Glial Neuroimmune Signaling in Opioid Reward

Abstract

The opioid epidemic is a growing public concern affecting millions of people worldwide. Opioid-induced reward is the initial and key process leading to opioid abuse and addiction. Therefore, a better understanding of opioid reward may be helpful in developing a treatment for opioid addiction. Emerging evidence suggests that glial cells, particularly microglia and astrocytes, play an essential role in modulating opioid reward. Indeed, glial cells and their associated immune signaling actively regulate neural activity and plasticity, and directly modulate opioid-induced rewarding behaviors. In this review, we describe the neuroimmune mechanisms of how glial cells affect synaptic transmission and plasticity as well as how opioids can activate glial cells affecting the glial-neuronal interaction. Last, we summarize current attempts of applying glial modulators in treating opioid reward.

1. Introduction

Opioids are among the most widely prescribed analgesics worldwide. From 2010 to 2012, 12,305 daily defined doses (DDDs) of opioids per million population were dispensed in the western European countries (van Amsterdam and van den Brink, 2015). In Canada, this number is almost tripled – approximate 30,000 DDDs per million population. Compared to all other countries, United States has the highest rate of opioid use during the same period, in which 51,873 DDDs per million were prescribed. Yet, as opioids possess a strong abuse potential due to their rewarding activity (Fields and Margolis, 2015), this high opioid prescription rate results in a wide range of Prescription opioid misuse and related harms, particularly in the United States. In 2017 alone, approximately 11 million Americans have misused opioids, over 2 million people have opioid use disorders and nearly 42,000 people died from overdose of prescription opioids, leading to a widespread opioid epidemic (Substance Abuse and Mental Health Services Administration, 2018; Seth et al., 2018). To solve this severe crisis, a better understanding of the pathophysiology behind opioid addiction must be achieved.

As the initial but critical step leading to opioid addiction, opioid-induced rewarding behavior is one of the main focuses for a considerable amount of preclinical and clinical studies. Classically, opioid reward is considered to be induced by modulation of neuronal transmission and plasticity (Fields and Margolis, 2015). However, accumulating evidence indicates that non-neuronal glial mechanisms and the associated neuroimmune signaling significantly contribute to this process (Bachtell et al., 2017; Lacagnina et al., 2017; Linker et al., 2019). In this review, we will introduce the current understanding of the mechanisms underlying opioid reward, present an overview of glial physiology, discuss opioid-induced neuroimmune signaling in the central nerve system (CNS) and their contribution to opioid reward. Finally, the clinical implications of regulating neuroimmune signaling to treat opioid abuse will be reviewed.

2. Mechanisms of opioid-induced reward

2.1. Neuronal mechanisms of opioid reward

Reward is a CNS process that is induced when the natural drives of an individual, such as the desire to eat, drink or mate has been satisfied (Fields, 2018). From an evolutionary perspective, this feeling of satisfaction guides individuals to perform specific behaviors continually that benefit their survival and reproduction (Fields, 2018).

Dopamine-dependent mechanism:

Current studies identified that ventral tegmental area (VTA) and nucleus accumbens (NAc) are two key brain loci belonging to the mesolimbic circuitry that play a critical role in processing reward-associated stimuli, such as food, water, sex and social dominance (Volkow and Morales, 2015). The dopaminergic neurons located in the VTA are thought to directly encode reward or a reward prediction signal by producing a rapid, phasic dopamine release in NAc (Volkow and Morales, 2015). However, the activity of these dopaminergic neurons is physiologically suppressed by tonic GABA input from VTA GABAergic neurons and other GABAergic neurons from the rostromedial tegmental nucleus (RMTg), NAc and ventral pallidum (Fields and Margolis, 2015; Volkow and Morales, 2015). Opioids suppress this GABAergic tone by either direct, hyperpolarization of neurons or inhibition of neurotransmitter release, which eventually disinhibits the activity of dopaminergic neurons and allows for dopamine release (Fields and Margolis, 2015). This process is commonly thought, at least partially, as the neural pathway controlling opioid-induced reward.

