The Periaqueductal Gray and Its Extended Participation in Drug Addiction Phenomena

Abstract

The periaqueductal gray (PAG) is a complex mesencephalic structure involved in the integration and execution of active and passive self-protective behaviors against imminent threats, such as immobility or flight from a predator. PAG activity is also associated with the integration of responses against physical discomfort (e.g., anxiety, fear, pain, and disgust) which occurs prior an imminent attack, but also during withdrawal from drugs such as morphine and cocaine. The PAG sends and receives projections to and from other well-documented nuclei linked to the phenomenon of drug addiction including: (i) the ventral tegmental area; (ii) extended amygdala; (iii) medial prefrontal cortex; (iv) pontine nucleus; (v) bed nucleus of the stria terminalis; and (vi) hypothalamus. Preclinical models have suggested that the PAG contributes to the modulation of anxiety, fear, and nociception (all of which may produce physical discomfort) linked with chronic exposure to drugs of abuse. Withdrawal produced by the major pharmacological classes of drugs of abuse is mediated through actions that include participation of the PAG. In support of this, there is evidence of functional, pharmacological, molecular. And/or genetic alterations in the PAG during the impulsive/compulsive intake or withdrawal from a drug. Due to its small size, it is difficult to assess the anatomical participation of the PAG when using classical neuroimaging techniques, so its physiopathology in drug addiction has been underestimated and poorly documented. In this theoretical review, we discuss the involvement of the PAG in drug addiction mainly via its role as an integrator of responses to the physical discomfort associated with drug withdrawal.

Introduction

The Diagnostic and Statistical Manual of Mental Disorders (DSM-5) uses the term "substance use disorders" to encompass 10 different classes of drugs of abuse: alcohol, caffeine, cannabis, hallucinogens, inhalants, opioids, sedatives (hypnotics or anxiolytics), stimulants, tobacco, and other (or unknown) substances [1]. All these drugs are similar in that they activate the reward circuit [2, 3] and, except for hallucinogens and inhalants, the rest induce withdrawal symptoms. The intake of a substance may result in a chronic addictive disorder if one of the following features is present: (i) compulsion to seek the substance; (ii) its uncontrolled intake; and (iii) the appearance of feelings of discomfort that include dysphoria, anxiety, pain, or irritability [4, 5]. During the establishment of drug addiction, the drug consumption behavior evolves differentially (i.e., not equally for every substance) from impulsiveness to compulsivity, especially with substances like alcohol [6]. The above suggests that drug addiction is not a simple sequence but instead, both impulsive and compulsive intake behaviors and the underlying biology are important [6]. The impulsive intake of substances is an early phase of the addiction cycle that generates a sense of pleasure and gratification via the brain reward system and the reinforcing effects of substance intake; whereas the compulsive intake is a late phase of the addiction cycle, in which the alleviation of physical discomfort, which may range from mild to severe distress, has been proposed to be elemental [2, 7]. Interestingly, specific environmental contexts such as visual and olfactory signals may promote craving and produce difficulties in controlling impulsive behaviors [8]. The establishment of a compulsivity intake behavior involves allostatic changes in the neurotransmission of the brain reward and anxiety systems such as alterations of dopaminergic function [4, 9–11]. Hence, drug addiction involves responses to psychiatric perturbations, stressful situations, and/or physiological challenges that are integrated to promote adaptation and coping and thus, homeostasis [12, 13]. Notably, compulsive behaviors induced by some drugs of abuse (alcohol, cocaine, morphine, and heroin) may develop before any evidence of dependency and tolerance [7, 14]; thus, neither dependence nor tolerance may solely explain the complexity of the drug addiction cycle.

Drug addiction can be cyclic and consists of at least three main stages: (i) binging/intoxication, in which there is a strong motivation to take drugs due to their reinforcing effects; (ii) withdrawal/negative affect, characterized by the presence of anxiety, irritability, widespread pain, dysphoria, and hyperkatifeia, among others; and (iii) anticipation/craving, that may drive to the binging stage again. Each of these stages encompasses brain circuits that are anatomically, neurophysiologically, and neurochemically specific [3, 15–19]. In animal models, addiction-like behaviors include a regular, predictable, and/or uninterrupted use of the substance [7].

The midbrain central gray, also referred to as the PAG or substantia grisea centralis, is a brainstem structure bordering the cerebral aqueduct [20]. The PAG, along with the amygdala, hypothalamus, and the bed nucleus of the stria terminalis (BNST), form the aversion system [21], which is activated during acute and chronic exposure to stressors [13] and is responsible for executing defensive behaviors against anxiety, pain, and fear, such as the consumption of a drug in order to avoid the discomfort associated with withdrawal of the drug [21–27]. During acute or prolonged withdrawal, the cerebral aversion system seems to be involved in the increased release of stress promotors such as glucocorticoids, corticotropin-releasing factor (CRF), norepinephrine, and dynorphin [17]. In normal conditions, factors such as neuropeptide Y (NPY), nociceptin, and endocannabinoids seem to be involved in maintaining a low stress response [17, 28].

