Circuit and Neuropeptide Mechanisms of the Paraventricular Thalamus across Stages of Alcohol and Drug Use

Abstract

The paraventricular nucleus of the thalamus (PVT) is a midline thalamic brain region that has emerged as a critical circuit node in the regulation of behaviors across domains of affect and motivation, stress responses, and alcohol- and drug-related behaviors. The influence of the PVT in this diverse array of behaviors is a function of its ability to integrate and convey information about salience and valence through its connections with cortical, hypothalamic, hindbrain, and limbic brain regions. While understudied to date, recent studies suggest that several PVT efferents play critical and complex roles in drug and alcohol-related phenotypes. The PVT is also the site of signaling for many neuropeptides released from the synaptic terminals of distal inputs and local neuropeptidergic neurons within. While there is some evidence that neuropeptides including orexin, neurotensin, substance P, and cocaine and amphetamine-related transcript (CART) signal in the PVT to regulate alcohol/drug intake and reinstatement, there remains an overall lack of understanding of the roles of neuropeptides in the PVT in addiction-related behaviors, especially in a circuit-specific context. In this review, we present the current status of preclinical research regarding PVT circuits and neuropeptide modulation of the PVT in three aspects of the addiction cycle: reward/acquisition, withdrawal, and relapse, with a focus on alcohol, opioids (particularly morphine), and psychostimulants (particularly cocaine). Given the PVT’s unique position within the broader neural landscape, we further discuss the potential ways in which neuropeptides may regulate these behaviors through their actions upon PVT circuits.

1. Introduction

The paraventricular nucleus of the thalamus (PVT) is a midline thalamic structure ventral of the third ventricle with a long extent across the anterior-posterior axis of the brain. While initial research hypothesized that the PVT primarily played a “nonspecific” role in arousal (Groenewegen & Berendse, 1994), it has gradually been acknowledged as an integral member of the neural circuitry involved in motivation (McGinty & Otis, 2020; Millan et al., 2017), affective behavior (Barson et al., 2020; Kirouac, 2021), integration of environmental stress information (Rowson & Pleil, 2021), and addiction-related phenotypes (Kirouac, 2015; Matzeu et al., 2014; McNally, 2014; Zhou & Zhu, 2019). Many of the PVT’s roles in behavior appear to be a function of its topographical organization. It projects to several critical limbic brain regions and receives modulatory inputs from many regions across the brain, including cortical, hypothalamic, hindbrain, and limbic structures. Notably, 85% of PVT neurons are glutamatergic projection neurons (Christie et al., 1987; Levine et al., 2021), and because there are almost no GABAergic interneurons within the PVT (Christie et al., 1987), modulation of the PVT projections is predominated by neurotransmitters and neuropeptides released from afferent terminals and neuropeptide-containing cells within the region (as neuropeptides can be released somato-dendritically to regulate the microcircuits in which they participate). Recent progress has led to a rapidly expanding characterization of the roles of specific PVT circuits, as well as the neuromodulators regulating PVT function, in alcohol and drug-related behaviors across the addiction cycle. However, both of these avenues are in their nascent stages and there is little data examining the overlap between them. In this review, we present the current status of preclinical research efforts to delineate PVT circuits and neuropeptide modulation of the PVT in three stages of alcohol, opioid, and psychostimulant use across the addiction cycle (reward/acquisition, withdrawal, and relapse/reinstatement), and we highlight the potential ways in which the roles of neuropeptides in the PVT may occur through circuit-specific modulation. Additionally, within this context, we touch upon the relative dearth of studies which have examined females or included sex as a biological variable.

2. Topographical organization of the PVT

The anterior (aPVT) and posterior (pPVT) subregions can be somewhat divided based on their functions and anatomical connections with a diverse set of brain regions, including structures/nuclei residing in the hindbrain, diencephalon, and telencephalon (Kirouac, 2015). Specifically, the PVT receives afferent inputs from the locus coeruleus (LC; Otake & Ruggiero, 1995), nucleus of the solitary tract (NST; Otake & Ruggiero, 1995), raphe nuclei (Chen & Su, 1990; Otake, 2005; Otake & Ruggiero, 1995), periaqueductal gray (PAG; Li et al., 2014; Otake, 2005), hypothalamic nuclei (Kirouac et al., 2005, 2006; Lee et al., 2015; Li et al., 2014; Otake, 2005; Peyron et al., 1998), thalamic reticular nucleus (Chen & Su, 1990; Li & Kirouac, 2012), bed nucleus of the stria terminalis (BNST; Dong et al., 2001; Otake, 2005; Otake & Nakamura, 1995), central nucleus of the amygdala (CeA; Otake & Nakamura, 1995; Otake et al., 1995), hippocampus (Chen & Su, 1990; Li & Kirouac, 2012), and prefrontal cortex (PFC; Li & Kirouac, 2012; Vertes, 2004). Overall, significantly more neurons within the PFC and hippocampus target the PVT than neurons from various nuclei contained within the hypothalamus and/or brainstem; the aPVT and pPVT preferentially receive inputs from the infralimbic (IL) and prelimbic cortex (PL), respectively, whereas the ventral hippocampus preferentially projects to the aPVT (Gao et al., 2020; Li & Kirouac, 2012). Nevertheless, the PVT receives substantial neuropeptidergic input from various hypothalamic nuclei, a topic which will be further discussed below.