Dopamine-independent mechanism:

In addition to the dopamine-dependent pathway, some studies also suggest the presence of a non-dopaminergic neuronal pathway that mediates opioid reward. Initial evidence includes a study showing that dopamine-deficient mice still maintained morphine-induced conditioned place preference (CPP) (Hnasko et al., 2005). Likewise, the administration of a dopamine receptor antagonist α-flupenthixol or the ablation of dopaminergic terminals in NAc did not affect heroin self-administration in rats (Ettenberg et al., 1982; Nader and van der Kooy, 1997; Pettit et al., 1984). These studies indicate the presence of a dopamine-independent pathway in opioid-induced reward and/or additional non-mesolimbic pathway(s). Further studies show that the function of this non-dopaminergic reward pathway is state-dependent, and it only mediates the opioid reward when animals are in an opioid-naïve or opioid-dependent but not withdrawal states through the tegmental pedunculopontine nucleus (TPP) located in pons (Nader and van der Kooy, 1997). Once the animals are in a withdrawal state after chronic exposure to opioids, the rewarding effect of opioids will shift and are dependent on the mesolimbic dopamine system (Laviolette et al., 2002; Nader and van der Kooy, 1997).

2.2. Evidence of non-neuronal actions that contributes to opioid reward

Although opioid-induced neuronal adaptations remain the major focus of current studies in opioid reward, accumulating evidence indicates that the non-neuronal glial cells, primarily microglia and astrocytes, and their associated immune signaling are also responsible for the rewarding effect produced by opioids. The first piece of evidence supporting this view was obtained by Narita, et al, who found that morphine-mediated CPP was enhanced by intra-NAc injection of astrocyte-conditioned medium (ACM) (Narita et al., 2006). Similarly, Arezoomandan et al. also found that administration of ACM in NAc facilitated maintenance and reinstatement of morphine CPP (Arezoomandan et al., 2016). In contrast, the application of glial activation inhibitors in animals significantly attenuated the acquisition and maintenance of morphine-induced CPP as well as dopamine release in NAc (Bland et al., 2009; Hutchinson et al., 2008; Narita et al., 2006; Zhang et al., 2012). Additionally, blocking activity of a pro-inflammatory cytokine IL-1β or promoting the expression of an anti-inflammatory cytokine IL-10 in the NAc of opioid-treated animals can effectively reduce morphine CPP as well (Lim et al., 2014; Schwarz et al., 2011). These studies demonstrated that glial cells and their mediated immune responses can directly affect the rewarding behaviors in opioid-treated animals, indicating the presence of a non-neuronal mechanism in the development of opioid reward. In the following sections of this review, we will focus on discussing how opioids influence glial cell activity and the potential mechanisms that glia and their associated neuroimmune signaling may have on regulating opioid-induced reward.

3. Basic physiology of glial cells

3.1. Microglia

Microglia are the major central immune cells comprising 10% of the total CNS population (Salter and Stevens, 2017). They are historically assumed to be derived from myeloid/mesenchymal progenitor cells or fetal macrophages that migrate into CNS during postnatal development (Kettenmann et al., 2011). Recently, definitive evidence suggests that they solely originate from yolk-sac progenitors that migrate to the developing CNS at embryonic day 8.5 in mice and at the 3rd gestational week in humans (Gomez-Nicola and Perry, 2015; Salter and Stevens, 2017). In a healthy mature CNS, microglia are in a ramified morphology and actively survey the local environment with their fine processes on the cell surface (Kettenmann et al., 2011). When danger signals are identified, such as bacteria, virus and other xenobiotics, microglia rapidly undergo a morphological change by reducing the complexity of the cellular processes and reverting to an amoeboid appearance (Kettenmann et al., 2011). At the same time, microglia also experience a profound change in their gene expression patterns and functional behaviors. This process was previously considered as a monolithic state, termed “microglial activation”. However, this idea was later identified to be oversimplified and does not reflect the functional plasticity of microglia. Today, studies with novel techniques examining the transcriptomes of microglia show that “microglia activation” is a highly dynamic process with quite distinct characteristics in different pathological conditions, which has been thoroughly described in several reviews (Gomez-Nicola and Perry, 2015; Ransohoff, 2016; Salter and Stevens, 2017).

