Prolonged cocaine consumption induces significant alterations of the number of Orx neurons in the HYP and it has been shown that chronic cocaine use increases the number of Orx-expressing cells (James et al., 2019; Matzeu and Martin-Fardon, 2021). These studies used two different models to induce addiction-like behavior: extended access to cocaine self-administration for 6 h/day for 21 days (Matzeu and Martin-Fardon, 2021) and intermittent access to cocaine self-administration for 5 min, followed by 25 min of cocaine non-availability (repeated 12 times/day for 14 days; James et al., 2019). The addiction-like behavioral phenotype that is induced by these animal models was accompanied by persistent increases in the number of Orx-immunoreactive hypothalamic neurons starting at 14–21 days and up to 150 days following the last cocaine exposure (James et al., 2019; Matzeu and Martin-Fardon, 2021). Importantly, this increase in expression was observed exclusively for Orx and not for other neuropeptides, such as melanin-concentrating hormone, in the same hypothalamic region (James et al., 2019). A study investigated the participation of Orx transmission in the posterior paraventricular nucleus of the thalamus (pPVT) in cocaine-related behaviors, showing that exposure to cocaine under extended access conditions increased OrxR2 immunoreactivity at 14–21 days of abstinence, which returned to basal levels after 35 days (Matzeu and Martin-Fardon, 2021). This observation was particularly important because it confirmed the participation of OrxR2 signaling in the pPVT in mediating the priming effect of intra-pPVT OrxA-induced cocaine-seeking behavior (Matzeu et al., 2016; Matzeu and Martin-Fardon, 2021). The persistent upregulation of Orx peptide during abstinence from cocaine, together with alterations of OrxR2 in the pPVT, suggests that cocaine compromises the Orx system, and this effect persists into abstinence and most likely affects brain regions beyond the pPVT that receive HYP Orx projections.

Similar to cocaine, alcohol consumption has been shown to dysregulate the Orx system, but chronic alcohol intake data have been somewhat controversial. For example, chronic home cage alcohol drinking was shown to reduce Orx mRNA expression in the HYP, whereas acute oral alcohol administration by gavage was associated with an increase in Orx expression (Morganstern et al., 2010). Moreover, alcohol dependence downregulated Orx mRNA expression at 12 h of withdrawal in rats that were made dependent via repeated intragastric (i.e., gavage) alcohol administration (Sharma et al., 2020). Consistent with these data, a clinical study reported that blood OrxA levels in patients with alcohol use disorder were inversely related to the severity of withdrawal symptoms (i.e., stronger withdrawal symptoms were associated with lower OrxA expression; Bayerlein et al., 2011). This observation might partially account for the exacerbation of lethargy during the day that is experienced by subjects who suffer from alcohol use disorder during alcohol withdrawal (for review, see Miller et al., 2021).

Opposing results were recently found in our study that investigated the participation of the pPVT in alcohol seeking. We found a significant increase in Orx mRNA expression in the HYP and OrxR1 and OrxR2 mRNA expression in the pPVT following extinction at 8 h of withdrawal in rats that were made alcohol dependent by chronic intermittent alcohol vapor exposure (Matzeu and Martin-Fardon, 2020a). Consistent with our findings, the Orx system was reported to be upregulated in adult rats following binge-like patterns of alcohol intoxication during adolescence (Amodeo et al., 2020) and in rats that exhibited high novelty-induced locomotor activity, a predictor of high alcohol consumption (Barson et al., 2013). Interestingly, pre-fertilization maternal alcohol consumption in zebrafish significantly increased the number of Orx neurons and alcohol consumption in offspring (Collier et al., 2020). The dependence induction procedure, time point of withdrawal, age of the animals, species, and other experimental manipulations might account for discrepancies in these preclinical studies.

The dysregulation of Orx neuropeptides has also been demonstrated following opioid abuse. An increase in the number of Orx-producing neurons was found in postmortem brains from individuals with heroin addiction (Thannickal et al., 2018). Similar increases in Orx-producing cells were induced in wildtype mice by long-term morphine administration that lasted up to 8 weeks after morphine cessation (Thannickal et al., 2018). Moreover, intermittent access to fentanyl was associated with an increase in the number Orx cells in the HYP (Fragale et al., 2021), suggesting that a common effect of chronic opioid use is the upregulation of Orx-producing cells.