Recently, based on anatomical and functional evidence, it has been proposed that the PAG plays a fundamental role in controlling motivated goal-directed behaviors of all types [29]. Therefore, in this review we aimed to discuss and propose the participation of the PAG in the pathophysiology of drug addiction.

Physiology of the Periaqueductal Gray

The PAG is a well-conserved midbrain area in chordate species [30, 31] and has a similar proportional size (up to 10% of the mesencephalon) in rodents, cats, and humans [32]. Its functions include food intake [29], pain modulation [33–35], anxiety/panic [23, 36], unconditioned, conditioned, as well as learned behaviors such as fear [37–39], vocalization [40], and sexual behavior via the integration of the lordosis reflex in coordination with the medullary reticular formation and the ventromedial nucleus of the hypothalamus [41] during mating stimulation [42], and the integration of autonomic responses [32, 43]. Furthermore, neurons in the PAG integrate negative phenomena such as anxiety, stress, and pain with the autonomic, neuroendocrine, and immune systems to facilitate responses to threat [44]. Pain, anxiety, and fear are normal emotions with great adaptive value in evolutionary selection in mammals [45]. Brain structures involved in processing pain, anxiety, and fear under natural circumstances are also related to the pain, anxiety, and fear associated with the intoxication/withdrawal induced by drugs of abuse [46–48]. While fear occurs in response to specific threats, the source of anxious behavior is usually undefined or unknown [45]. There is substantial evidence that the PAG is a key midbrain structure involved in the processing of pain, anxiety, and fear [49].

The PAG is partitioned into various structural subdivisions disposed like the neuroaxis itself: dorsomedial, dorsolateral (DL), lateral, and ventrolateral (VL). These subdivisions also project in other planes among themselves (anterior and posterior) with different participation around the integration of defensive behaviors such as pain, anxiety, and fear [32, 50, 51]. The dorsal and lateral PAG (D/L-PAG) play a role during combative defensive reactions and non-opioid-based analgesia [52, 53]. The D/L-PAG also receives inputs from the amygdala, the ventromedial hypothalamus, and the medial prefrontal cortex (mPFC) [54]; whereas the VL-PAG seems to be important for evoking passive behaviors and opioid analgesia via the anterior insula and the medial and dorsomedial PFC [50, 55]. Indeed, tonic immobility-elicited analgesia is mediated by mu opioid receptors in the VL-PAG via inhibition of the GABAergic projections to the rostral ventromedial medulla (RVM) and dorsal raphe (DR) [56]. On the other hand, glutamatergic neurons in the VL-PAG, when activated under physiological conditions, cause freezing behavior that is also regulated by mu opioid receptors [57]. Interestingly, hypofunction of glutamatergic transmission in the VL-PAG has been proposed to be associated with depressive-like behaviors [58].

Some studies have proposed the PAG as a structure that plays a role in the integration of other complex behaviors such as sadness, fury, pleasure, worry, and fear of painful stimuli through neuronal processes that involve the participation of other brain areas including the amygdala and hypothalamus [59–61]. In fact, one of the main functions of the PAG is related to the integration of peripheral and central afferent inputs, which in turn may result in homeostatic defensive reactions mainly via activation of the sympathetic autonomic nervous system (ANS) [59, 62], which modulates the activity of visceral tissues (e.g., blood flow and adrenaline release) via its monoaminergic receptors, α and β adrenoceptors. In this sense, the ANS prepares the organism for the classic fight or flight response by regulating visceral activity (cardiovascular, metabolic, and respiratory adaptations) through sympathetic fibers, which are finely modulated by monoaminergic receptors [63, 64]. Indeed, the interruption of sympathetic activity seems to involve both monoaminergic auto- and hetero-receptors [65, 66]. Interestingly, the endocannabinoid [67–69] and opioid systems [70] are also involved in the modulation of cardiovascular sympathetic and sensory drives.

Several preclinical and clinical studies have suggested that stressful experiences that occur throughout life may contribute crucially to the development and pathogenesis of several psychiatric disorders such as schizophrenia, mood disorders, anxiety, erroneous defensive responses, and affect reward-seeking [44, 71–73], all of which can promote drug abuse. Moreover, most of the symptoms of anxiety disorders are accompanied by activation of the hypothalamic-pituitary adrenal axis [71, 74, 75]. Due to similarities between the behavioral and autonomic responses induced by D-PAG stimulation and the semiology of panic attacks, it has been suggested that the D-PAG is deeply involved in the genesis of panic-related disorders in humans [76]. In support of this hypothesis, chemical or electrical stimulation of the D-PAG induces panic attacks in rodents [76]. Likewise, panic-like behaviors can be evoked by systemic cholecystokinin 2 (CCK₂) receptor agonists, an effect that is prevented by intra-PAG microinjection of CCK antagonists [77–79]. Hence, the physiology of the PAG suggests that this structure plays a key role in the integration of actions primarily evoked to modulate discomfort (e.g., anxiety, fear, and painful situations that are commonly experienced during withdrawal in the addiction cycle [80–84]). Moreover, there is substantial evidence (see section 2) for the role of the pain circuit and its impact in producing negative reinforcement that may contribute to sustained habitual drug intake, as for the PAG-pain circuit and cocaine addiction) [5, 85, 86]. Importantly, the diminutive size and form of the PAG have made its functional analysis very challenging when using standard noninvasive magnetic resonance imaging (MRI) techniques [87]. Notably, with the advent of better imaging tools, this kind of structure will be better understood. In support of this notion, a very recent functional MRI (fMRI) study has proposed a cortex-PAG connection that appears to be essential in alcohol abuse [88].