3.2. Astrocytes

Astrocytes are specialized glial cells with star-shaped processes extending from their cell soma (Ponath et al., 2018). As the most abundant glial cells in CNS, they make up about 30% of the whole glial population and outnumber neurons by over 5-fold (Ponath et al., 2018; Sofroniew and Vinters, 2010). Astrocytes were initially thought of as the supportive cells providing inert scaffold for the distribution and communication of neurons (Sofroniew and Vinters, 2010). However, as the studies advanced, the concept of astrocytes has changed dramatically especially in the past thirty years. Currently, we know that astrocytes participate in a large variety of complicated and important functions in the brain, including regulating blood flow, energy supply and metabolism, maintaining the homeostasis of fluid, ions, pH, and transmitter, organizing and maintain the blood brain barrier, and reuptake of neurotransmitters (Sofroniew and Vinters, 2010).

Like microglia, astrocytes are also immune-competent cells and are capable of inducing neuroinflammation in CNS (Tian et al., 2012). When stimulated by chemical or electrical signals that may represent damage or danger, astrocytes undergo a series of structural and functional changes known as reactive astrogliosis, resulting in the secretion of diverse cytokines, chemokines and neurotrophic factors (Sofroniew and Vinters, 2010; Tian et al., 2012). These released factors could promote the leakage of the blood brain barrier and recruit immune cells to the impaired spots, mediating the elimination of injurious or diseased insults. Reactive astrocytes themselves can have enhanced functions in scavenging excessive glutamate, free radicals and ammonia, protecting CNS cells and tissues from cytotoxic materials (Sofroniew and Vinters, 2010). In some cases when the CNS is suffering from severe damage or infection, reactive astrocytes can induce the formation of glial scars, which limit the spread of inflammatory cells or infectious agents into healthy CNS areas (Sofroniew and Vinters, 2010).

4. Effects of opioids on glial cell activation

4.1. Opioids influence glial activation

As a class of xenobiotics that are derived from the plant opium or are synthesized chemically, opioids can directly stimulate glial cells and lead to their functional conversion. Previous studies showed that morphine exposure increases the expression of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) in rodents, both of which are the activation markers of astrocyte and microglia, respectively (Beitner-Johnson et al., 1993; García-Pérez et al., 2016, 2014; Goins and Bajic, 2018; Hutchinson et al., 2009; Marie-Claire et al., 2004; Zhang et al., 2012). Morphine also promotes the brain distribution of [¹⁸F]DPA-714, a PET biomarker of glial activation in non-human primates (Auvity et al., 2017). In addition, morphine administration alters the gene expression profiling in glial cells and promotes their release of various cytokines and chemokines, including IFN-γ, IL1-β, IL-6, IL-10, CCL4 and CCL17 (García-Pérez et al., 2016, 2014; Hutchinson et al., 2009; Schwarz et al., 2011; Suder et al., 2009; Zhang et al., 2017).

