Addictive drugs, such as cocaine and heroin, have well-documented actions on behavior. The mechanisms that these drugs employ have been under investigation for decades although the investigation of how gonadal and pituitary hormones impact these mechanisms is a relatively new focus. Here, we have assembled a group of primary-literature papers solicited from many of the leading addiction laboratories in the world. The papers in this compellation highlight different drug classes, hormones, levels of analysis, and animal models. Our hope is that this “wide angle” approach provides us some overarching conclusions on common mechanisms. In this editorial, we present an overview of each paper and then speculate about general principles.

The first paper in this issue is a landmark study that validates a new translational model to assess self-administration (SA) of fentanyl, a drug that is currently a leading cause of death in young adults [1]. In this article, Towers et al. [2] demonstrated that, as with other addictive drugs such as cocaine, estradiol enhances vulnerability to the development of addictive-like features with fentanyl. They examined effects in ovariectomized females with and without estradiol and across multiple phases of SA behavior, including initial drug use during acquisition, extended-access SA, and the subsequent development of addiction-like features following withdrawal (i.e., physical dependence, an enhanced motivation for the drug, and enhanced drug-seeking/vulnerability to relapse). They found that estradiol moderately to markedly impact each of these behaviors. Specifically, females with estradiol were more likely to acquire SA within the first 5 days of access, and following acquisition, self-administered markedly more fentanyl than females without estradiol under extended-access conditions. With or without estradiol, rats lost weight during the withdrawal period, responded at high levels during cue-induced relapse testing, and developed an enhanced motivation for the drug (relative to baseline prior to extended-access SA); however, these features were more robustly displayed and/or persisted longer (weight loss) in rats with estradiol versus without estradiol. Another striking difference was that females with estradiol were more likely than females without estradiol to develop severe health complications over withdrawal as compared to females without estradiol. These important results extend the well-documented vulnerability enhancing effects of estradiol observed with other addictive drugs to fentanyl. These data pave the way for more in-depth analysis of mechanisms underlying estradiol’s impact on vulnerability and fentanyl-associated toxicity.

The neuropeptide oxytocin is associated with affiliative behavior [3]. The hypothesis that drug use is associated with lack of social bonds leads to the prediction that oxytocin may reduce drug taking/seeking [4]. This hypothesis is tested in the second paper by Carter et al. [5] using heroin as the addictive drug. Oxytocin is produced by neurons in the periventricular (PVN) region of the hypothalamus. These neurons project to sites throughout the brain and to the posterior pituitary where their terminals release oxytocin into the peripheral vasculature. Steroid hormone receptors, particularly estrogen receptors, regulate the transcription of both oxytocin peptide and its receptor [6‒8]. Carter et al. [5] tested the actions of oxytocin, given peripherally, after forced abstinence for one or 30 days. At both time points, oxytocin reduced heroin seeking, as predicted. The team also enhanced or reduced oxytocin neuronal activity in PVN neurons more directly with oxytocin specific DREADDS. Using this central method, after 1 day of abstinence, neither inhibition nor excitation of oxytocin neurons in PVN changed behavior. However, after 30 days, inhibition of oxytocin activity decreased heroin seeking. This same treatment also reduced interest in social interaction. In addition, they employed immunohistochemistry for c-Fos (an early immediate transcription factor) and oxytocin. There was an increase in co-localization of these proteins when peripheral oxytocin was given. And they found increased c-Fos in the central amygdala but not in other oxytocin-projection sites. The team primarily used male rats but indicate that their findings from a subset of gonad-intact females, included in the DREADD study, were similar to those obtained in males. In summary, these tantalizing data require follow-up but could suggest either indirect actions of peripheral oxytocin injections and/or that administration systemically depresses oxytocin neuronal activity.

