A hallmark of cocaine use disorder (CUD) is dysfunction of dopamine signaling in the mesolimbic pathway, including impaired dopamine 2 (D2) receptor signaling. This was first reported in humans by Volkow and others (1990) using positron emission tomography (PET) and showed that individuals with CUD had lower striatal D2 receptor availability compared to healthy controls. Since the original report with cocaine (Volkow et al., 1990), this neuroadaptation has been observed in many subsequent studies for cocaine and other drugs such as methamphetamine, nicotine, opioids, and alcohol (Volkow et al., 1993, 2001; Martinez et al., 2004, 2005, 2012; Fehr et al., 2008). Low levels of striatal D2 receptor availability have also been shown to be associated with greater drug craving and relapse vulnerability in individuals with substances use disorders (Heinz et al., 2004; Wang et al., 2011). Moreover, high levels of striatal D2 receptor availability has been observed in unaffected members of families with a history of alcohol use disorder, suggesting that higher striatal D2 receptor availability is protective against the development of substance use disorder (Volkow et al., 2006).

In parallel, preclinical studies have shown that subordinate male cynomolgus monkeys have lower D2 receptor availability in the basal ganglia, which includes the ventral striatum (nucleus accumbens; NAc), and are more sensitive to the reinforcing effects of cocaine as compared to dominant monkeys (Morgan et al., 2002). Studies in male rats further indicate that changes in D2 receptors occur as the result of chronic self-administration. For example, Conrad and colleagues (2010) showed that surface expression of D2 receptors in the NAc decreased following prolonged cocaine self-administration (4h/day for 3 weeks). Similarly, Mateo et al., 2005 showed that NAc dopamine terminals and presynaptic D2 receptors (autoreceptors) are less sensitive to cocaine following extended-access (ExA; 24-h/d for 10 days) cocaine self-administration versus acute cocaine administration (1.5 mg/kg, i. v.). Our recent findings in male rats further indicate that there is a functional shift in D2 receptor signaling in the reward pathway following the development of an addiction-like phenotype. We have used an enhanced motivation for cocaine, as assessed under a progressive-ratio (PR) schedule, to define the development of an addiction-like phenotype since this feature develops following withdrawal from ExA (24-h/day for 10 days), but not ShA (20 infusions/day for 5 days) cocaine self-administration, and like in humans, once this feature emerges, it represents a relatively permanent shift to a higher motivational state (Ramôa et al., 2013, 2014; Doyle et al., 2014; Bakhti-Suroosh et al., 2019). The development of an enhanced motivation for cocaine also corresponds to the development of other addiction-like features, including compulsive cocaine use despite negative consequences (Towers et al., 2021) and enhanced cocaine-seeking (e.g., Peterson et al., 2014; Towers et al., 2023). Furthermore, we showed that in males the development of an enhanced motivation for cocaine is accompanied by a decreased sensitivity to NAc D2 receptor antagonism (relative to ShA controls), indicating that the role of D2 receptor signaling in motivating cocaine use becomes diminished with the development of addiction (Lynch et al., 2021).

One major caveat is that the evidence for changes in D2 receptor signaling with the development of addiction has been derived almost entirely from men and male animals. This is important particularly considering an accumulating body of evidence indicating that the mechanisms that underlie addiction are different in males and females. For example, while both male and female subordinate cynomolgus monkeys are less vulnerable to the reinforcing effects of cocaine as compared to their dominant counterparts, in females, this protection corresponds to lower D2 receptor availability (Nader et al., 2012), suggesting that the relationship between D2 receptor availability and vulnerability to cocaine is opposite in males versus females. Even more notable for this study are data from human smokers showing that males have reduced striatal D2 receptor availability compared to healthy controls, but within females, striatal D2 receptor availability is comparable between smokers and health controls (Brown et al., 2012). Similar sex differences have also been reported for cortical D2 receptor availability (Zakiniaeiz et al., 2019). These findings are intriguing, particularly considering that this neuroadaptation is thought to reflect poorer treatment outcomes and greater addiction severity (Volkow et al., 1999), yet it is not observed in females who show worse treatment outcome and greater addiction severity than males (for review see Towers et al., 2023). Therefore, sex differences in the molecular shifts that occur with D2 receptor signaling with the development of addiction needs to be further investigated.

Thus, the purpose of this study was to determine whether there are sex differences in the role of NAc D2 receptors in motivating cocaine use prior to and following the development of an addiction-like phenotype (defined by an enhanced motivation for cocaine relative to the ShA group). As in our previous study in males, shifts in D2 receptor signaling were determined by comparing effects of intra-NAc D2 receptor antagonism on PR responding for cocaine following withdrawal from either ShA cocaine self-administration (which does not induce an enhanced motivation for cocaine from baseline; Roberts et al., 2007) or ExA cocaine self-administration (which induces an enhanced motivation for cocaine from baseline; Towers et al., 2021). Based on the clinical finding in smokers, we predicted that the role of NAc-D2 receptors would become diminished with the development of an addiction-like phenotype in males, but not females.

We previously showed that an addiction-like phenotype, defined as the development of enhanced motivation for cocaine, develops over withdrawal following ExA cocaine self-administration and, in males, corresponds with a diminished role of NAc-D2 receptor signaling (Lynch et al., 2021). In the present study, we expanded the investigation of D2 receptor mechanisms motivating cocaine use to include females. Based on findings in human smokers, we predicted that the role of NAc-D2 receptor signaling would vary between males and females and in females, would not become diminished with the development of an addiction-like phenotype. Our findings support this hypothesis as we found that eticlopride was less effective at decreasing motivation for cocaine in ExA males versus ShA males, particularly at low doses; whereas, in females, there were no differences in the effectiveness of the eticlopride between ExA and ShA rats. These findings indicate that males, but not females, become less sensitive to NAc-D2 receptor antagonism with the development of an addiction-like phenotype.