4.2. Mechanisms underlying opioid-induced glial activation

Opioids activate glial cells by binding to the different receptors expressed on the cell surface. A class of receptors that may mediate this response is the opioid receptors. This receptor family currently includes four members - μ, δ, κ and nociceptin receptors, all of which are heterotrimeric Gi/o protein-coupled receptors (GPCRs) (Corbett et al., 2006). However, only μ opioid receptors (MOR) were thought to associate with the rewarding effect of opioid drugs. Previous studies have shown that all these opioid receptors were found to be expressed by both microglia and astrocytes (Chao et al., 1996; Dobrenis et al., 1995; Eriksson et al., 1992; Fu et al., 2007; Gurwell et al., 1996; Mduna et al., 2018; Meyer et al., 2017; Mika et al., 2014; Nam et al., 2018; Shrivastava et al., 2017; Woo et al., 2018). Surprisingly, very few studies have explored if the activation of these opioid receptors may stimulate glial cell activities or not. One possible reason for this lack of investigation is the inconsistent view about whether opioid receptors are truly expressed on glial cells since some reports, in contrast, show that no opioid receptors are expressed on glial cells (Corder et al., 2017; Kao et al., 2012). Another possible reason may be due to the traditional notion that the activation of opioid receptors is immunosuppressive, which is opposite to the observation of opioid-mediated glial activation (Y. Liang et al., 2016). This view is supported by several studies demonstrating the activation of opioid receptors can functionally inhibit glial cells (Chao et al., 1997; Hansson et al., 2008; Hu et al., 2000, 1998; Stiene-Martin and Hauser, 1993, 1990). Nevertheless, other studies indicate that opioid receptors exert stimulatory effects on the immune system (X. Liang et al., 2016; Muscoli et al., 2010). In addition, a recent study found that the activation of astrocytic MOR (possibly via Gi/o protein-mediated signaling) induced glutamate release from astrocytes in hippocampus and resulted in CPP, demonstrating the stimulatory role of Gi/o protein-mediated signaling by MOR and other GPCRs in immune response (Nam et al., 2019). Although the mechanisms of this action have not been clarified, it could relate to the distinct downstream signaling pathways coupled with opioid receptors under different physiological and pathological conditions (Al-Hasani and Bruchas, 2011).

Recently, increasing evidence suggests that Toll-like receptor 4 (TLR4) is a potential site for opioid triggering an innate immune response. TLR4 is a member of the pattern recognition receptors (PRRs) that have evolved to detect components of foreign pathogens with conserved structures (Kawai and Akira, 2010). TLR4 itself was found to selectively recognize and respond to bacterial lipopolysaccharide (LPS), a component in the outer membrane of Gram-negative bacteria. Because of its critical role in innate immunity, TLR4 is primarily and widely expressed by cells of innate immune system, including microglia and astrocytes (Vaure and Liu, 2014). On the cell surface, TLR4 forms a complex with the Myeloid differentiation factor 2 (MD2), and together they work as the primary binding site for LPS (Kawai and Akira, 2010) (Fig. 1). Opioids were recently identified as another class of substances that can bind to and activate TLR4. In silico docking simulations predicted that opioids occupy the same binding site as LPS on TLR4-MD2 complex (Hutchinson et al., 2012; M R Hutchinson et al., 2010; Mark R. Hutchinson et al., 2010), which was later verified by a series of in vitro assays (Mark R. Hutchinson et al., 2010; Wang et al., 2012). Opioid administration can stimulate the mitogen-activated protein kinases (MAPKs) signaling downstream to TLR4, including p38, JNK and ERK, while genetic depletion of TLR4 blocks this effect (Wang et al., 2012; Zhang et al., 2012) (Fig. 1). Additionally, opioid-induced TLR4 activation also promote the production of activation markers and pro-inflammatory mediators in glial cells, all of which can be suppressed by the treatment of glial activation inhibitors or TLR4 selective antagonists (Eidson and Murphy, 2013; M R Hutchinson et al., 2010; Y. Liang et al., 2016; Wang et al., 2012; Zhang et al., 2012). It is worth noting that the abilities to bind and activate TLR4 are also present in morphine-3-glucuronide (M3G), a morphine metabolite with no opioid receptor activity, while the opioid receptor active metabolite, morphine-6-glucuronide (M6G), lacks such properties (Due et al., 2012; Mark R. Hutchinson et al., 2010; Lewis et al., 2010). This provides further evidence that the opioid effect of inducing central immune signaling cannot be attributed to the actions of classical opioid receptors.