Corbett et al. [9] describe cocaine’s actions on synaptic transmission in males versus females as well as in females over the estrous cycle. Most drug studies using electrophysiology focus on the nucleus accumbens [10], an important hub for the reward system and terminal site of dopamine neurons. In this paper, the pyramidal neurons in the basal lateral amygdala (BLA) were probed. Rats were trained to SA cocaine under extended-access conditions (−6; h/day) over a 10-day period along with controls that received all the same procedures, but without access to cocaine. Female estrous cycles were monitored throughout the study. Electrophysiology was conducted two to 4 weeks after SA, conditions known to enhance BLA pyramidal neuron excitability in males. No sex differences were detected in the SA data. Nor were sex differences present for excitatory synaptic transmission or intrinsic excitability in the BLA. However, both measures of neuronal activity were enhanced by prior cocaine experience. When female data were examined with regard to cycle stage at the time of data collection, females with a history of cocaine SA had higher frequencies of excitatory transmission on estrous or proestrus days (higher estrogens) as compared to females in diestrus (lower estrogens) or any of the controls. When intrinsic action potentials were assessed, the same result was noted for action potential frequencies in cocaine-exposed estrous-stage females versus all other females. This study demonstrates the importance of the estrous cycle, a factor that must be considered in studies using ovary-intact females.

The same experimental approach used by Corbett et al. [11] electrophysiology quantified using whole-cell patch clamp, was employed by Miller et al. [10]. In the nucleus accumbens core, the physiology of the medial spiny neuron (MSN) population is sexually dimorphic [11], and in rats this sex difference is produced by estradiol [13]. Here, the authors asked which estrogen receptors (ERs) are responsible for its actions. They used young adult female rats. Slices including the nucleus accumbens core were prepared and challenged with a specific estrogen agonist; the agonists were either specific for ERα (agonist PPT), ERβ (agonist DPN), or the G-protein ER. The results are straightforward, showing that only the ERα agonist was able to rapidly decrease miniature excitatory postsynaptic current frequency in female rat nucleus accumbens core MSNs. This result implies that the sexually dimorphic behavioral responses to addictive drugs are regulated by this important estrogen receptor. The next step would be to do the study using cocaine-exposed and unexposed females. One of the caveats of the work is that estrous cycle was not controlled. Although the authors have acknowledged this, it is possible that during non-estrous phases other estrogen receptors may have increased expression and activity.

Martz et al. [14] examined the relationship between the medial preoptic (mPOA) efferents to the ventral tegmental area (VTA). Both regions are essential parts of the reward pathway in both sexes. Male and female rats were injected with a tract-tracer in the VTA, and immunochemistry was used to locate the cell bodies that projected from the mPOA to the VTA. In addition, the phenotypes of these neurons were revealed using antibodies to gamma-aminobutyric acid (GABA), ERα, and androgen receptor (AR). In the subpopulations of cells projecting to the VTA, a sex difference was present. In females, co-localization with ERα was greater than in males, and in males, the reverse was true for the efferents containing AR. In both sexes, these differences were most striking in the central part of the mPOA. No sex differences were noted for GABA-containing neurons per se, but the percent of cells containing both AR and GABA was significantly larger in the male central POA than in the female. In males, virtually all GABA neurons also expressed AR. This contribution moves us forward in our thinking about how and where sex differences in reward are translated into behavior.

The final two papers in the issue switch models from rats to mice. One traditional advantage of mice over other laboratory models is that genetic variation can be utilized. In Chapp et al. [15], the authors have leveraged mouse genetics and compared cocaine-induced spontaneous activity and sensitization. Both sexes were used, although estrous cycles were not monitored in the females. The group used three lines of mice from Jackson Labs, two of the most commonly employed inbred mouse strains (C57BL/6J and B6129SF2/J), and Diversity Outbred (DO) mice. While both C57BL/6J and B6129SF2/J have been used previously in cocaine-induced activity studies, most studies have included only males or have not found and/or explored sex differences [16]. The B6129sF2/J is a hybrid second generation cross between C57BL/6J females (B6) and 129S1/SvImJ males (129S). The virtue of F2 hybrids is that they can be used to map genetically based traits. The DO mice are an extremely genetically diverse laboratory strain which amplifies individual differences in behavior. During sensitization, sex differences were apparent in C57BL/6J mice, i.e., males displayed more activity than females did. The reverse pattern was noted in the B6129sF2/J mice. This information shows that there is a genetic component to the sex difference that could be evaluated and lead to novel genes that have sexually dimorphic roles in behavioral responses to cocaine. It is also highly likely that steroid hormones interact with some of the genes responsible for these sex effects. Finally, as expected, the DO mice display large variation in individual behaviors. This likely masks any potential sex differences but presents an opportunity for genomic analyses of behavioral differences.