The Link Between the PAG and the Reward Circuit

The possible participation of the PAG in the impulsive intake of substances of abuse should be related to its interaction with nuclei that are known to be involved in the reward, such as the nucleus accumbens (NAc), VTA, and hypothalamus [16], among others such as the substantia nigra [89–91]. The reward system is defined as a circuit whose activation leads to positive reinforcement with a positive hedonic overlay [19]. In such systems, chemical messengers including serotonin, cannabinoids, opioid peptides, enkephalin, and GABA are involved, directly or indirectly, in the actions that modulate the activation of the reward system via dopamine (DA) release in the NAc [89, 92, 93]. The PAG sends glutamatergic and GABAergic inputs directly to DA-containing and GABA-containing cells [94] in the VTA [95]. As the VTA is a key element in the reward system [96, 97], the PAG-VTA circuitry could also play an indirect role during the early phases of drug addiction [44]. Laurent et al. [98] found that activation of the GABAergic synapses between the VL-PAG and the VTA increases the immobility response that is blocked in the presence of opioids.

Another fundamental structure in the reward circuit is the NAc, which is vital for integrating reward, drug reinforcement, and motivated behavior [99, 100]. Moreover, this small nucleus is important for evaluating the cost-benefit decision-making in situations involving reward [99, 100]. It has been proposed that the NAc, together with the basolateral amygdala (BLA) and D-PAG, form a circuit in charge of responding to a prolonged stressful situation [101–103]. The NAc is anatomically separated into shell and core portions [104], both of which are involved in decision-making when seeking reward [100] and receive inputs from numerous areas such as the anterior cortex and the lateral hypothalamus (LH) [105]. The NAc shell increases the operant response to a reward-associated cue [106], participates in the expression of appetitive-type behaviors [107], and is more sensitive to the rewarding effects of cocaine and D1/D2 receptor agonists than the NAc core [108–110]. The NAc core is essential for the execution of Pavlovian behaviors since its inactivation causes indiscriminate responses during a cue-guided risk/reward paradigm in rats [100].

Another region important for the drug addiction cycle is the hypothalamus (conformed by lateral, medial lateral, medial periventricular, and medial zones), which is located ventrally in the brain and plays an important role in the regulation of the endocrine system [111]. The LH is involved in the control of feeding behavior [112]. Interestingly, the L-PAG [113] influences reward and reinforcement processes such as seeking appetitive reward by activation of the orexinergic cell group of the LH [114]. In addition, the LH projects to the ventrolateral neurons of the PAG and extended amygdala (EA) and receives afferent connections from the LH to modulate drug reward and neuro-adaptive changes that occur with chronic drug exposure [43, 115]. The ventromedial hypothalamus has reciprocal projections with the D-PAG that seem to be involved in the execution of panic-like behaviors [116]. The arcuate nucleus of the hypothalamus has NPY-containing projections towards the D- and V-PAG. Besides, the PAG is densely innervated by NPY fibers [117, 118] and contains very high levels of NPY [119, 120]. Among the NPY receptor subtypes identified so far (Y₁–Y₅), only two have been reported to have functional effects on the PAG: Y₁ and Y₂ [121]. These receptors have been shown to be involved in depression [122] and alcohol consumption [123]. Likewise, microinjection of NPY into the D-PAG and the VL-PAG has anxiolytic/analgesic effects [124] and may modulate alcohol intake [28].

The PFC can be divided into the mPFC, orbitofrontal cortex, ventrolateral PFC, dorsolateral PFC, and caudal PFC [125]. Cortical projections to the PAG originate primarily from the mPFC [126]. The interaction of the L- and D-PAG with the PFC involves executive functions [127]. The mPFC is mainly involved in cognitive functions [128], including reward-related activity [129]. The projections of the mPFC end in both the DL [130] and VL PAG [131] and it has been suggested that this mPFC-DL/VL projection participates in aversive and compulsive behavioral responses [132] as well as pain modulation [125, 133].

The Link Between the PAG and the Anti-reward Circuit

The anti-reward system includes the EA, which is composed of the central nucleus of the amygdala (CeA), BNST, and NAc [19]. The coordinated activity of these regions is involved in the integration of the negative affective states: anxiety, irritability, pain, and others [18]. It is important to note that the function of the above structures is not limited to the anti-reward system as they participate in other actions including yawning [134], impulsive behavior [135], and feeding behavior [136], as well as pain [137] and others.