The PVT projects to a variety of brain regions/structures, including the nucleus accumbens (NAc; Dong et al., 2017; Keyes et al., 2020; Parsons et al., 2007; Su & Bentivoglio, 1990; Zhu et al., 2016), CeA (Dong et al., 2017; Keyes et al., 2020; Li & Kirouac, 2008; Vertes & Hoover, 2008), BNST (Dong et al., 2017; Levine et al., 2021; Vertes & Hoover, 2008), hypothalamic nuclei (Csáki et al., 2000; Moga & Moore, 1997; Moga et al., 1995; Vertes & Hoover, 2008), hippocampus (Su & Bentivoglio, 1990; Vertes & Hoover, 2008), and PFC (Bubser & Deutch, 1998; Gao et al., 2020; Hoover & Vertes, 2007; Huang et al., 2006; Vertes & Hoover, 2008). Overall, the densest PVT projections are to the NAc, BNST, and CeA, and the PVT exhibits some topographical subregion organization regarding these projectors’ source. For example, the pPVT preferentially targets the CeA, BNST, and ventromedial NAc shell, while the aPVT projects preferentially to the dorsal NAc shell (Dong et al., 2017; Li & Kirouac, 2008); however, some recent evidence from our lab suggests a fairly similarly robust projection to the BNST across the A-P extent of the PVT (Levine et al., 2021). In addition, recent work has shown that some discrete projections (with diverging behavioral roles) may be distinguished by their molecular profiles. For example, Gao et al. (2020) described two PVT neuronal subtypes based on relative density of D2 dopamine receptors, with “type I” Drd2-positive neurons biased toward expression in the pPVT and preferentially innervating the PL and “type II” Drd2-negative neurons biased toward expression in the aPVT and preferentially innervating the IL (Gao et al., 2020). These types of distinctions may exist in other PVT outputs and extend to neuropeptide modulation (described below).

Interestingly, subsets of PVT efferents have been shown to exhibit significant levels of collateralization, including NAc projections from the PVT that bifurcate to the PFC, CeA, and/or BNST (Bubser & Deutch, 1998; Dong et al., 2017; Freedman & Cassell, 1994; Otake & Nakamura, 1998). Such collateralization potentially enables the PVT to effectively coordinate its widespread output among its projections to influence downstream behavioral effects. However, subpopulations of PVT efferents based on their overlapping projection targets to one, two, or more distal regions has yet to be quantified and characterized in a comprehensive manner. Further, the broader circuit relationships between the many afferents and efferents of the PVT has yet to be studied in detail. The early interpretation that the primary role of the PVT was nonspecific arousal was likely due to its overwhelming number of afferent and efferent connections and the technological limitations of the time to tease them apart. Further, because the PVT both receives input from and/or projects to areas of the brain associated with energy homeostasis (i.e., brainstem, hypothalamus), affective regulation (i.e., limbic regions), and “higher-order cognition” (i.e., telencephalon), it is positioned to serve as a central node mediating behavior via both bottom-up and top-down modulation. In turn, the relative strength and source of neurons that target the PVT, as well as neurons targeted by the PVT, may provide a finer representation of the PVT’s role in a particular behavioral phenotype. For examples, the PVT participates in arousal through its reciprocal connection as “type II” PVT neurons (Drd2−) of the aPVT project to the IL to signal arousal whereas “type I” PVT neurons (Drd2+) of the pPVT project to the PL and respond to stimulus valence (Gao et al., 2020).

3. Involvement of the PVT in alcohol- and drug-related phenotypes

Early studies consistently observed activation of the PVT in response to an acute bolus of varying drugs of abuse. For example, after acute administration of cocaine (30 mg/kg; Deutch et al., 1998), morphine (10 mg/kg; Garcia et al., 1995; Gutstein et al., 1998), or ethanol (1.5 or 4 g/kg or 4h vapor inhalation; Ryabinin et al., 1997; Ryabinin & Wang, 1998), induction of c-fos mRNA and/or c-Fos protein were robustly observed in the PVT of rats within the time course of several hours. These findings stimulated subsequent investigation into the PVT’s potential involvement in the conceptualized stages of addiction-related phenotypes: reward/acquisition, withdrawal, and relapse/reinstatement (Koob & Volkow, 2016) (Table 1). Here, we discuss what has been explored regarding the PVT’s role in each stage of this framework, with a particular emphasis on circuit manipulation experiments when available.

Table 1.

Effects of PVT manipulations on alcohol/drug phenotypes.

Species/Strain	PVT site/circuit	Manipulation	Drug paradigm	Effect	Reference
Reward/Acquisition
Rats / Long-Evans	PVT	Pharmacological inactivation prior to acquisition	Alcohol operant self-administration	No effect	Hamlin et al., 2009
Mice / C57BL/6J (M & F)	PVT-BNST	Acute chemogenetic inhibition	Alcohol binge drinking (DID)	↑ intake in females No effect in males	Levine et al., 2021
Mice / C57BL/6	PVT-CeA	Chemogenetic inhibition: daily morphine conditioning sessionsa, test dayb	Morphine CPP	↓ CPPa No effectb	Keyes et al., 2020
Mice / C57BL/6	PVT-NAc	Chemogenetic inhibition: daily morphine conditioning sessionsa, test dayb	Morphine CPP	No effecta ↓ CPPb	Keyes et al., 2020
Rats / Sprague-Dawley	PVT	Pharmacological inactivation on test day	Cocaine CPP	↓ CPP	Browning et al., 2014
Rats / Sprague-Dawley	PVT-NAc	Pharmacological inactivation prior to acquisition	Cocaine operant self-administration	↓ cocaine infusions	Neumann et al., 2016
Withdrawal
Mice / C57BL/6	PVT-NAc	Optogenetic inhibition during naloxone-precipitated withdrawal	Naloxone-Precipitated morphine withdrawal (CPA)	↓ acute somatic symptoms ↓ CPA (acute & 7 days later)	Zhu et al., 2016
Mice / C57BL/6	PVT-NAc	Chemogenetic inhibition of PVT-NAc during daily saline conditioning sessions	Spontaneous morphine withdrawal (CPA)	↓ CPA	Zhu et al., 2016
Relapse/Reinstatement
Rats / Long-Evans	PVT	Pharmacological inactivation prior to acquisition	Context-induced alcohol reinstatement	↓ reinstatement	Hamlin et al., 2009
Mice / C57BL/6	PVT-NAc	Acute chemogenetic inhibition on prior drug-free test	Morphine-primed CPP reinstatement	↓ reinstatement	Keyes et al., 2020
Rats / Sprague-Dawley	PVT	Acute pharmacological inactivation	Cocaine-primed reinstatement	↓ reinstatement	James et al., 2010
Rats / Wistar	pPVT	Acute pharmacological inactivation	Cue-induced cocaine reinstatement	↓ reinstatement	Matzeu et al., 2015
Rats / Sprague-Dawley	PVT-NAc	Acute pharmacological inactivation	Cue-induced cocaine reinstatement	No effect	Neumann et al., 2016