The downregulation of Orx gene expression in blood was reported in nicotine-dependent cigarette smokers and subjects with Δ⁹-tetrahydrocannabinol dependence (Rotter et al., 2012), suggesting that Orx system dysregulation is a common factor across several classes of drugs of abuse.

Orexin neuropeptides facilitate drug-directed behavior, especially under motivationally salient, high-effort conditions (Borgland et al., 2009; Mahler et al., 2014). Particularly significant is a study that used a short-hairpin RNA-encoding adeno-associated viral vector to knock down Orx expression throughout the HYP in adult rats and found lower motivation for cocaine intake under both progressive-ratio (PR) and fixed-ratio (FR) schedules in rats that were given extended access to cocaine self-administration. Interestingly, knocking down Orx also reduced the intake of a palatable food reward (i.e., sweetened condensed milk). Importantly, Orx silencing did not affect food or water consumption and had no effect on general measures of arousal or stress reactivity (Schmeichel et al., 2018). These findings support the hypothesis that Orx neuropeptides promote operant responding for both drug and non-drug rewards, specifically under conditions that require a high degree of motivation (e.g., PR schedules, extended access to cocaine, and a highly palatable food reward).

Much evidence indicates that OrxR1 is critical for driving highly motivated responding for cocaine, particularly in rats with high motivation for the drug. The blockade of OrxR1 by systemic SB334867 administration decreased cocaine self-administration in rats under limited access conditions under an FR5 timeout 20 s (TO20 s) schedule of reinforcement (Hollander et al., 2012) or PR schedule of reinforcement (Hollander et al., 2012; Prince et al., 2015) but did not alter food self-administration under an FR5 TO20 s schedule of reinforcement (Hollander et al., 2012). OrxR1 knockout mice self-administered significantly less cocaine than wildtype mice (Hollander et al., 2012). Another study described the lack of an effect of peripheral OrxR1 and OrxR2 antagonist administration on reducing cocaine self-administration under an FR1 TO20 s schedule in rats with limited access to cocaine (Smith et al., 2009). These findings suggest that Orx signaling is unnecessary for already established cocaine self-administration under low-effort conditions (e.g., low FR combined with short cocaine self-administration sessions). The blockade of OrxR1 preferentially reduced cocaine self-administration under high-effort conditions (e.g., low cocaine dose and PR schedule; Brodnik et al., 2015), in rats with extended access to cocaine (Schmeichel et al., 2017), and in rats that were given intermittent access to cocaine and exhibited an addiction-like phenotype (James et al., 2019). These data demonstrate that OrxR1 plays an important role in regulating the reinforcing and reward-enhancing properties of cocaine and that Orx transmission is important for establishing and maintaining cocaine intake under high motivation conditions.

Brain regions need to be identified where Orx neuropeptides can mediate drug-directed behavior. For example, Orx inputs to the ventral tegmental area (VTA) are particularly interesting. The majority of Orx axons in the VTA are passing fibers, with only a small portion of Orx fibers in direct synaptic contact on VTA dopamine and γ-aminobutyric acid neurons (Balcita-Pedicino and Sesack, 2007). Intra-VTA OrxA increases the firing rate of VTA dopamine neurons (Korotkova et al., 2003; Muschamp et al., 2007) and increases extracellular dopamine levels in the prefrontal cortex (PFC) and nucleus accumbens shell (NAcSh; Narita et al., 2006, 2007; Vittoz and Berridge, 2006; Vittoz et al., 2008), key brain structures of the neurocircuitry of addiction. The administration of OrxA directly in the VTA increased the motivation to self-administration cocaine, reflected by an increase in cocaine self-administration in discrete trials and under PR schedules of reinforcement but not under an FR schedule of reinforcement (Espana et al., 2011) as previously reported following intracerebroventricular OrxA administration (Boutrel et al., 2005). Altogether, these data confirm that the Orx system may not influence cocaine self-administration when conditions to obtain cocaine require low effort. Orexin inputs to the VTA appear to play a pivotal role in controlling cocaine intake when conditions to obtain the drug require a high level of motivation. Additional studies confirmed the importance of Orx transmission in the VTA during cocaine self-administration. Intra-VTA administration of the OrxR1 antagonist SB334867 reduced cocaine self-administration (Muschamp et al., 2014). These findings were subsequently extended to the central nucleus of the amygdala (CeA), showing that intra-CeA SB338467 administration decreased cocaine self-administration in rats under extended access conditions (Schmeichel et al., 2017; Figure 1).