The PAG provides inputs to the BNST [138, 139] which is a critical structure for stress, anxiety [140–142], and fear responses [143]; and it is enriched mainly in NPY and CRF neurons [144]. Release of the neurotransmitter NPY has anti-drinking effects mediated through the Y₁ receptor, which inhibits BNST-CRF neurons [145], and is a key structure in the withdrawal/negative affect and anticipation/drug craving [82, 146, 147]. The BNST receives 50% of its DA contribution from the D- and V-PAG [139, 148] (Fig. 1), and this could be involved in pain regulation and anxiety [149]. Furthermore, it has been reported that the BNST and VL-PAG interaction is involved in the modulation of eating behavior through GABAergic pathways [136]. The amygdala is a connecting structure in the limbic system and can be divided into three groups: (i) the BLA, which includes the lateral, basal, basomedial and basoventral nuclei; (ii) the centromedial amygdala, which includes the CeA and medial nuclei (M) (the CeA nuclei have four subdivisions: the capsular, lateral (CeL), intermediate, and medial (CeM) subdivisions) [150, 151]; and (iii) the superficial or cortical-like region that includes the cortical nuclei and the nucleus of the lateral olfactory tract [152, 153]. The CeA is the main output area from the amygdala to the PAG at different points of the antero-posterior axis. For example, the CeM projects mainly to the rostral and caudal PAG, while the CeL projects only to the caudal PAG. Further, the D-PAG is selectively targeted by the CeM and the VL-PAG by the CeL [154]. Lesions in the BLA have been shown to block flight responses evoked by stimulation of the D-PAG [102]. In addition, it has been reported that the CeA/BLA–mPFC–VL-PAG circuit is involved in pain processing [133, 155], cataplexy [156], and pain symptoms correlated with depression [157].

Fig. 1.

Interaction of the PAG with other brain nuclei potentially involved in drug addiction. A Inputs to the PAG from different nuclei. The dorsal and ventral PAG (orange lines) receive glutamatergic inputs from the mPFC; the D-PAG and VL-PAG receive GABAergic inputs (blue lines) from both CeA and VTA (blue lines); the VL-PAG receives GABAergic projections from both the BNST and the hypothalamus; the arcuate nucleus of the hypothalamus has NPYergic projections to the D- and V-PAG (black lines); the VTA projects to the VL-PAG via glutamatergic neurons (orange line); the Pn has a cholinergic projection to the VL-PAG (purple line). B Outputs from the PAG to different nuclei. The BNST and CEA receive dopaminergic inputs from both the dorsal and ventral PAG (red lines); the VL-PAG sends glutamatergic projections to the VTA (orange lines). BNST, bed nucleus of the stria terminalis; CeA, central nucleus of the amygdala; D-, L-PAG, dorsal and lateral periaqueductal gray; mPFC, medial prefrontal cortex; Pn, pontine nucleus; VL-PAG, ventrolateral periaqueductal gray; VTA, ventral tegmental area.

Chronic exogenous activation of the reward system may drive decreases of DA, serotonin, and opioid peptides and their actions in the ventral striatum-VTA as an adaptive response to lessen the effects of drugs. If this exogenous activation occurs, negative reinforcement such as intrusive thinking and anxiety may increase the probability of searching for drugs [11, 17, 158]. It has been suggested that these negative reinforcement actions are integrated by an anti-reward system [17] that would be complementary to the reward system to achieve coordinated activity under normal conditions, but that could also contribute to the seeking and binging of drugs during the drug addiction process. These reward and anti-reward circuits do not work independently; both are necessary for the establishment of addiction.

The PAG is involved in the execution of various emotional conditions/behaviors such as pain, anxiety, and fear; but it has been more recently addressed as a brain area participating in drug addiction. However, further understanding of its contribution during the negative affective state seen during drug-withdrawal is required [28, 85, 86, 159]. The PAG afferent and efferent nerve fibers with the nuclei involved in drug addiction are summarized in Fig. 1.

Links Between the PAG and Substances of Abuse and Appetitive/Consummatory Behaviors

As noted above, the PAG is a key integrating structure for executing defensive responses via opioid receptors [160]; and some groups have reported possible participation of the PAG in the expression of several signs of withdrawal from diverse drugs of abuse such as morphine [14, 80, 161, 162]. In fact, morphine injections into the PAG produce conditioned place preference, suggesting that the PAG may be involved in the reinforcing actions of opioids during the early (impulsive) phase [163]. Moreover, the PAG has been reported to mediate reward information that promotes food intake, whereas PAG inhibition has anorexic effects in hungry rats [164]. In the next section, we discuss the preclinical and clinical evidence available involving the role of PAG activity in the actions of the major pharmacological classes of drugs of abuse (Table 1). In addition, we extend the discussion to some appetitive consummatory behaviors.