3.1. Reward/Acquisition

Reward/acquisition of alcohol/drug use is initially based upon the principles of positive reinforcement and can be evaluated via several different assays, most notably conditioned place preference (CPP) and self-administration paradigms (Koob & Le Moal, 2008). CPP is a Pavlovian conditioning protocol (for review, see Bardo & Bevins, 2000; McKendrick & Graziane, 2020; Tzschentke, 2007) in which distinct environmental cues/contexts are explicitly paired with noncontingent administration of either drug or vehicle. After repeated intermittent conditioning trials, preference for the drug vs. vehicle-paired environments is assessed, while in a drug-free state, essentially probing the established strength of association between environmental cue/context and previously experienced “pleasurable” pharmacological effects. In fact, CPP is often used in the pharmaceutical industry as a screening tool for abuse liability of newly synthesized drugs, since all drugs of abuse reliably elicit CPP (Tzschentke, 2007). The PVT appears necessary for expression of cocaine-induced CPP, as transient inactivation of the PVT in rats via intra-PVT microinfusion of baclofen/muscimol on test day prevented cocaine-induced CPP (Browning et al., 2014). More recently, the role of two major PVT efferent pathways, PVT-CeA and PVT-NAc, were recently shown to have differential effects on morphine-induced CPP acquisition and expression (Keyes et al., 2020). In mice expressing an inhibitory designer receptor exclusively activated by designer drugs (DREADD) in the PVT, local microinfusion of the DREADD agonist clozapine-N-oxide (CNO) into the CeA prior to each daily morphine-paired conditioning session to inhibit the PVT-CeA pathway blocked morphine-induced CPP tested one day later in a drug-free state, while microinfusion into the CeA on test day did not affect CPP expression. In contrast, local microinfusion of CNO into the NAc to inhibit the PVT-NAc pathway during each morphine-paired conditioning session had no effect on acquisition of morphine-induced CPP, while microinfusion on test day abolished expression of CPP. Additionally, PVT-NAc suppression on test day was sufficient to permanently block all future expression of morphine CPP, even reinstatement attempts via a priming dose of morphine. These results demonstrate the differential necessity of distinct PVT efferents in acquisition and expression of morphine CPP and the enduring behavioral effects they can exert.

Self-administration paradigms in laboratory animals likely best model human drug consumption, however, they are technically difficult and laborious to perform, particularly in mice. In operant self-administration, animals are trained to perform a task (e.g., press a lever or nose poke) to receive contingent delivery of a drug (typically through a jugular catheter). In cued self-administration paradigms, discrete or discriminative cues signal the availability of the drug, and the animal learns to perform the operant behavior upon presentation of the paired cue to receive the drug (Venniro et al., 2016). Following acquisition, seeking behavior can be tested in the absence of drug availability. A single study in male rats, utilizing this paradigm, demonstrated that the PVT-NAc pathway is necessary for cocaine self-administration but not the subsequent seeking behavior. Tetanus toxin-mediated inhibition of the PVT-NAc pathway prior to training suppressed lever pressing (cocaine infusions) in rats from their first exposure to cocaine and all the way through the 5-day acquisition paradigm (Neumann et al., 2016). However, 24 hours after the last training session, in the absence of cocaine availability, these same rats showed a high level of operant responding similar to control rats. These results have important implications for the role of the PVT-NAc projection in drug-related behaviors. First, they suggest that disruption of the PVT-NAc pathway did not affect rats’ cocaine seeking behavior. While not an analogous paradigm to CPP, this result is somewhat similar to the lack of effect of PVT-NAc inhibition during training on morphine-CPP observed in Keyes et al. (2020), also assessed in the absence of drug after the last drug training session. Second, these results implicate a role for the PVT-NAc circuit in the self-administration acquisition behavior when the drug is available that may be distinct from the subsequent seeking behavior. Other studies have shown that the PVT-NAc circuit plays a role in the acquisition and maintenance of naturally rewarding substances such as sucrose and high fat diet (Christoffel et al., 2021; McGinty & Otis, 2020), indicating there may be a role for this pathway more generally in reward consumption. Notably, these studies examined the PVT-NAc pathway’s role in behavior in animals exposed to the reward and paradigm prior to circuit manipulation, perhaps suggesting that inhibition prior to reward (natural or drug) exposure is not required for PVT-NAc manipulations to affect consummatory behavior. Unlike self-administration procedures, acquisition of CPP cannot be quantified during conditioning, an aspect that perhaps contributes to the sometimes incongruent interpretations of results obtained by the two paradigms (Green & Bardo, 2020). Thus, it is possible that the PVT-NAc plays some role in the animals’ behavior during drug conditioning that is difficult to directly measure.