Fig. 1.

Molecular mechanism underlying opioid-induced glial activation. Opioids can activate glial cells by direct actions on the classical opioid receptor, MOR, and the innate immune receptor, TLR4. The activation of TLR4 by opioids induces the activity of intracellular MAPKs, which regulate the production of glial activation markers and pro-inflammatory mediators. Opioid-induced glial activation also involves other receptors, including TLR2 and P2X4R as well as intracellular signaling molecules, JAK/STAT and HDAC6/HSP90.

In addition to opioid receptors and TLR4, there are other receptors and signaling molecules that have been postulated as being involved in opioid-induced glial activation (Fig. 1). TLR2, another member of toll-like receptors, was identified to facilitate chronic morphine-induced expression of CD11b, TNFα, IFNγ and IL6 in mouse NAc (Zhang et al., 2011). P2X4 receptor, an ATP-gated cation channel, was shown to enhance chronic morphine-induced Iba1 and GFAP expression and can be blocked by the selective antisense oligonucleotide of P2X4 receptors (Horvath et al., 2010). JAK/STAT and HDAC6/HSP90 signaling pathways when activated individually can also mediate morphine-induced glial activation (Chen et al., 2017; Narita et al., 2006; Tsai et al., 2015). However, whether these receptors and signaling pathways that directly interact with opioids is unclear. Further research is required to characterize their roles in opioid-induced glial activation.

5. Glial neuroimmune signaling in the modulation of opioid reward

As described previously, glial cells when activated by opioids will alter their gene expression profiling and increase the production of multiple pro-inflammatory cytokines and chemokines, which contribute to opioid reward. Based on this observation, one may ask how these glial-derived immune-related molecules influence the neuronal transmission and plasticity mediating the perceptions of opioid reward. Although many studies have demonstrated that glial-derived immune factors can modulate synaptic transmission and plasticity, only a few of them have directly associated these actions with opioid-induced reward. As synaptic plasticity was shown to make significant contribution to addictive behaviors by opioids (Langlois and Nugent, 2017), those immune factors with the abilities to regulate synaptic functions may also participate in the development of opioid reward. Therefore, in this review we discuss the glial-derived immune factors that have been demonstrated or have the potentials to regulate synaptic transmission and plasticity in order to have a comprehensive understanding of glial neuroimmune signaling in opioid reward.

5.1. Microglia

Microglia are the primary focus of current studies in the modulation of synaptic regulation via neuroimmune signaling. Previously we described that the pattern recognition receptor TLR4 expressed on microglia is a potential target of opioids and contributes to opioid-induced glial activation. Based on this observation, we could expect that the manipulation of TLR4 would modulate opioid-induced reward behaviors. Indeed, genetic and pharmacological blockade of TLR4 impaired the development of opioid-induced DA release in NAc, conditioned place preference and self-administration (Chen et al., 2017; Hutchinson et al., 2012). Further studies found that TLR4 modulates the rewarding effect of opioids via a MyD88-dependent pathway (Fig. 1) as evidence by oxycodone-induced CPP being reduced in MyD88 knockout mice (Hutchinson et al., 2012). Similarly, intra-NAc administration of a p38 inhibitor, SB203580, effectively suppressed the acquisition and maintenance of morphine-induced CPP (Zhang et al., 2012). Furthermore, the depletion of TLR4 significantly prevented the phosphorylation of p38 and JNK, which are the downstream transducers of MyD88-dependent pathway (Hutchinson et al., 2012). Interestingly, a recent study found that selective knockout of MyD88 in microglia did not change the acquisition of morphine but prolonged the extinction of the reward memory and enhanced reinstatement in mice, which contradicts the results from whole-body knockouts of TLR4 and MyD88 (Rivera et al., 2019). This result may suggest that MyD88 plays distinct roles in microglia and other cell types and highlights the importance of acquiring a higher degree of cell specificity in the research in order to know the actual effects of glial-derived TLR4 and its associated signaling in opioid reward.