Table 1.

Role of the PAG in the actions of the major pharmacological classes of drugs of abuse.

Drug of abuse	Substance or receptor involved	PAG subdivision involved/outcome	References
Alcohol	NMDA or AMPA receptor antagonists	Intra-D-PAG injection of NMDA/AMPA receptor antagonists is anxiolytic and inhibits alcohol intake	[169]
	carboxy-PTIO and L-NAME	NO inhibition in DL-PAG decreases the anxiogenic effects of alcohol withdrawal	[172]
	NPY-Y1	Intra-D-PAG injection of NPY-Y1 is anxiolytic and reduces alcohol consumption	[28]
Caffeine	Caffeine	fMRI shows that the anxiogenic actions of caffeine are associated with PAG activation	[179]
Cannabis	CB1	Activation of CB1 in D-PAG has panicolytic-like effects, analgesia, anti-aversive effects, and prevention of hyperlocomotion	[190, 194–197]
Opioids	Morphine	VL-PAG exhibits hyperexcitation of GABAergic neurons, astrocyte activation, and release of pro-inflammatory cytokines during opioid withdrawal	[194, 197, 201, 202]
Sedatives	Bicuculline or flumazenil (GABAA)	Intra D-PAG injection of benzodiazepines has anti-panic effects	[203]
Stimulants	Cocaine	Cocaine withdrawal produces hyper-responsiveness of VL-PAG neurons	[204]
	Cocaine	fMRI images show that PAG is involved in cocaine craving	[86]
Tobacco	Nicotinic acetylcholine receptors	Anti-depressive effects of nicotine involve the NAc-DR-PAG circuit; its anxiolytic effects include actions on the DR and PAG	[205–207]

Alcohol

Alcohol intake produces euphoria, disinhibition, anxiety-reduction, sedation, and hypnosis that are associated with its role as a positive allosteric modulator of the GABA_A receptor [165]. Interestingly, the alcohol hangover involves severe physical discomfort which includes anxiety, pain (mainly headache), and nausea [166]. It is popularly believed that the physical discomfort induced by the alcohol hangover decreases with low alcohol consumption on the next day [167]. In fact, the alcohol hangover may slightly promote a faster recurrence of alcohol consumption [81]. The above suggest that, during alcohol relapse in some consumers, escaping from the physical discomfort induced by withdrawal may be a promoting stimulus. Remarkably, there is increased anxiety after alcohol consumption that can be explained by a decrease in the inhibitory control of GABA over the PAG, accompanied by an increase in the excitatory glutamatergic tone [168–170]. Microinjections of NMDA (N-methyl-D-aspartate) or AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) antagonists into the D-PAG during withdrawal decrease alcohol consumption [169]. Likewise, in brain slices from animals in a state of alcohol withdrawal, the glutamatergic transmission mediated by NMDA or AMPA receptors is altered in the PAG, as spontaneous excitatory postsynaptic potentials are decreased compared to controls [171]. Bonassoli et al. [172] injected carboxy-PTIO [a nitric oxide (NO) scavenger] or L-NAME, a nonselective NO synthase inhibitor, into the D-PAG and both drugs decreased the anxiogenic effects of alcohol withdrawal, suggesting that the NO pathway plays an important role during this stage [172]. In addition, it has been reported that during alcohol withdrawal, the blockade of NPY-Y₁ receptors in the D-PAG induces anxiety and alcohol relapse [28]. Indeed, it has been reported that acute exposure to alcohol increases the activity of dopaminergic neurons in the VL-PAG, while chronic exposure to alcohol does not modify the activity of these neurons [173]. McClintick et al. [174] has suggested that early-age drinking of alcohol, specifically during adolescence, leads to changes in the expression of key genes in the PAG. They reported alterations in 1,670 of 12,123 detected genes in the PAG. The main decreased alterations caused by alcohol intake were in the GABAergic, serotoninergic, cholinergic, dopaminergic, and opioid systems, while the expression of hypocretin (orexin) neuropeptide precursor (Hcrt) increased in these neuronal populations [174]. Together, these changes may increase the susceptibility for developing anxiety, pain, and fear (i.e., physical discomfort) [174] which could facilitate compulsive alcohol intake.

Interestingly, alcohol intake is used by some patients as a pain reliever [175]. Egli et al. [176] have proposed a genetic and functional link between pathological pain and the development of alcohol dependence, as the neurocircuitry related to alcohol dependence is closely connected to the neurocircuitry for pain. For example, the PAG is not only involved in the direct spinothalamic processing of pain as well as in indirect processing via the amygdala tract [176]; but also, in the negative emotional states of drug addiction via its projections to the BNST and CeA (Fig. 1). In addition, Avegno et al. [155] reported that chronic alcohol exposure decreases GABAergic signaling from the CeA to the PAG and alters the melanocortin system in the CeA, phenomena that could be related to the hyperalgesia induced during alcohol withdrawal.