Additional evidence for the role of the PVT when drugs are onboard comes from its role in the locomotor effects of drugs, which can also be assessed during CPP training. Some drugs of abuse, notably psychostimulants and opioids, reliably induce hyperlocomotion even only after a single dose (Marie et al., 2019), and repeated use (either experimenter- or self-administered) can result in an increase in the drug’s ability to enhance locomotion, a phenotype termed locomotor or behavioral sensitization (Valjent et al., 2010) that can be related to the rewarding effects of the drug. Previous work has demonstrated that both acute hyperlocomotion and locomotor sensitization rely on mesolimbic dopaminergic input to the NAc (Pijnenburg et al., 1976); however, the role of glutamatergic input to the NAc in these locomotor phenotypes has more recently been acknowledged as well (Takahata & Moghaddam, 2003). For example, electrolytic lesioning of the PVT abolishes cocaine-induced locomotor sensitization in rats (Young & Deutch, 1998). Consistently, activation of dopamine D2 receptors (D2Rs) inhibits tonically active PVT neurons, and overexpression of D2Rs in the PVT attenuates cocaine-induced locomotor sensitization in mice (Clark et al., 2017). Altogether, several studies suggest that the PVT, perhaps through its projections to the NAc and other limbic structures, mediates many of the acute effects of drugs that play a role in the interoceptive effects and longer-term consequences of drugs. Further, endogenous activation of neuromodulators that decrease PVT activity and/or synaptic glutamate release at PVT axon terminals serve to dampen the locomotor effects of cocaine and possibly other drugs of abuse. Future studies will need to be conducted to characterize the contributions of PVT circuits to alcohol reward and intake behaviors.

Aside from the few studies described, there remains a lack of information regarding the role PVT circuits play in alcohol- or drug-related behaviors. No studies have examined the roles of the PVT projections to the NAc or CeA in alcohol drinking behavior, and no studies have probed the role of the PVT-PFC pathway in alcohol- or drug-related phenotypes at all. However, our lab has recently examined the role of the PVT-BNST pathway in voluntary binge alcohol consumption (Levine et al., 2021). Using a multiplexed chemogenetic approach, we found that inhibition of this pathway via activation of an inhibitory DREADD promoted alcohol intake in female, but not male, mice; in these same mice, activation of an excitatory DREADD in the pathway suppressed drinking in a subset of male mice without affecting females. Taken together, these results suggest that the PVT-BNST pathway suppresses alcohol intake, and further that it is tonically engaged in females. Notably, BNST-projecting neurons were similarly tonically active in males and females, but the probability of synaptic glutamate release in the BNST was higher in females, suggesting that presynaptic modulation of PVT axon terminals is an important endogenous regulator of the PVT’s role in alcohol/drug intake. Together with the studies on the NAc and CeA projections, the BNST projection may play an opposing role in modulating alcohol/drug reward and intake, however more studies will need to be conducted to delineate the roles of various PVT projections in these behaviors. Importantly, it must be noted that none of these other studies has examined females or included sex as a biological variable, so it is possible that the characterized roles of PVT-NAc and PVT-CeA circuits in behavior do not extend to females.

3.2. Withdrawal

Cessation of alcohol or drug intake is typically met with acute somatic symptoms and/or longer-term affective disturbances during withdrawal that contribute to the likelihood of eventual relapse. The increasing severity of these withdrawal effects contributes to a shift in the underlying reinforcement mechanisms driving intake behavior from positive to negative (Koob & Le Moal, 2001,2008), promoting an increase in the dose and frequency of substance intake required to alleviate withdrawal symptoms. Therefore, plasticity during acute, repeated, and protracted withdrawal plays a key role in the development of alcohol and substance use disorders and has therefore been a focus of much research in the addiction field. Several studies have examined the activation of the PVT across varying time points of alcohol withdrawal. After induction of dependence via either 4-day intragastric or 14-day oral maintenance on an ethanol liquid diet (Lieber & DeCarli, 1982), Fos-like protein activation was observed within the PVT of rats during acute abstinence, specifically between 8-14 hours postcessation (Knapp et al., 1998). More recently, cFos activation in the PVT in mice has been shown to dynamically fluctuate across the temporal spectrum of alcohol abstinence (Smith et al., 2020). After utilizing chronic intermittent ethanol (CIE) vapor exposure to induce dependence (Becker et al., 1997; Becker & Hale, 1993), mice were sacrificed at five different withdrawal time points spanning from 2 hours to 7 days. Compared to air-exposed controls, CIE-exposed mice exhibited decreased c-Fos expression at the 2-hour, 72-hour, and 7-day post-CIE time points, but no difference at 10-hours post-CIE and an increase in c-Fos expression at 26-hours. These temporal adaptations in PVT c-Fos expression implicate a complex role of the PVT in the emergence and stabilization of affective-behavioral disruptions during alcohol withdrawal, which often persist across even prolonged periods of abstinence.

While no circuit perturbation analysis involving the PVT and alcohol withdrawal has yet been performed, Zhu et al. (2016) comprehensively examined the contribution of the PVT-NAc circuit in male mice to morphine withdrawal using an optogenetic approach. First, they used a real-time place preference assay in naive mice while optically stimulating glutamate release from channelrhodopsin (ChR2) expressing PVT terminals in the NAc. They observed a robust reduction in the time spent in the light stimulation-paired compartment, suggesting that robust activation of the PVT-NAc pathway is aversive. They then probed whether this pathway was involved in the emergence of somatic symptoms during morphine withdrawal as well as the exhibition of withdrawal-induced conditioned place aversion (CPA). After induction of morphine dependence through a 5-day home cage injection paradigm, mice underwent naloxone-precipitated withdrawal two hours following the last morphine injection while being confined to one side of a two-compartment locomotor box. In morphine-dependent mice, naloxone elicited significant somatic symptoms of morphine withdrawal (e.g., jumping, tremors), induction of c-Fos in NAc-projecting PVT neurons, and subsequent CPA (at days 1 and 7 of withdrawal) for the naloxone-paired side. Interestingly, optogenetic inhibition of the PVT-NAc pathway via stimulation of archaerhodopsin-3 (ArchT) in PVT terminals during naloxone-precipitated withdrawal reduced the expression of both somatic symptoms and CPA in morphine-dependent mice. They also found that daily chemogenetic inhibition of the PVT-NAc pathway during saline conditioning sessions (presumably during a withdrawal state) prevented CPA. Taken together with the results from Keyes et al. (2020), the PVT-NAc pathway may be essential for retrieval, but not acquisition, of morphine-context associations, perhaps by playing a specific role in the somatic and affective effects during the withdrawal phase that predict future seeking or intake behavior. This role for the PVT in drug withdrawal may similarly extend to withdrawal of alcohol and other drugs, however, that has yet to be experimentally tested. Further, the roles of other PVT projections in withdrawal behavior have yet to be explored, and no studies to date have examined females or the role of the PVT in acute withdrawal prior to dependence. Therefore, many gaps remain in our current understanding.