Similar to preclinical research on cocaine, there is extensive interest in investigating the effects of OrxR1 antagonism on alcohol intake. SB334867 has been shown to reduce alcohol intake under conditions that are characterized by high impulsivity (i.e., binge-like consumption; Olney et al., 2015). SB334867 decreased alcohol consumption and preference exclusively in rats that had high preference or motivation to drink alcohol (Moorman and Aston-Jones, 2009; Lei et al., 2016a; Moorman et al., 2017) and in genetically alcohol-preferring rats (Lawrence et al., 2006; Jupp et al., 2011). Moreover, OrxR1 blockade selectively decreased the escalation of alcohol drinking in mice that were alcohol-dependent, without altering lower levels of alcohol intake in non-dependent mice (Lopez et al., 2016).

In contrast, the blockade of OrxR2 with TCSOX229 did not alter alcohol intake (Lei et al., 2016a). However, other studies showed that OrxR2 blockade with subcutaneous JNJ10397049 administration dose-dependently reduced alcohol self-administration in rats (Shoblock et al., 2011). Intraperitoneal administration of the OrxR2 antagonist LSN2424100 lowered breakpoints on a PR schedule and reduced alcohol consumption in alcohol-preferring rats (Anderson et al., 2014). Corroborating these data, intracerebroventricular TCSOX229 administration reduced alcohol self-administration (Brown et al., 2013), confirming that OrxR2 signaling mediates some aspects of alcohol drinking. Other evidence suggests that the involvement of OrxR1 and OrxR2 activity in alcohol intake may be attributable to a general effect that is not specific to alcohol consumption. In fact, it was shown that blockade of both OrxRs reduced water intake (Anderson et al., 2014). Nevertheless, these data indicate that Orx neuropeptides via OrxR1 and OrxR2 signaling regulate the motivation for alcohol, and targeting OrxRs might be one approach to prevent compulsive, highly motivated behaviors that are directed toward alcohol drinking that is characteristic of alcohol use disorder.

Studies of central effects of Orx transmission on alcohol drinking have identified the involvement of the NAcSh, medial PFC (mPFC), CeA, and VTA (Lei et al., 2016b,2019; Olney et al., 2017). OrxR1 antagonism in the NAcSh was particularly effective in reducing high levels of alcohol drinking in mice, with no significant effect in mice that exhibited only a moderate level of drinking (Lei et al., 2019, 2020; Kwok et al., 2021). The blockade of OrxR2 with TCSOX229 in the VTA and CeA did not alter alcohol drinking (Olney et al., 2017), but TCSOX229 administration in the nucleus accumbens core (NAcC) decreased alcohol intake (Brown et al., 2013; Figure 1). Barson et al. (2015) reported that OrxA and OrxB administration in the anterior PVT (aPVT) but not pPVT increased alcohol intake, an effect that was reversed by the Orx2R antagonist TCSOX229 (Barson et al., 2015, 2017), supporting the hypothesis that the aPVT plays a role in mediating alcohol drinking via OrxR2 signaling (Figure 1). Overall, alcohol consumption data implicate Orx neuropeptides in the pathological motivation for alcohol drinking and demonstrate that both OrxR1 and OrxR2 play an important role in controlling voluntary alcohol consumption.

Several studies have investigated the involvement of OrxR antagonists in animal models of opioid use disorder. In the last two decades, the abuse of prescription opioids (e.g., oxycodone, remifentanil, and fentanyl) has received increasing attention because of the increase in the misuse of these medications and increase in overdoses (Seth et al., 2018). One of the first studies that tested the effects of OrxR antagonists on opioid self-administration showed that systemic SB334867 administration reduced heroin intake under both FR and PR schedules of reinforcement (Smith and Aston-Jones, 2012). OrxR2 antagonism with NBI80713 was subsequently shown to decrease heroin self-administration in rats that were given extended access to heroin (Schmeichel et al., 2015). However, when the effects of a different OrxR2 antagonist, TCSOX229, on oxycodone self-administration were tested under extended access conditions, no effect was observed (Matzeu and Martin-Fardon, 2020c). Systemic SB334867 administration significantly reduced oxycodone self-administration. Consistent with the efficacy of SB334867 in reducing oxycodone intake, other studies showed that this compound reduced the consumption of two other prescription opioids, remifentanil (Porter-Stransky et al., 2017) and fentanyl (Mohammadkhani et al., 2019, 2020). Interestingly, the efficacy of SB334867 in reducing remifentanil self-administration was preserved when the compound was injected in the ventral pallidum (VP; Mohammadkhani et al., 2019, 2020; Figure 1). These findings add to the growing body of evidence that targeting the Orx system may be beneficial for the treatment of opioid use disorder.