A recent fMRI study has suggested that the PAG is negatively involved in the physical and social pleasure expectancy in alcohol drinkers exposed to alcohol cues, whereas the medial orbitofrontal cortex (mOFC) is positively involved [177]. More recently, another fMRI study proposed that the connection between the mOFC and D-PAG is predominantly involved in alcohol abuse [88].

Hence, different neurotransmitters, neuropeptides, and gasotransmitters modulate the PAG during alcohol withdrawal, while alcohol exposure may induce functional, molecular, and genetic changes in the PAG, suggesting that this structure is a key modulator of alcohol intake behaviors. This idea has been recently confirmed using fMRI [88].

Caffeine

Caffeine is an extremely popular psychostimulant drug consumed all over the world in diverse presentations that include fresh or hot beverages, even mixed with cola-drinks and taurine. Exposure to caffeine increases DA in the NAc-shell [178], which explains its acute reinforcing effects. Caffeine also causes diastolic hypertension and exerts anxiogenic actions in humans [179]. It is popularly consumed early in the morning to generate a sensation of alertness (increased attention) and to avoid somnolence [180], actions that are associated with its anxiogenic effects. By fMRI, it has been shown that the anxiogenic action of caffeine involves activation of the PAG [179]. It is possible that this anxiogenic effect, induced via PAG activation, contributes to the desired effects in consumers (inhibition of somnolence and increased alertness and attention) [179]. However, it remains to be determined whether PAG inhibition also decreases caffeine intake.

Cannabis

Cannabinoids (phyto, endogenous, and synthetic) interact with type 1 (CB₁) and 2 (CB₂) cannabinoid receptors. Although the behavioral effects of cannabinoids have been classically associated with CB₁ receptors mainly expressed in the brain [181], diverse studies have reported a potential role for CB₂ [182]. Other putative receptors strongly associated with the endocannabinoid system (e.g., GPR12, GPR18, and GPR55) have also been associated with multiple cognitive processes such as learning and memory and food intake. [183–188].

Given that mainly glutamatergic and to a lesser extent GABAergic PAG projections to the VTA induce stimulation and DA release [94], substances that increase the activity of these neurons would be expected to trigger reinforcing actions through possible integration with the reward system. Interestingly, cannabinoids acting at an area that receives PAG inputs and projects PAG outputs (i.e., the tail of the VTA/rostromedial tegmental nucleus) may also facilitate CB₁-mediated DA release from the VTA [189] to the NAc [92]. Casarotto et al. [190] reported that CB₁ and transient receptor potential vanilloid 1 receptor (TRPV1; responsible for the painful sensation caused by capsaicin or heat) are expressed in the D-PAG and are frequently co-localized at the same synapses [190, 191]. In Casarotto’s study, stimulation of CB₁ in the D-PAG had panicolytic actions, whereas stimulation of TRPV1 had the opposite effect [190]. The above opposing effects may explain, at least partially, why some consumers report a subjective feeling of tranquility and others develop panic attacks [192, 193], and both actions may involve the cannabinoid targets CB₁ and TRPV1, respectively, in the PAG.

Exposure to stressful conditions or electrical stimulation of the PAG raises endocannabinoid levels [194, 195]. Moreover, CB₁ receptor agonist injection and increased anandamide in the D-PAG alleviate physical discomfort by analgesia [195, 196], anxiolysis, and anti-aversive actions [194]. Other studies have indicated that the CB₁ receptor agonist HU210, administered either systemically or specifically in the D-PAG, increases plasma corticosterone levels and prevents the hyperlocomotion induced by aversive stimuli [197]. CB₁ receptors seem to be involved only in the anti-hyperlocomotion action of HU210, while the increase in plasma corticosterone levels is not blocked by rimonabant (a non-selective CB₁ antagonist) [197]. Rimonabant per se increases corticosterone [197], suggesting a non-cannabinoid effect that remains to be elucidated. As GPR55 is a target for rimonabant [198] and is a receptor expressed in several areas (including the PAG) involved in learning, pain, and anxiety [183, 199, 200], its participation in the reward and anti-reward systems needs further analysis.

In summary, cannabinoids reach the PAG to produce analgesia, anxiolysis, fear inhibition, and anti-aversive affects (alleviation of physical discomfort). In addition, cannabinoids may modulate DA release in the NAc via activation of CB₁ receptors in areas with reciprocal connectivity with PAG that regulate VTA neurons. All these actions may influence both the impulsive and compulsive behaviors of drug addiction.