3.3. Relapse/Reinstatement

Alcohol and substance use disorders are chronic diseases with high rates of recidivism. Within the addiction literature, “reinstatement” is defined as restoration of drug-seeking behavior following extinction/abstinence and then acute exposure to the drug itself, acute stress, or drug-associated cues/contexts (Namba et al., 2018; Venniro et al., 2016), with operant self-administration and CPP paradigms being the most common models of drug relapse behavior in humans (Bossert et al., 2013). In these paradigms, following the establishment of self-administration behavior or CPP (both of which are conditioned in a specific context and/or with specific cues present, as described above), behavioral responding or CPP is extinguished by removal of the drug across sessions/days in the presence of the same context and cues in which acquisition occurred. Notably, this behavioral extinction requires the formation of a new, stronger memory, rather than degradation of the original (Myers & Davis, 2002). In CPP paradigms, during the subsequent reinstatement phase, the animal is again placed into the chamber in which preference was established and extinguished, sometimes following acute exposure to a stressor (such as foot shock, forced swim, or restraint) or a noncontingent priming dose of the drug, and preference for the initially drug-conditioned compartment is assessed to measure drug seeking behavior; these are referred to as stress-primed and drug-primed reinstatement, respectively. In operant self-administration models, during the subsequent reinstatement phase, the animal’s propensity to re-establish operant responding to the previously drug-associated cue is examined; this is termed cue-induced reinstatement. Additionally, a variant of cue-induced reinstatement, termed context-induced reinstatement, is identical except that extinction occurs in a different context (context B) than where acquisition occurred (context A). During the context-induced reinstatement phase, the animal is placed back into context A and re-establishment of the initially trained cue-induced operant behavior is assessed. This probes how the context indirectly modulates cue-induced extinction and reinstatement. Interestingly, because abstinence in humans is often voluntary and initiated due to the negative consequences stemming from excessive drug use (Rosansky & Rosenberg, 2020), some studies have added punishment (i.e., mild foot shock) on half of the operant behavioral responses during the extinction phase in context B in an effort to improve face validity of the model, a variant model termed “punishment-imposed abstinence” (Marchant et al., 2013).

Using these various paradigms, a handful of studies have examined the role of the PVT in the reinstatement of alcohol and drug-related behaviors using c-Fos activation and/or behavioral pharmacology measures. The PVT has been shown to exhibit increased c-Fos activation following cue-induced reinstatement of both alcohol (Dayas et al., 2008) and cocaine (James, Charnley, Flynn, et al., 2011; Matzeu et al., 2017) seeking behavior. Moreover, transient inactivation of the PVT (via intra-PVT microinjections of baclofen/muscimol) blocks cocaine cue-induced reinstatement behavior (Matzeu et al., 2015). Together with this study’s finding that the same manipulation blocks the expression of CPP (prior to extinction), the PVT may be broadly involved in CPP across the many stages of the paradigm. In contrast, Hamlin et al. (2009) utilized the context-induced reinstatement paradigm and found that pharmacological inactivation of the PVT does not disrupt acquisition of alcohol self-administration in rats, but it abolishes context-dependent reinstatement. In addition, this study found that in intact animals, NAc-projecting PVT neurons specifically exhibited increased c-Fos activation in response to re-exposure to the previously alcohol-paired context, pointing to a potential role for this circuit in reinstatement behavior (Hamlin et al., 2009). Other studies have employed the punishment-imposed abstinence variant of the context-induced reinstatement model and shown PVT c-Fos activation in response to context-induced reinstatement of both alcohol (Marchant et al., 2016; Marchant et al., 2014) and cocaine (Pelloux et al., 2018) seeking behavior.

In addition to cue- and context-induced drug reinstatement, several studies have examined the PVT’s role in drug-primed reinstatement. Inhibition of the PVT via intra-PVT microinfusion of tetrodotoxin blocks cocaineprimed reinstatement (James et al., 2010). Further, chemogenetic inhibition of the PVT-NAc pathway via local CNO delivery in the NAc just prior to a CPP expression test blocks CPP not only while CNO is on board but prevents all future expression of a preference when tested in the absence of CNO, including during subsequent morphine-primed reinstatement tests (Keyes et al., 2020). The similarity of results obtained using distinct reinstatement models solidify the PVT’s contribution to reinstatement behavior and, collectively, these studies suggest PVT recruitment transcends drug classes, at least in terms of reinstatement models.

