effect was relatively modest, and only observed in one of the two experiments we performed. In females, drug exposure increased impulsivity in the adult-exposed group only. The reason for this somewhat surprising result is not clear, but a contributing factor may have been the shorter time that elapsed between drug exposure and testing in the adolescent- compared to the adult-exposed groups. In studies that have used non-contingent exposure to amphetamine, but that utilized only male rats, there is evidence for drug exposure causing effects that are dependent on age of exposure (for review, Hankosky & Gulley, 2016; Westbrook et al., 2020a). It remains unclear whether there are sex differences in many of the age-dependent neuroadaptations caused by amphetamine exposure that have been demonstrated in the literature.However, we very recently reported that adolescent females exposed to 3.0 mg/kg methamphetamine daily from P40-P48 had fewer parvalbumin-positive interneurons compared to saline-treated controls, whereas females exposed earlier during adolescence (P30-38) or during young adulthood (P60-68) had more than controls (Brinks, Carrica, Tagler, Gulley & Juraska, 2023). This study, which found no effects of methamphetamine exposure in males, suggests the timing of drug exposure in adolescence may interact with sex to influence the impact of methamphetamine on the brain. Recent work has identified the importance of puberty onset for the developmental changes seen during adolescence, and this timing differs substantially between males and females (Juraska and Willing, 2017). It is possible that existing sex differences, or the lack thereof, that have been reported to date may be influenced by testing male and female animals at the same chronological age, when indeed, males and females are at distinctly different points in their neurobiological development. To address this, future studies should take pubertal status, as well as demonstrated stages of cortical development, into account when deciding on timepoints for treatment and analysis in males and females.

Laboratory animals: self-administration

The most clinically relevant work studying the effects of drug use in laboratory rodents utilizes procedures that allow animals to self-administer their drugs, usually via the intravenous route of administration. This type of experiment requires a training procedure for animals to learn to consume drugs by performing a behavior (e.g., lever press), and it allows researchers to investigate drug seeking and taking behaviors, as well as the impact of volitional drug exposure on the brain. Numerous studies have described self-administration of amphetamines and its consequences in adult rats and mice, whereas considerably fewer have done this in adolescent subjects. Also limited in number are studies that investigate self-administration in both sexes of rodents, or with comparison groups of subjects taking the drug during adulthood. Self-administration studies tend to be longer and more technically challenging, not to mention more costly to run, than those using forced (i.e., non-contingent) drug exposure, but their greater face validity and potential to tap into drug taking behaviors in these distinct groups makes them hugely valuable to our understanding of the effects of adolescence and sex on drug use.

Some work suggests that there are age differences in the motivation for amphetamine or methamphetamine. One study in our lab utilized a progressive ratio (PR) schedule of reinforcement to assess the amount of effort adult male and female rats were willing to expend for a variety of methamphetamine doses. Across all doses tested (0.02 – 0.1 mg/kg/inf), male and female rats that initiated drug use in adulthood (> P90) reached higher breakpoints than their adolescent-onset counterparts (>P40), and adolescent males were most likely to miss the acquisition criteria (Hankosky, Westbrook, Haake, Marinelli & Gulley, 2018a). A study by Shahbazi and colleagues similarly found that male rats that acquired self-administration as adults and were tested on a PR schedule had higher breakpoints than their adolescent-onset counterparts when responding for either 0.0125 or 0.05 mg/kg/inf amphetamine. However, this effect was reversed in females (Shahbazi, Moffett, Williams & Frantz, 2008). One important aspect of these studies is that animals were trained to acquire and use amphetamine or methamphetamine in what are commonly referred to as “short access” (ShA) sessions. These allow drug access for ≤ 2 h/day and they engender stable responding and relatively consistent drug intake across self-administration sessions. Long access (LgA) procedures, in contrast, involve much longer session durations of ≥ 4 h/day that typically lead to an increase in intake, termed escalation, across drug taking sessions. This pattern of escalated intake better models amphetamine use behavior in humans (Vanderschuren & Ahmed, 2021), and has revealed unique differences in drug taking between different age and sexes.

One study that investigated age differences in methamphetamine self-administration (Anker, Baron, Zlebnik & Carroll, 2012) found that during ShA sessions, there was no difference in intake between adolescent- (~P27) and adult (~P90)-onset male rats, but when tested under LgA conditions adolescent-onset males took a greater cumulative amount of drug than their adult-onset counterparts. They also escalated their intake over the 15 days of LgA, unlike the adults (Anker et al., 2012). Work in our lab has expanded upon this finding by including female animals, and found that following 7 days of ShA, adolescent- (P40) and adult (P90)-onset rats were allowed to self-administer at a dose of 0.1 mg/kg/inf under LgA conditions for 14 days. Results showed that adolescent-onset rats of both sexes took more methamphetamine than their adult-onset counterparts (Westbrook and Gulley, 2020). We also demonstrated that female rats escalated to a greater extent than their male counterparts (Westbrook, Dwyer, Cortes & Gulley, 2020b), although this effect was not seen in a subsequent experiment (Westbrook & Gulley, 2020). This difference in the degree of escalation was not due to differences in in the number of animals who escalated their intake, as similar proportions of each group (~76%-83%) escalated (> 5% increase) from the first to the final session (Westbrook et al, 2020a).