Decreased striatal D2 receptor binding in addiction is one of the most replicated findings in human imaging research (see Trifilieff et al., 2017 for review); however, this molecular shift may not translate to females (Brown et al., 2012; Zakiniaeiz et al., 2019). Our findings in the present study build on this possibility by showing that while low doses of eticlopride were less effective at decreasing motivation for cocaine in ExA versus ShA males, there was no difference in its efficacy between ExA and ShA females (Lynch et al., 2021). Based on the assumption that low doses are more selective and induce fewer off target effects than high doses, our findings indicate that males, but not females, develop a decrease in sensitively to D2 receptor antagonism with the development of an addiction-like phenotype. One alternative possibility, however, is that this shift does occur in females, but perhaps sooner/more readily, such that it was already present in females tested following ShA self-administration. Females are known to transition from initial drug use to meeting the criteria for CUD/seeking treatment faster than males (for review see Towers et al., 2023). Additionally, in our present study, ShA females were more motivated to obtain cocaine than ShA males. Thus, it is possible that at least some of the females in the ShA group had already developed an addition-like phenotype and a shift toward a diminished role of D2 receptors and this explains why we did not see a difference between ShA and ExA females. It is notable that no differences were observed for the effects of eticlopride between ExA males and females. Further research using a within subject design to determine the development of an enhanced motivation for cocaine (relative to baseline prior to ShA versus ExA self-administration) is necessary to resolve this question.

It is also important to note that findings in this study along with ours (Doyle et al., 2014; Ramoa et al., 2014) and others previous studies (e.g., Castner et al., 2000; Henry et al., 2009) provide support for similarities in the mechanisms underlying addiction in males and females. More specifically, here we showed NAc-D2 receptor blockade with eticlopride dose-dependably decreased motivation for cocaine in both ShA and ExA males and females. This indicates that D2 receptors remain critical for motivating cocaine use prior to and following the development of an addiction-like phenotype in both sexes. This is in contrast to our previous findings for NAc dopamine 1 (D1) receptor signaling, which showed NAc-D1 receptor blockade with SCH-23390 dose-dependably decreased motivation for cocaine in ShA, but not ExA, males and females. These findings indicate that D1 receptors are critical for motivating cocaine use prior to, but not following, the development of an addiction-like phenotype in both sexes (Doyle et al., 2014; Ramoa et al., 2014). Preclinical studies in non-human primates have also shown stimulant-induced dopamine release from presynaptic terminals is decreased in males and females with repeated drug use (Castner et al., 2000; Henry et al., 2009). Thus, blunted dopamine transmission in the striatum likely renders males and females vulnerable to continued drug use, and there are likely similarities (D1R signaling) and differences (D2 receptor signaling) in the mechanisms underlying these striatal dopamine deficits associated with addiction in males and females.

There is an urgent need to disentangle the differences and similarities in the mechanisms underlying CUD in males and females. While males having higher rates of cocaine use than females, the number of females using cocaine and other addictive drugs in their lifetime is on the rise, and females are more vulnerable to many aspects of CUD compared to males. For example, females develop CUD and/or seeking treatment for the disorder after few years of cocaine use, report longer periods of cocaine use after relapse, have greater stress-induced cravings, and show an enhanced vulnerability to cocaine-related medical consequences compared to males (Griffin et al., 1989; White et al., 1996; McCance-Katz et al., 1999; Haas and Peters, 2000; Gallop et al., 2007; Waldrop et al., 2010; Moran-Santa Maria et al., 2018). Preclinical studies have similarly shown that females have an enhanced sensitivity to reinforcing effects of cocaine, and display a greater vulnerability during the transition from initial cocaine use to the development of an addiction-like phenotype, including self-administering more cocaine under ExA conditions and developing an enhanced motivation for cocaine/preference for cocaine over other reinforcers after less drug exposure and/or shorter periods of withdrawal than males (see Towers et al., 2023 for review). Although the present study was not designed to assess behavioral sex differences, similar to the findings mentioned above, we observed higher levels of cocaine intake under ExA conditions and motivation for cocaine use following ShA cocaine self-administration in females than males. Thus, females and males also differ in several key features of addiction, and understanding sex differences in the mechanisms underlying these features will hopefully lead to the development of sex-specific treatments and the first FDA approved treatment for CUD.

In summary, despite the severity of addiction in females and an accumulating body of evidence indicating that females and males differ in several features of addiction, clinical and preclinical studies have historically used male subjects, resulting in a male-centric neurobiological basis of addiction. The current study builds on the literature showing that findings in males do not necessarily translate to females. Future studies are needed to and to determine whether striatal D2 receptor signaling becomes diminished with the development of other addiction-like features in females, such as compulsive use despite negative consequences and preferential use of cocaine over other natural rewards, as well as with the development of other substance use disorders such as opioid use disorder. Future studies are also needed to determine the impact of ovarian hormones on D2 signaling in females especially considering that ovarian hormones, and particularly estradiol, is known to impact cocaine’s reinforcing effects and the development of addiction-like features in females (see Towers et al., 2023 for review).