Although the exact role of microglial-derived TLR4 in opioid reward remains to be investigated, it is definitive that microglial-derived TLR4 signaling affects opioid reward processing (Jacobsen et al., 2014). Current studies believe that this influence of TLR4 is partially mediated by the proinflammatory cytokine TNFα. TNFα is a final product of MyD88 pathway and the sequestration of soluble TNF prevents TLR4-mediated morphine-induced neuroinflammation (Eidson et al., 2017; Kawai and Akira, 2010). TNFα can regulate the homeostasis of synaptic transmission by altering the trafficking of GABA_A receptors and Ca²⁺-permeable AMPA receptors (Fig. 2A) (Lewitus et al., 2014; Stellwagen et al., 2005; Stellwagen and Malenka, 2006). TNFα was also found to regulate synaptic transmission presynaptically by triggering astroglial release of glutamate via TNF receptor I and then activating presynaptic metabotropic glutamate receptors (Bezzi et al., 2001; Domercq et al., 2006; Pascual et al., 2012) (Fig. 2A). Previous evidence suggests that TNFα may be involved in the modulation of rewarding properties of opioids since it may contribute to the alteration of opioid sensitivity in humans revealed by a genetic association study (Reyes-Gibby et al., 2008). Additionally, TNFα was also shown to affect the conditioned behaviors induced by opioids (Niwa et al., 2007). However, in contrast to our previous discussion that glial activation promotes opioid reward, the presence of TNFα significantly suppressed morphine CPP while the genetic depletion of TNFα facilitated this process (Niwa et al., 2007). The exact reasons for these results are unclear but may be partially explained by the sickness behaviors induced by TNFα (Palin et al., 2007). It is also possible that TNFα may modulate the reward circuitry in a distinct way compared to other pro-inflammatory cytokines and chemokines although further studies are required to support this idea.

Fig. 2.

Glial neuroimmune signaling modulates synaptic transmission and plasticity. (A) Synaptic strength is regulated by TNFα secreted from microglia. Microglial TNFα activates TNFRI on neurons and regulates homeostasis of the surface expression of AMPA receptors and GABA_A receptors. Microglial TNFα also promotes the release of astrocytic glutamate, which activates pre-synaptic metabotropic glutamate receptors and modifies synaptic transmission. Glu, glutamate. (B) Microglia regulate the activity of GABAergic neurons via BDNF. When activated by opioids, microglia can secrete BDNF, which suppresses the expression of a K⁺-Cl⁻ co-transporter KCC2. KCC2 downregulation leads to the accumulation of Cl⁻ in the cytoplasm, shifting the inhibitory signaling produced by the GABA_A receptors to an excitatory one. (C) Glia regulate synaptic elimination. Microglia, as the phagocytic cells in CNS, are recruited to the unnecessary synapses by the “find-me” signal, CX3CL1, secreted from neurons. When approaching synapses, microglia recognize the “eat-me” signal C1q and C3 on the surface of synapses and perform engulfment. The expression of C1q on the synapse can be promoted by the TGFβ released by astrocytes.