Only limited data are available on the involvement of Orx neuropeptides in nicotine addiction. For example, Hollander et al. (2008) showed that systemic SB334867 administration or a site-specific injection of SB334867 in the insular cortex reduced nicotine self-administration (Figure 1) and abolished the stimulatory effects of nicotine on brain reward systems, reflected by reversal of the nicotine-induced lowering of intracranial self-stimulation thresholds. The blockade of OrxR1 appears to effectively reduce nicotine intake. Uslaner et al. (2014) reported that OrxR2 blockade had no effect on nicotine self-administration. Moreover, somatic signs of nicotine withdrawal were attenuated by the OrxR1 antagonist SB334867 but not by the OrxR2 antagonist TCSOX229 in mice and were also attenuated in Orx knockout mice (Plaza-Zabala et al., 2012). Additionally, local SB334867 infusion in the paraventricular nucleus of the hypothalamus decreased the expression of nicotine withdrawal (Plaza-Zabala et al., 2012). Overall, these data demonstrate that OrxR1 signaling plays a role in the reinforcing effects of nicotine and expression of nicotine withdrawal.

For nearly two decades, our group and others have contributed significantly to characterizing the role of Orx neuropeptides in cocaine seeking. Intracerebroventricular OrxA administration led to the dose-dependent reinstatement of cocaine seeking without altering cocaine intake in rats, an effect that depended on corticotropin-releasing factor, suggesting that Orx and stress systems may closely interact to regulate cocaine-seeking behavior (Boutrel et al., 2005). Intracerebroventricular OrxA administration elevated intracranial self-stimulation thresholds, suggesting that OrxA negatively regulates the activity of brain reward circuitry. Furthermore, the selective OrxR1 receptor antagonist SB334867 blocked the stress-induced reinstatement of extinguished cocaine-seeking behavior (Boutrel et al., 2005; Zhou et al., 2012), confirming a role for Orx neuropeptides in driving drug seeking through the activation of stress pathways in the brain. When injected systemically, SB334867 but not the OrxR2 antagonist 4PT also reversed conditioned reinstatement that was induced by cocaine-related stimuli (Smith et al., 2009; Zhou et al., 2012; Martin-Fardon and Weiss, 2014a) but not reinstatement that was triggered by a priming injection of cocaine (Zhou et al., 2012). Another study found that pretreatment with SB334867 significantly attenuated cocaine-seeking behavior that was elicited by a drug-associated context following either extinction or abstinence (Smith et al., 2010), further demonstrating that OrxR1 signaling is critical for conditioned reinstatement. Altogether, these findings suggest that OrxR1 is necessary for cocaine seeking that is elicited by previously drug-paired cues/contexts and stress.

Studies have investigated discrete brain regions that are critically important for cocaine-seeking behavior. The PVT, especially the pPVT, was identified as one such key brain structure (Figure 1). A recent electrophysiological study that used a brain slice preparation for cellular recordings found that the superfusion of OrxA onto pPVT neurons increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) but had no effect on miniature EPSCs (mEPSCs), suggesting a network-driven effect of OrxA. The amplitudes of s/mEPSCs were unaffected by OrxA, indicating a presynaptic action on glutamate release (Matzeu et al., 2018). Thus, the effect of OrxA in the pPVT appeared to be presynaptic and solely target glutamate release, with no involvement of postsynaptic efficacy. Microinjections of OrxA in the pPVT reinstated extinguished cocaine-seeking behavior, and this reinstatement was abolished by a concomitant injection of the OrxR2 antagonist TCSOX229 (Matzeu et al., 2016; Figure 1). Interestingly, this priming effect of OrxA in inducing cocaine-seeking behavior was not long lasting (Matzeu and Martin-Fardon, 2021), implying that the HYP (Orx)↔pPVT circuit undergoes neuroadaptive changes during abstinence, reflected by alterations of the efficacy of OrxA in inducing reinstatement when injected in the pPVT. In addition to the PVT, other brain regions that receive Orx inputs, such as the VTA and CeA, have been investigated because of their known role in cocaine-seeking behavior. Similar to observations in the pPVT, local injections of OrxA in the VTA reinstated cocaine-seeking behavior (Wang et al., 2009). However, in contrast to the pPVT, this priming effect of OrxA was reversed by a selective OrxR1 antagonist but not by an OrxR2 antagonist (Wang et al., 2009). Supporting the importance of OrxR1 signaling in the VTA during cocaine-seeking behavior (Figure 1), another study showed that SB334867 administration in the VTA attenuated the conditioned reinstatement of cocaine-seeking behavior, an effect that was not observed when the same OrxR1 antagonist was injected in the PVT (James et al., 2011). Finally, intra-CeA SB334867 administration significantly reduced the stress-induced reinstatement of cocaine-seeking behavior (Schmeichel et al., 2017). Together with findings in the VTA and pPVT, these results suggest the brain region-dependent involvement of OrxR1 vs. OrxR2 in cocaine-seeking behavior (e.g., OrxR1 in the VTA and CeA and OrxR2 in the pPVT; Figure 1).