Opioids

As a key region in the pain circuit, the PAG is known for its role in mediating negative emotions [208]. The PAG is rich in opioid receptors and enkephalins [55]. Consequently, opioid addiction (e.g., morphine) critically involves enkephalins and the PAG area. In support of this notion, several studies have shown that: (i) there are several alterations in the PAG during morphine withdrawal, including hyperactivation of GABAergic neurons in the VL-PAG and astrocyte activation, and the release of pro-inflammatory cytokines such as TNFα [201, 202]; (ii) intra-PAG administration of an enkephalin analog suppresses the signs of morphine withdrawal [209]; (iii) intra-PAG infusion of morphine produces physical dependence after a naloxone challenge (an opioid receptor antagonist) [14]; and (iv) intra-PAG injection of morphine produces conditioned place preference [163]. Thus, the PAG is important in both the physical dependence and reinforcing actions of opioids. The needed dose of morphine to produce conditioned place preference via PAG injections was ten-fold higher (i.e., 5 µg vs. 0.2 µg, respectively) than via VTA [163]. This observation perhaps explains why in previous reports, animals did not exert self-injections into the PAG, but do into the VTA [14].

An extensively studied (and thus, not further discussed here) link between analgesia and addiction involves the opioid system [210–212]. Indeed, the great risk in prescribing opioid analgesics is the rapid development of addiction [210, 212]. Both analgesia mediated by opioids [149] and by placebo require opioid receptors in the PAG [213, 214]. Intriguingly, morphine withdrawal produces extreme anxiety accompanied by a plethora of physically uncomfortable disturbances, phenomena linked to PAG sensitization [215]. It has been classically reported that painful stressors are strong factors in relapsing opioid-related addiction [216]. Moreover, chronic use of opioids drives hyperalgesia and negative emotional states which have been proposed to contribute to the compulsive phase of drug addiction [84].

Hence, PAG is a key region highly altered during opioid withdrawal and may be responsible for some of the compulsive actions in the drug addiction. But also, simple intra-PAG injections of opioids (e.g., morphine) produce reinforcing actions (e.g., conditioning place preference).

Sedatives, Hypnotics, or Anxiolytics

Electrical stimulation of D-PAG evokes fear, panic attacks, intrusive ideas about death, among other negative feelings in humans [37]. Notoriously, tThe panicolytic, but not the anxiolytic action of alprazolam (a benzodiazepine) is prevented by intra-D-PAG injection of bicuculline or flumazenil, suggesting the participation of GABA_A receptors in this brain structure [203]. Moreover, a serotoninergic circuit integrating the DR nucleus and VL-PAG, and controlled by the BLA, has been proposed as a natural anti-panic system that is facilitated by chronic antidepressants [217]. Therefore, part of the alleviative actions of this kind of compounds may be directly and/or indirectly associated with its actions in the PAG.

Stimulants

Cocaine is a non-selective inhibitor of monoamine transporters; thus, this drug potentiates monoaminergic transmission (DA, serotonin, and norepinephrine) and increases DA levels in the NAc [218] and amygdala [219, 220]. Recently, Li et al. [149] characterized the DA/glutamate neurons in the VL-PAG that are involved in pain integration and opiate anti-nociception. The reciprocal PAG-EA connections [138, 139, 221] are dopaminergic and could be involved in the expression of withdrawal/negative affect symptoms of methamphetamine [222].

It is interesting to consider that social stress seems to strongly promote cocaine intake [223, 224]. In fact, stress induced by social defeat or cocaine intake produces Fos-like immunoreactive (Fos-LI) augments in PAG, DR and locus coeruleus [223]. These changes may be related to cocaine sensitization [224]. Prolonged use of cocaine attenuates the dopamine efflux in the NAc and promotes dose-dependent anxiety and anhedonia [204, 225]. Moreover, after a cocaine binge, the PAG neurons are hyper-responsive to tactile stimulation in rats [204]. In humans, Zhang et al. [85] reported alterations in functional connectivity between the PAG, hypothalamus, and D-mPFC in cocaine-dependent subjects compared to healthy controls. Furthermore, a higher activation of the PAG and connectivity of the PAG with the vmPFC was detected been reported during exposure to cocaine cues [86].

The PAG-vmPFC connectivity is correlated with tonic cocaine craving and biological sex differences have been confirmed by a slope test [86]. While PAG-vmPFC connectivity reflects tonic cocaine craving in men, the ventromedial PAG appears to play a role in influencing the PAG response to alleviate cocaine craving in women. These suggest that PAG may be a key structure involved in cocaine withdrawal and cocaine craving [86].

Self-administration of other stimulants such as methamphetamine produce biochemical adaptations in DA neurons in the PAG, but when methamphetamine-seeking is combined with exercise (e.g., wheel-running) there is a reduction in the biochemical alterations in the PAG and in the methamphetamine-seeking, suggesting that exercise plays a neuroprotective role via the prevention of PAG alterations [222]. For stimulants, the PAG seems to be involved in substance-seeking, while biochemical changes in DA neurons in the PAG may contribute to the consolidation of the drug addiction cycle.

Tobacco

Nicotine interacts with high affinity with the α4 subunit [226] of the nicotinic acetylcholine receptor (nAChR), which is ubiquitously expressed in the brain [227]. nAChRs are expressed in the mesocorticolimbic dopaminergic system, which is mainly implicated in the reinforcing action of tobacco consumption [228, 229]. The background of nicotine addiction has several components, including pharmacological, genetic, and environmental factors [230]. Pharmacologically, nicotine interacts as an agonist for the different subunits of nAChRs [226]. The nAChRs consist of an assembled complex with five subunits selected from nine α subunits (α2 to α10) and three β subunits (β2 to β4) [230].