Finally, Le et al. [18] use a genetic model that has been employed to study sex differences for over 20 years [19], the four core genotype (FCG) mouse. This model is used to test the hypothesis that genes on X- and/or Y-chromosomes are involved in sexually dimorphic responses to cocaine. The four genotypes include: males with XY or XX sex chromosomes, and females with either XX or XY chromosomes. Because of this the effects of sex chromosome complement can be assessed independently of gonadal sex (testes vs. ovaries). A previous study from this group [20] tested this hypothesis in gonad-intact male and female FCG mice and showed that, as with findings in gonad-intact rats, XX females acquire cocaine SA sooner and at a higher rate than XY males. Interesting XY females were equivalent to XX females but XX males were intermediate between XY males and females. In the current study, all mice were gonadectomized to block potential effects of gonadal hormones and any interactions they may have with sex chromosome genes. In addition, estradiol (E2) replacement was given to half the mice in each genotype. The results show effects of sex chromosome complement on vulnerability to cocaine SA, but surprisingly, in this case, chromosomal XY males acquired more readily than all the other groups. It is also notable that E2 implants blocked the enhanced vulnerability observed in gonadectomized XY males. Unfortunately, in the current study a modified SA schedule was used that eliminated the highest dose of cocaine (1.0 mg/kg). Thus, the previous data (from gonad-intact FGC mice) were not directly comparable. The conclusion is that in mice tested under identical gonadal conditions, sex chromosome complement does modify vulnerability to cocaine. There are several candidate genes, on the X-chromosome, that escape X-inactivation and their message and protein are present in higher amounts in females than in males. Le et al. [18] are currently working to determine which of these genes are critical for sex differences in addictive behaviors.

There are many take-home messages from these articles. First of all several papers provide either direct [2] or indirect [5] evidence for a mediating role of estradiol in sex differences related to both opioids (heroin and fentanyl) and cocaine. Specifically, estradiol directly enhances addictive behavior and electrophysiological phenotypes in the female MSN in the nucleus accumbens. Indirect actions implicate estradiol signaling between the VTA and mPOA and in neuronal activity in the BLA in females. The second implication of these data is that since both the mPOA and BLA are well studied in the context of other behaviors they should receive more attention in addiction studies. Third, the two studies that target specific estrogen receptors [10] show that the ERα is critical. Taken together the studies strongly suggest a role for estradiol throughout the reward circuitry. The interface between circulating estradiol and addiction starts with an ER. Discovery of novel genes regulated by ERα deserves more investigation.

Another important topic is sex differences in reward pathways, which are revealed in Martz et al. [14]. The medial part of the POA is one of the best defined sexually dimorphic areas of the brain. The projections to the VTA, many of which are GABAergic, may be regulated by different gonadal hormones in females and males. This contribution confirms the importance of ERα in females but as importantly suggests that work in males should focus on AR, particularly since the majority of GABA cells in male mPOA also express AR. In general, the role of gonadal hormones in male addictive behavior has received relatively little attention and should prove to be extremely important since ultimately more men than women become addicted to drugs.

The last two papers highlight the potential of mice for genetic approaches to addiction. Chapp and colleagues [15] report that in one commonly used inbred mouse strain activity in response to cocaine is greater in males and in another strain, the sex differences are reversed. The final paper in the issue asks about sex differences from the perspective of sex chromosome complement. When gonadal hormones are eliminated the mice in the FCG closest to normal XY males are far more vulnerable to cocaine SA than either XX females or the discordant groups (XXM and XYF). These findings taken together hint that estradiol may interact with genetic factors to influence vulnerability to addiction.

The authors declare no conflicts of interest.

This work was funded by NIDA grant R01 DA048638 (WJL and EFR).

This work was equally written by both Emilie F. Rissman and Wendy J. Lynch.

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