4. Neuropeptide modulation of the PVT’s effects on alcohol-related phenotypes

The PVT receives modulatory inputs from many sources rich in neuropeptide and biogenic amine expression (Kirouac, 2015). Adrenergic fibers from the LC, NST, and thalamic reticular nucleus (Otake & Ruggiero, 1995; Otake et al., 1995), serotonergic fibers from the raphe nuclei (Otake & Ruggiero, 1995), and dopaminergic fibers from the PAG and several hypothalamic nuclei (Li et al., 2014) have all been shown to innervate the PVT. Additionally, the PVT receives robust neuropeptidergic signaling (Fig. 1) in the form of corticotropin-releasing factor (CRF) from the CeA and BNST (Otake & Nakamura, 1995), cholecystokinin (CCK) from the dorsomedial hypothalamus and PAG (Otake, 2005), neuropeptide Y from the arcuate nucleus of the hypothalamus (Lee et al., 2015), substance P (SP) from multiple brainstem nuclei (including the Edinger-Westphal nucleus, mesopontine tegmentum, and medullary raphe nuclei) (Otake, 2005), cocaine and amphetamine-related transcript (CART) from the lateral, paraventricular, and arcuate nucleus of the hypothalamus (Kirouac et al., 2006; Lee et al., 2015), and orexin (OX) from the lateral hypothalamus (Kirouac et al., 2005; Peyron et al., 1998). The latter three neuropeptides have been shown to signal in the PVT to modulate alcohol or drug-related behaviors (discussed below).

Figure 1.

Circuit model depicting major PVT glutamatergic projections and the known (solid lines) and putative (dotted lines) neuropeptidergic afferents to the PVT that modulate alcohol- and drug-related behaviors.

In addition to sources of neuropeptides from distal inputs, PVT neurons themselves are a rich source of neuropeptide expression. A recent analysis of in situ hybridization data from the Allen Brain Atlas identified the expression of 41 unique neuropeptides within the PVT that are known to be involved in a variety of behavioral, cellular, or reproduction roles (Curtis et al., 2020), including SP, CART, and neurotensin (NTS), some of the few neuropeptides whose actions in the PVT have been examined for the roles of their actions in addiction-related phenotypes. As nearly all PVT neurons are glutamatergic projections, it is likely that most neuropeptides are expressed within subpopulations of PVT efferent neurons. Thus, neuropeptide signaling at PVT terminals in distal regions, as well as somato-dendritic neuropeptide release within the PVT, may play a large modulatory function in the role of the PVT in alcohol and drug phenotypes (Curtis et al., 2020; Kolaj et al., 2014). Here, we focus our discussion on neuropeptide signaling within the PVT (as distal peptidergic effects are unknown) on three aspects of addiction-related phenotypes, how these actions may stem from local and distal origins, and the PVT efferents that may be most affected by these modulatory actions (Table 2). Notably, most of the anatomical and behavioral evidence for the organization and function of neuropeptide in the PVT is from male rodents, so it remains to be determined whether the research discussed extends to females.

Table 2.

Effects of intra-PVT neuropeptide manipulations on alcohol/drug phenotypes.

Species/Strain	Neuropeptide	Drug paradigm (drug history prior to manipulation)	Acute PVT Manipulation	Effect	Reference
Reward/Acquisition
Rats / Long-Evans	Orexin	Alcohol binge drinking (IA,4-wk history)	OX-A or OX-B in aPVTa or pPVTb	↑ intakea No effectb	Barson et al., 2015
Rats / Long-Evans	Orexin	Alcohol binge drinking (IA,4-wk history)	OXR1a antagonist or OXR2b antagonist in aPVT	No effecta ↓ intakeb	Barson et al., 2015
Rats / Long-Evans	Substance P	Alcohol binge drinking (IA, 2-wk history)	SP in aPVTa or pPVTb	↑ intakea No effectb	Barson et al., 2017
Rats / Long-Evans	Substance P	Alcohol binge drinking (IA, 2-wk history)	NK1R antagonist in aPVTa or pPVTb	↓ intakea No effectb	Barson et al., 2017
Rats / Long-Evans	Neurotensin	Alcohol binge drinking (IA,4-wk history)	NTS in aPVT in lowa and highb drinkers	No effecta,b	Pandey et al., 2019
Rats / Long-Evans	Neurotensin	Alcohol binge drinking (IA,4-wk history)	NTS in pPVT in lowa and highb drinkers	No effecta ↓ intakeb	Pandey et al., 2019
Rats / Long-Evans	Neurotensin	Alcohol binge drinking (IA,4-wk history)	NTS receptor antagonist in pPVT in lowa and highb drinkers	↑ intakea No effectb	Pandey et al., 2019
Withdrawal
Rats / Long-Evans	Neurotensin	Light-Dark Box during acute withdrawal from alcohol binge drinking (IA, 10-wk history)	NTSR2 antagonist in aPVT in low drinkers	↑ withdrawal-induced exploratory behavior	Pandey and Barson, 2020
Rats / Sprague-Dawley	Orexin	Morphine withdrawal CPA	OXR1 and OXR2 antagonist in PVT: conditioning sessiona, test dayb	No effecta ↓ CPAb	Li et al., 2011
Relapse/Reinstatement
Rats / Wistar	Orexin	Cue-induced cocaine reinstatement	OX-A in PVT	↑ reinstatement	Matzeu et al., 2016
Rats / Sprague-Dawley	Orexin	Cue-induced cocaine reinstatement	OXR2 antagonist in PVT	No effect	James et al., 2011
Rats / Sprague-Dawley	CART	Cocaine-primed reinstatement	CART in PVT	↓ reinstatement	James et al., 2010