Different combinations of these subunits produce the different subtypes of nAChR, the α4β2 subtype being the main receptor that mediates nicotine addiction [231, 232]. Activation of nAChRs modulates synaptic transmission, since it increases the probability of release of neurotransmitters, including DA, GABA, glycine, glutamate, norepinephrine, and ACh [233–236]. In fact, activation of nAChRs in midbrain DA neurons mediates DA release in the NAc shell [237] which explains (at least in part) the reinforcing actions of nicotine [238]. The ACh that endogenously regulates midbrain DA is released from the mesopontine nuclei in a circuit that involves PAG neurons [238]. The PAG receives cholinergic inputs from the pontine tegmentum [239] and they contribute to maintaining the tonic activity of its GABAergic neurons [233]. On the other hand, α7 nAChRs have been reported to be expressed in VL-PAG cholinergic neurons that project to the RVM [240, 241]. Activation of these cholinergic projections is involved in pain inhibition [233]. Interestingly, the VL-PAG neurons are also involved in opioid-mediated analgesia [62], which is mediated via inhibition of GABAergic neurons in the PAG [242]. The above dichotomy strongly supports the integrative role of the PAG in the physiology of pain and analgesia via stimulation and inhibition of different nuclei by its cholinergic and GABAergic projections, respectively [233, 241].

Some emotions, such as fear and anxiety induce functional changes in the PAG [32, 243]. Likewise, depression can modify the genetic profile of the VL-PAG via mechanisms that remain to be elucidated [244]. Interestingly, nicotine has been shown to have antidepressant effects in several animal models [245–248]. The link between the antidepressant effects induced by nicotine and the nAChRs in the PAG is unknown. However, depression is a sign of nicotine withdrawal [249]. A plausible explanation of this link may be the actions mediated by the important cholinergic control of serotoninergic neurons from the DR to the NAc [205]. It is important to note that DR neurons are under dual modulation by PAG DA/glutamate neurons with repercussions for anxiety and analgesia [207]. Moreover, nicotine induces anxiolytic effects via DR neurons [206].

Although the participation of the PAG in the reinforcing mechanisms of nicotine are obscure, like other main pharmacological classes of addictive drugs, its direct and/or indirect actions in the PAG drives pain inhibition and anxiolysis, which contribute to the alleviation of physical discomfort induced by several conditions including nicotine withdrawal.

PAG involvement in appetitive behaviors and food intake

PAG activation promotes food intake and reward processing [159], as it has extensive reciprocal connections with brain circuits that mediate appetitive processes and consummatory behaviors (prefrontal cortex, LH, BNST, amygdala, parabrachial nucleus, VTA, and DR) [29, 136, 250, 251]. The food reward depends on the AL, DL, and VL PAG [136, 164]. It has been reported that these columns of the PAG contain synaptic terminals that release relaxin and oxytocin [252], peptides with a modulatory effect on feeding [253, 254]. There is a reciprocal relationship between the DR and the PAG since they play roles in multiple behaviors such as food consumption, anxiety, withdrawal, and depression [29]. On the other hand, it has been shown that the suppression of GABAergic neuronal activity in the VL-PAG, either directly or by long-projection GABAergic inputs from the BNST or LH, is sufficient to induce feeding behavior and, on the contrary, activation of these cells suppresses this behavior [136].

A link between drug-seeking and food-seeking has been reported [255, 256]. DA, which is essential for activation of the reward system, is also key for activating the "motivation system" [255, 256]. An imbalance in the dopaminergic system has been associated with both the compulsion for a drug and the compulsion to eat food [256, 257]. Interestingly, both food intake and reward/aversion processes are under fine modulation by the endocannabinoid system and putative cannabinoid receptors [186, 187, 258–260]. Under these circumstances, the PAG might serve and participate as an interface between the endogenous opioid system and the hedonic aspects of food reward [261–264]. Actually, the PAG is required for the normal consumption of food [29, 136] and its participation in appetitive and consummatory behaviors was recently discussed by Silva and McNaughton in an excellent critical review [29]. Our view completely agrees with Silva and McNaughton about the key role of the PAG in motivated behaviors and we support their proposal with the inclusion of the drug addiction cycle among the phenomena in which the PAG is involved.

Conclusions

Especially due to its small size, anatomical location, and its indirect participation in multiple kinds of motivated behaviors, the contribution of the PAG in the consolidation of drug addiction remains elusive and underestimated. We propose that, mainly by promoting the alleviation of physical discomfort (mainly pain, anxiety, and fear) linked to substance-withdrawal, the PAG may contribute to the mechanisms underlying relapse (compulsive intake). However, the participation of the PAG in the initial phase (impulsive intake) of drug intake cannot be excluded and requires further investigation.

Conflict of interest

The authors declare no conflicts of interest.