A potential explanation for these disparate patterns of methamphetamine self-administration is that they arise from differences in sensitivity to the drug between adolescents and adults. Our previous work utilizing a PR schedule is not consistent with the hypothesis that adolescent animals are less sensitive to lower doses of methamphetamine (Hankosky, Westbrook, Haake, Willing, Raetzman, Juraska, et al, 2018b); however, we have found no differences in acquisition (Hankosky et al., 2018a) or cumulative intake (Westbrook et al, 2020a; Westbrook & Gulley, 2020) of several doses (0.05-0.1 mg/kg/inf) of methamphetamine. When data are collapsed across session, adolescent rats take significantly more methamphetamine during hours 2-5 of the 6-h LgA session (Westbrook et al., 2020b), suggesting that they may indeed be less sensitive to the drug and thus require more of it to reach the same level of effect as their adult counterparts. This reduced sensitivity, and subsequent increase in the amount of drug taken, may drive more rapid and robust escalation in the adolescent population by making them more susceptible to developing compulsive drug-taking. Indeed, Anker et al. demonstrated that adolescent males had more non-reinforced lever presses following LgA, which is suggestive of compulsive-like responding for the drug (Anker et al, 2012). Work in adult male rats has shown differential patterns of escalation of intake, depending on the dose animals are allowed to respond for (Kitamura, Wee, Specio, Koob & Pulvirenti, 2006). Future work in adolescent and adult animals is needed to determine if these patterns observed in the previous studies is dependent on dose.

In addition to these behavioral differences in drug use patterns, studies have shown differential neurobiological effects of self-administered amphetamines in adolescents versus adults, as well as differences in the response to pharmacological manipulations of drug self-administration. For example, Zlebnik et al. found that selective antagonists of both the orexin-1 and orexin-2 receptors reduced methamphetamine intake in both male and female rats, regardless of age. However, the orexin-2 antagonist only reduced methamphetamine-primed drug seeking in the adult animals (Zlebnik, Holtz, Lepak, Saykao, Zhang, & Carroll, 2021). In studies from our lab, we found that a history of methamphetamine self-administration was associated with deficits in a cognitive flexibility task for adolescent-onset female rats only (Hankosky et al., 2018b). In adult-onset males from this study, we observed a significant increase in serotonin 5-HT_2C receptors that were co-localized with parvalbumin interneurons in the orbitofrontal cortex. These results suggest that there are age- and sex-dependent impacts of methamphetamine self-administration on both neurobiological adaptations and behavior, as well as in what treatment options may be most successful in altering drug taking behavior. It is also possible that dose may play a role in drug taking and seeking behavior, and thus it is critical that a range of doses be tested in these experiments to pinpoint which of these behaviors are dose-specific. Because of the dearth of literature that includes groups of both ages and both sexes, a great deal of work remains to be done to fully elucidate the biological mechanisms that underly differences in drug-taking behavior.

Conclusion

Age and sex appear to play an important role in the outcomes of amphetamine and methamphetamine use in humans, with females and those who begin using earlier in life experiencing unique and often worse outcomes. Both drugs are used therapeutically at low doses to treat ADHD in children and adults, and recent literature examining the outcomes of these treated individuals has not revealed an increased risk for later development of SUD or other drug-related problems. Due to the ethical concerns of testing even low amounts of these drugs in young humans, animal models are a necessary tool to investigate the potential effects of age and sex upon substance use outcomes in an empirical manner.

There have been major strides in the preclinical field towards better elucidating the effect of exposure to amphetamines during adolescence, but it remains the case that there are relatively few studies that incorporate animals of both sexes, and at adolescent and adult ages, that are allowed to freely self-administer these drugs. These shortcomings make it difficult to draw complete conclusions about what effects may be specific to age or sex. While cumbersome, these types of experiments are critical to teasing apart what may be subtle differences that compound to lead to the more adverse outcomes that have been highlighted in the human literature. It is critical that work examining the effects of drug taking during adolescence utilizes comparison groups of adult subjects, and that researchers consider the potential confounds of differing withdrawal lengths and/or testing ages when deciding upon their study design. Utilizing what existing work that reveals sex-specific timelines of both cortical and subcortical development, and considering how these timelines may equate to human development, may help researchers decide upon more appropriate exposure windows to tap into these issues, rather than using chronological age alone. Lastly, a factor that has been historically overlooked is the impact of stress during sensitive developmental time points. Stress is a major risk factor in substance misuse, not only for initiation of use but for escalation and relapse (Aguilar, Garcia-Pardo, Montagud-Romero, Minarro , & Do Couto, 2013). Just as the neurobiological changes taking place during adolescence may render them more vulnerable to exogenous insults like drugs of abuse, so too may stress impact adolescents differently than adults. There may also be differential impacts of stress between males and females, with some studies showing females have higher sensitivity compared to males to certain types of stress (Pankevich and Bale, 2008). Indeed, a review of how stress affects reinstatement of drug seeking in rodents emphasizes the need for including both sexes and those of different ages, as certain stressors have greater effects on different populations (Mantsch, Baker, Funk, Lê, & Shaham, 2016). One common issue in laboratory studies is that animals are often single-housed during treatment and/or testing, a practice that has been shown to be highly stressful for adolescent animals (for review, see Vanderschuren, Achterberg, & Trezza, 2016). Notably, a recent review reported that social isolation during adolescence increased locomotor activity in rodents following amphetamine use (Noschang, Lampert, Krolow, & de Almeida, 2021). Additional stress may be introduced by shipping animals at sensitive time periods (e.g., near weaning) versus breeding them in-house, differences in restraint techniques and overall handling of the animals, and whether animals are tested during the light or dark cycle. All of these variables can play a major role in the results of drug use studies, and must be carefully considered by researchers trying to tap into the specific effects of age and sex.

Abbreviations:

ADHD

Attention deficit hyperactivity disorder

LgA

Long access

P

Post-natal day

PR

Progressive Ratio

ShA

Short access

SUD

Substance use disorder