4.1. Reward/Acquisition

Behavioral pharmacology studies have begun to investigate the effects of neuropeptide modulation of the PVT on voluntary alcohol consumption. There has been a particular interest in the role of OX in the PVT during drug consumption because OX is made exclusively in the lateral hypothalamus (LH), modulates a variety of behaviors such as food and drug intake, and the PVT is one of the most densely innervated targets of OX neurons (Kirouac et al., 2005). Further, pharmacological activation of OX receptors in the PVT increases downstream NAc dopamine levels (Choi et al., 2012), suggesting that OX may selectively modulate PVT-NAc neurons to affect drug reward. To probe the role of PVT OX signaling in alcohol intake, studies have primarily used an intermittent-access (IA) two-bottle paradigm in male rats, which is a model of “binge-like” drinking where intervals of 20% ethanol access and deprivation are alternated every 24 hours under ad libitum access to water (Simms et al., 2008; Wise, 1973). After four weeks of IA alcohol drinking, microinfusion of either orexin-A (OX-A), with affinity for both orexin receptors 1 and 2 (OXR1 and OXR2, or orexin-B (OX-B), with five times higher affinity for OXR2 than OXR1), into the aPVT, but not the pPVT, enhances alcohol drinking (Barson et al., 2015). Moreover, local aPVT antagonism of OXR2 but not OX1R decreases alcohol consumption, suggesting hypothalamic OX projections promote voluntary alcohol consumption through the actions of OX2R in the aPVT.

Substance P (SP) released from mid/hindbrain afferents (Otake, 2005) or within the PVT (Curtis et al., 2020) may also play a role in promoting alcohol consumption. Systemic antagonism of the SP receptor, neurokinin 1 receptor (NK1R), results in reduced alcohol intake (Steensland et al., 2010), and mice deficient in NK1R expression exhibit attenuated alcohol consumption at high concentrations (George et al., 2008). Microinfusion of SP in the aPVT, but not the pPVT, enhances IA alcohol drinking in rats whereas antagonism of NK1R, only in the aPVT, attenuates alcohol drinking (Barson et al., 2017). Interestingly, microinfusion of OX into the aPVT, but not pPVT, results in increased SP mRNA and peptide expression while OX-induced enhancement of IA alcohol drinking can be prevented through SP receptor antagonism (Barson et al., 2017). Thus, the effects of OX may require local SP synthesis and signaling, highlighting the possibility of many neuropeptide interactions that could regulate intake of alcohol and other drugs of abuse.

In contrast to SP, Neurotensin (NTS) has been shown to decrease alcohol intake, as mice deficient in NTS receptor type 1 (NTS1) or type 2 (NTS2) display increased alcohol intake (Lee et al., 2010; Lee et al., 2011). Local delivery of NTS to the pPVT, but not aPVT, reduces IA alcohol drinking, but only in rats classified as high drinkers (Pandey et al., 2019). Moreover, researchers found that inhibition of pPVT NTS signaling through local antagonism of the NTS receptors enhances IA alcohol drinking only in rats classified as low drinkers, suggesting that NTS tone in the pPVT inhibits voluntary alcohol drinking. As the aPVT is typically associated with reward-related behaviors and the pPVT with stress responses (Zhou & Zhu, 2019), high and low drinking phenotypes may reflect some distinction between the activity of aPVT and pPVT projection neurons and tonic signaling of neuropeptides, including OX, SP, and NTS, that exert differential effects on PVT subregions to influence voluntary alcohol consumption.

4.2. Withdrawal

As the role of the PVT in withdrawal-related phenomena has been under-investigated, so too has neuropeptide modulation of the PVT in these behaviors. Using the IA paradigm and high vs. low drinker classification as described above, one study found that during acute withdrawal (24-27 hours post-alcohol access) following 11 weeks of IA, high drinkers displayed an anxiolytic phenotype/increased exploratory behavior as measured on in the light-dark box and elevated plus maze (Pandey & Barson, 2020). Interestingly, these phenotypic differences between high and low drinkers were absent prior to initiation of alcohol access but emerged as early as four weeks into IA alcohol access, suggesting either an effect of cumulative alcohol consumption or interaction between drinking phenotype and alcohol consumption on withdrawal-induced anxiety. Additionally, high drinkers showed increased NTS receptor type 2 (NTS2R) levels in the aPVT compared to low drinkers at this acute withdrawal time point, and local agonism of aPVT NTS2R in low drinkers produced the amplified exploratory phenotype akin to that observed in high drinkers, suggesting that NTS signaling in the aPVT promotes abstinence-induced exploratory behavior (Pandey & Barson, 2020). Altogether, findings from NTS pharmacology studies suggest that NTS signaling in the aPVT and pPVT modulate distinct alcohol-related phenotypes (with aPVT NTS signaling decreasing anxiety-like behavior and pPVT NTS signaling inhibiting alcohol drinking).

Although the role of the PVT in alcohol withdrawal-related associations has not been explored, previous work has shown differential effects of local OX on the acquisition and expression of morphine withdrawal-induced CPA. Antagonism of OX1R or OX2R within the PVT prevents the expression, but not acquisition, of morphine withdrawal-associated CPA as well as expression of somatic withdrawal symptoms (Li et al., 2011). As the PVT-NAc pathway has been shown to be necessary for retrieval, but not acquisition, of morphine context associations (Keyes et al., 2020; Zhu et al., 2016), OX could perhaps be a key component driving such effects. However, the possibility for the actions of many other independent and interaction neuropeptides remains open.

4.3. Relapse/Reinstatement

In addition to the role of some neuropeptidergic modulation of the PVT in voluntary alcohol drinking behavior, some attention has been paid to its role in the reinstatement of this behavior following extinction. For example, Dayas et al. (2008) found that reinstatement of cue-induced alcohol seeking behavior elicited c-Fos activation in the PVT, and these neurons were closely associated with OX and CART terminal fields (with OX from the LH and CART from the LH and/or PVT neurons), suggesting these specific neuropeptides contribute to this activation. Although the roles of these neuropeptides have not directly been probed in cue-induced alcohol reinstatement, OX’s role in cue-induced reinstatement of cocaine seeking behavior has been examined. Intra-PVT OX-A promotes this reinstatement behavior (Matzeu et al., 2016), while local antagonism of OX1R exerts no effect (James, Charnley, Levi, et al., 2011). These findings suggest that either OXR2 mediates the effects of OX, or that OX signaling is sufficient, but not necessary, for cocaine cue-induced reinstatement behavior. Given the aPVT-specific effects of OX in alcohol self-administration (Barson et al., 2015), it is also possible that there are subregion-specific effects of OX receptor modulation of cocaine-related phenotypes playing a role in the results of these studies. These many possibilities demonstrate the need for future research to determine the roles of OX signaling in the PVT in the reinstatement behavior for alcohol or for other drugs of abuse that could speak to the generalizability of the role of OX in reinstatement.

