Our RNA-seq study provides, for the first time, an exploration of the sex-dependent differential effects of ACE on the striatal transcriptome. We will focus our discussion on reward processes, response to drugs, and substance use disorders, which is the aim of the present work. However, given the importance of some of the transcriptional signatures obtained for schizophrenia, an important comorbid condition of substance used disorders (Khokhar et al., 2018), we also provide some discussion of the relevance of our findings to this disorder in the supplementary Discussion.

In male rats, the upregulated gene with the highest fold change was Satb2 (SATB homeobox 2), involved in transcription regulation and chromatin remodeling. CB₁ receptors are coupled to the regulation of the Ctip2–Satb2 transcriptional regulatory code (Diaz-Alonso et al., 2012), and in so doing, they guide corticospinal motor neuron differentiation. The alteration of the Satb2 gene in the NAcc of our animals could also have developmental consequences in the morphology or function of accumbal neurons as suggested by the ontologies affected by THC treatment. Moreover, Satb2 in the paraventricular thalamus is also sensitive to cocaine-rewarding actions (Salti et al., 2018), so it could be speculated that this upregulated gene in accumbal cells may affect the rewarding actions of cocaine under specific circumstances (such as progressive ratio schedules; see below). Another gene with potential implications for our behavioral results was Notch3 (notch receptor 3), which was downregulated. This gene belongs to the notch signaling pathway that is also involved in brain development (Androutsellis-Theotokis et al., 2006). Interestingly, Notch3 is downregulated in striatal territories in spontaneously hypertensive rats treated with methylphenidate during adolescence (and that further self-administered methylphenidate as adults) (dela Peña et al., 2014), suggesting that this gene is responsive to several pharmacological challenges during adolescence (not just cannabinoids), with dopamine acting as a potential common link (Gottlieb, 2001; Bossong et al., 2009; Wahlstrom et al., 2010), and also that its downregulation may predispose to psychostimulant consumption.

In addition, we found transcriptional and translational alterations in adult animals exposed to adolescent cannabinoids that may influence, in a sex-dependent manner, elements of the dopaminergic signaling pathway and shape drug-related behaviors. In this regard, the transcriptional factor Zinc Finger Homeobox 3, Zfhx3 (downregulated in THC males, upregulated in THC females and associated with neurogenesis and ion-binding Gene Ontologies extracted from the list of differentially expressed genes in the males), is distinctively present in a subtype of D₂-expressing neurons of the adult midbrain (Poulin et al., 2014). Thus, this difference may suggest a potential modulation of this specific subtype of D2-expressing neurons in the NAcc shell of rats exposed to THC. Additionally, THC males showed an upregulation of the nuclear receptor gene Nr4a2 involved in behavior and neuron differentiation gene ontologies within the male differentially expressed genes. Nr4a2 can be modulated by neuronal firing and dopamine signaling; moreover, the loss of D₂ signaling also contributes to Nr4a2 upregulation (Tseng et al., 2000).

ACE also altered several elements belonging to glutamate, GABA signaling, and other ion channels relevant for the expression of motivated behaviors and drug use. Among the glutamatergic alterations, we find it relevant to highlight the upregulation in both males and females of the solute carrier Slc17a6, which encodes the vesicular glutamate transporter 2(VGlu2) protein involved in glutamate uptake into synaptic vesicles at presynaptic nerve terminals. Noteworthy, dopamine neuronal subtypes express this protein, and the presence of VGlu2 seems to be required for psychostimulant-induced behavioral activation (Birgner et al., 2010).

In addition, there are several relevant changes in the male NAcc shell ion channel expression profile. In this regard, THC produced a protracted downregulation in the male NAcc shell of the solute carrier Slc1a2, a glial transporter that clears glutamate from the synaptic cleft. The expression of this gene is altered by many drugs of abuse (cocaine, amphetamines, nicotine, opioids, ethanol, and cannabinoids), and it has received attention as a potential target for pharmacological interventions in substance use disorders (Roberts-Wolfe and Kalivas, 2015); the voltage-gated potassium channel subunit beta-2 Kcnab2, which is similarly depleted after chronic morphine exposure (Mazei-Robison et al., 2011) and has been involved in motivated behaviors (O’Donovan et al., 2019); the ATP-sensitive inward rectifier potassium channel 10 Kcnj10, also involved in substance use disorders and ethanol preference (Zou et al., 2009); the sodium channel protein type 8 subunit alpha, Scn8a, which plays an important role in regulating excitability in the brain; and the potassium-chloride transporter member 5, Slc12a5, associated with the formation and maturation of glutamatergic and GABAergic synaptic connections (Medina et al., 2014).

In the females, ACE was associated with upregulation of the GABA A Receptor Epsilon Subunit, Gabre. In the context of substance use disorders, rats with a genetic predisposition to alcohol consumption showed a Gabre upregulation (Spence et al., 2018). The hormone activity gene ontology was enriched in the female subset of DEGs, and, noteworthy, the neuropeptides included in this subset may be determining the dopaminergic activity in the NAcc shell of THC-treated females. Thyrotropin-releasing hormone Trh, upregulated by THC in females, participates in energy metabolism and affects different hormonal functions but also enhances dopamine release in the NAcc (Puga et al., 2016). We also detected an upregulation of Agt, which encodes angiotensinogen, the precursor protein of angiotensin I, which is further converted to the peptide angiotensin II. Reductions of angiotensin II, and consequent less activation of the angiotensin II type 1 and type 2 receptors are associated with lower levels of dopamine in the ventral tegmental area and linked to lower alcohol consumption (Maul et al., 2005). Similarly, the cocaine- and amphetamine-regulated transcript seems to exert a neuromodulatory role in the NAcc attenuating dopamine release (Rakovska et al., 2017), and cocaine- and amphetamine-regulated transcript injections into NAcc inhibit the behavioral effects of cocaine (Yu et al., 2017).

We have also detected an upregulation of the Gal gene, encoding the neuropeptide galanin. Noteworthy, galanin has been involved in pathological food consumption and addiction (Gosnell et al., 1986a, 1986b; Sandi et al., 1988). In this regard, an overabundance of galanin has been shown to decrease the sensitivity to amphetamine-induced behavioral effects (Clarke et al., 1988). It is also important to mention that agonists of the galanin receptor can reduce reinstatement of cocaine-seeking (Ogbonmwan et al., 2015) and cocaine-conditioned place preference (Narasimhaiah et al., 2009), which is important considering the sex-specific effects in cocaine SA after ACE (Higuera-Matas et al., 2008). Finally, THC produced a sex-dependent change in the expression of the Cck gene (upregulated in males and downregulated in females treated with THC), which encodes the peptide hormone cholecystokinin. Cholecystokinin (Cck) signaling pathways have been related to food intake, but also with reward and anxiety and even panic (Bradwejn and Vasar, 1995; Rotzinger and Vaccarino, 2003), and have also been studied in the context of drug-related behaviors (Lu et al., 2001, 2002; Wunderlich et al., 2004). Moreover, cocaine behavioral sensitization is accompanied by increasing levels of Cck in the NAcc shell (Beinfeld et al., 2002).