Several studies have shown that the Orx system is strongly engaged during alcohol-seeking behavior (e.g., Millan et al., 2010). OrxR1 blockade reduced alcohol cue-induced reinstatement (Lawrence et al., 2006; Martin-Fardon and Weiss, 2014b; Moorman et al., 2017), supporting the hypothesis that Orx neuropeptide transmission at OrxR1 specifically regulates high levels of motivation for alcohol. SB334867 has also been reported to effectively reduce the stress-induced reinstatement of alcohol-seeking behavior, but this effect was not specific for alcohol and instead generalized to the stress-induced reinstatement of sucrose seeking (Richards et al., 2008).

Investigations of specific brain regions where Orx neuropeptides are implicated in the control of alcohol-seeking behavior revealed that the pPVT plays a pivotal role in the stress-induced reinstatement of alcohol-seeking behavior (Matzeu and Martin-Fardon, 2020a; Figure 1). An intra-pPVT injection of the dual OrxR antagonist TCS1102 prevented stress-induced reinstatement selectively in alcohol-dependent rats (Matzeu and Martin-Fardon, 2020a). Another study showed that an injection of an OrxR2 antagonist in the NAcC did not affect alcohol seeking that was triggered by cue presentation (Brown et al., 2013). Similar to cocaine, these findings suggest that the involvement of OrxR1 vs. OrxR2 in alcohol-seeking behavior is brain region-dependent.

The pharmacological manipulation of OrxR1 as a potential treatment to prevent opioid relapse has received much research attention. SB334867 attenuated the reinstatement of heroin-seeking behavior that was elicited by discrete cues but not reinstatement that was elicited by a heroin priming injection (Smith and Aston-Jones, 2012). Several studies corroborated the efficacy of SB334867 in preventing prescription opioid seeking. For example, a recent study from our group found that systemic administration of the OrxR1 antagonist SB334867 but not OrxR2 TCSOX229 prevented the conditioned reinstatement of oxycodone-seeking behavior (Matzeu and Martin-Fardon, 2020c). Consistent with these findings, other studies of different opioids showed that OrxR1 blockade reduced the cue-induced reinstatement of remifentanil seeking (Porter-Stransky et al., 2017) and fentanyl seeking (Fragale et al., 2019). Studies that investigated the neurobiological basis of remifentanil seeking found that the VP is a key brain region where Orx neuropeptides that act through OrxR1 (Figure 1) can influence the cue-induced reinstatement of remifentanil seeking (Mohammadkhani et al., 2019, 2020).

Limited and somehow controversial data are available on the effects of OrxR antagonists on nicotine-seeking behavior. Plaza-Zabala et al. (2013) showed that pretreatment with the OrxR1 antagonist SB334867 but not OrxR2 antagonist TCSOX229 decreased the cue-induced reinstatement of nicotine seeking. In contrast, Uslaner et al. (2014) showed that OrxR2 blockade significantly prevented nicotine seeking that was triggered by environmental cues. Specifically, the selective OrxR2 antagonist 2-SORA 18 reduced the cue-induced reinstatement of nicotine-seeking behavior but not drug-induced reinstatement (Uslaner et al., 2014). Although in apparent disagreement, these two studies demonstrate the importance of OrxR signaling for nicotine relapse.