In addition to OX, other neuropeptides have been shown to act in the PVT to modulate reinstatement behavior. Context-induced reinstatement of alcohol seeking behavior is inhibited by either intracerebroventricular infusion of CART (King et al., 2010) or intra-PVT microinfusion of a kappa opioid receptor (KOR) agonist (Marchant et al., 2010), and intra-PVT microinfusion of CART has been shown to block cocaine-primed reinstatement (James et al., 2010). These CART findings suggest a potential role for CART signaling in the PVT in inhibiting reinstatement across alcohol and other drugs. Further, ex vivo electrophysiological evidence demonstrates CART significantly suppresses excitatory synaptic drive of aPVT neurons in mice, regardless of whether they were naive or had a history of cocaine exposure (Yeoh et al., 2014). Therefore, CART signaling in the aPVT may be sufficient to suppress the reinstatement of cocaine and perhaps alcohol and other drug self-administration, through inhibition of aPVT neurons, such as those that project to the NAc. As this electrophysiology experiment was performed just 24 hours after the last cocaine exposure, whether CART signaling tone (due to decreased CART levels and/or receptor expression) is decreased at a time point reflective of reinstatement is not yet known; however, it is possible that there is plasticity in CART signaling that contributes to the expression of reinstatement behavior.

5. Conclusions

5.1. PVT circuitry in drug-related behavior

Here, we reviewed the current progress of preclinical research efforts to elucidate the PVT efferents involved in the reward/acquisition, withdrawal, and relapse stages of alcohol/drug use, and we highlighted what is known regarding neuropeptide modulation of the PVT’s effects on these stages, particularly in terms of alcohol use. As discussed, the central positioning of the PVT within neural circuitry networks involved in addiction-related phenotypes implies the ability of the PVT to exert both bottom-up and top-down modulation of downstream behavior. Currently, the overwhelming evidence of the PVT’s role in these behaviors is derived from c-Fos activation observations and nonspecific intra-PVT manipulations and does not span the various stages of addiction. Initial studies have explored the specific roles of several major PVT efferent pathways (e.g., projections to CeA, NAc, and BNST) in alcohol- and drug-related phenotypes. For example, our lab has found that the PVT-BNST pathway inhibits alcohol intake (Levine et al., 2021). Furthermore, two studies have characterized the differing contributions of PVT-CeA and PVT-NAc pathways in morphine-context associations (Keyes et al., 2020; Zhu et al., 2016), but whether these pathways similarly modulate alcohol-context associations remains to be investigated. Moreover, PVT-PFC reciprocal circuits have recently been shown to play important roles in salience and valence (Gao et al., 2020), thus future investigation of this circuit in alcohol- and drug-related phenotypes is imperative given the known significance of drug-context associations. Further, the role of neuropeptides in the PVT in these behaviors is extremely limited, and it is completely absent in a circuit-specific context. Thus, there exists a tremendous opportunity for circuit-level dissection approaches, such as optogenetic and chemogenetic methods, in these behaviors, particularly with regards to neuropeptide modulation.

5.2. Sex differences in the role of the PVT

To our knowledge, apart from Levine et al. (2021) and Clark et al. (2017), all work discussed above has been conducted only in male rodents (or sex is not described); therefore, there exists little data regarding the interaction of neuropeptides and the PVT in modulation of addiction-related phenotypes in females. This absence of evidence is especially crucial given that female mice generally exhibit greater voluntary alcohol consumption (g/kg) compared to male mice (Yoneyama et al., 2008). And, across different classes of drugs, females display a “telescoping” effect in which the likelihood of dependence escalates more rapidly across increased alcohol, opioid, or cocaine use than in males (Brady & Randall, 1999; Hernandez-Avila et al., 2004; Keyes et al., 2010; Lewis & Nixon, 2014; Sartor et al., 2014; Stoltman et al., 2015). Given the PVT’s emerging role in drug reward, aversion, and memory, the PVT may display basal and/or drug-induced changes in function that contribute to escalation of consumption, severity of withdrawal, and the transition to dependence and relapse potential.

5.3. The PVT’s role in withdrawal

There has been comparatively more PVT-focused studies investigating opioids and psychostimulants despite, by certain accounts, that alcohol has greater overall harmful effects (i.e., harm to oneself, friends, family, and/or society) than any other drug class (Nutt et al., 2010). Likewise, the current studies which have examined neuropeptide modulation of the PVT’s effects on alcohol-related phenotypes have focused more heavily on voluntary alcohol consumption and reinstatement behavior than withdrawal-related phenomena. Although all stages are vital to our comprehensive understanding, longer-term affective–behavioral disruptions, which can emerge and persist long after initial detoxification, are likely the key contributors to high rates of relapse (Hartmann et al., 2020; Heilig et al., 2010; Holleran & Winder, 2017). Keyes et al. (2020) highlights the circuit-specific nature of the role of the PVT in morphine-context associations based on acute drug status (intoxication vs. withdrawal) and repeated use. The transitioning role of the PVT may be coordinated through the activation and interactions of neuropeptides within the PVT. Overall, future mechanistic research into the neuropeptide modulation of PVT circuitry during the various stages of alcohol use is necessary for ultimately elucidating the neural correlates driving the development of addiction-related symptomatology.