Interleukin-1β (IL-1β) is another proinflammatory cytokine that is thought to mediate TLR4-induced alteration in neuronal plasticity. Like TNFα, IL-1β is also a product of TLR4 signaling since its production requires the activation of TLR4 to prime the inflammasome and increase pro-IL-1β expression (Hutchinson et al., 2011; Latz et al., 2013). In the CNS, IL-1β is considered the primary immune player in regulation of long-term potentiation (LTP) - a cellular process that is thought to underlie learning and memory (Rizzo et al., 2018). Indeed, both pharmacological and genetic inhibition of IL-1β or its corresponding receptor IL-1R significantly altered the induction and maintenance of LTP (Avital et al., 2003; Coogan et al., 1999; Goshen et al., 2007; Schneider et al., 1998). A possible mechanism for this modulation may involve he alterations of NMDA receptor conductance by the phosphorylation of receptor subunits (Viviani et al., 2003). IL-1β was found to play a role in the perceptions of opioid reward. A previous study showed that central administration of IL-1β increased dopamine levels in rat midbrain (Song et al., 2006). In addition, the single nucleotide polymorphisms in IL1B gene, which increased IL-1β production, were found to be associated with an increased risk of opioid dependence in humans (Liu et al., 2009). Furthermore, systemic administration of IL-1β was shown to modulate morphine CPP in a bidirectional manner (Hymel et al., 2016). Interestingly, some studies revealed that opioid addiction shares some similar characteristics with chronic pain, indicating the common neuroadaptations between these two disorders (Elman and Borsook, 2016). A recent study reported that opioid use may mediate the initiation and maintenance of a chronic pain state via stimulating the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome-induced IL-1β release (Grace et al., 2016). This may suggest a role of NLRP3 inflammasome in the modulation of opioid reward by IL-1β but further studies are required to validate this idea.

Currently, brain-derived neurotrophic factor (BDNF) is the best-characterized microglial-derived immune factor affecting opioid reward. BDNF is a member of the neurotrophin protein family that plays an essential role in the regulation of synaptic plasticity in the CNS (Sasi et al., 2017). Recent studies found that the polymorphisms of BDNF are associated with age of onset of substance abuse in heroin-dependent patients, suggesting a role of BDNF in opioid dependence (Cheng et al., 2005; Haerian, 2013; Jia et al., 2011; Meng et al., 2012). In the previous chapter, we discussed that chronic administration of opioids shifts the production of opioid rewarding effect from dopamine-independent system to the mesolimbic dopamine system. Studies show that this process is probably mediated by BDNF (Taylor et al., 2016; Vargas-Perez et al., 2009) (Fig. 2B). Taylor et al found that chronic opioid exposure activates the microglia and promotes them to release BDNF in VTA (Taylor et al., 2016). This upregulation of BDNF decreases the expression of the K⁺-Cl⁻ co-transporter KCC2 on the VTA GABAergic neurons, altering the transmembrane Cl⁻ concentration in VTA. As a result, the conductance property of the GABA_A receptors on VTA GABAergic neurons is transformed and shifts the inhibitory signaling of these GABAergic neurons to an excitatory one, leading to the activation of dopamine-dependent pathway (Vargas-Perez et al., 2009). In consistent with this finding, the authors directly infused a single dose of BDNF into the VTA of naive animals, which recapitulated the similar transformation exhibited in opioid-dependent and withdrawn animals (Vargas-Perez et al., 2009). This further demonstrates the critical role of microglia-derived BDNF in the regulation of opioid reward.

In addition to synaptic transmission, microglial-derived immune factors can regulate neuronal activities via synaptic elimination. Synaptic elimination is a process of brain development that selectively removes unnecessary synaptic connections to refine circuits and connections in the nervous system (Wilton et al., 2019). Recent studies reported that this process also occurs after opioid treatment, suggesting a potential role in the development of opioid addiction (Dong, 2016; Graziane et al., 2016). Synaptic elimination was identified to be mediated by the CX3CL1/CX3CR1 signaling pathway. CX3CL1 is a chemokine expressed by neurons, while its corresponding receptor CX3CR1 is exclusively expressed on microglia (Harrison et al., 1998). Studies found that genetic deletion of CX3CR1 resulted in a transient reduction of microglia number as well as decreased entry of microglia into the developing brain regions (Hoshiko et al., 2012; Paolicelli et al., 2011). Simultaneously, the densities of dendritic spines and immature synapses were significantly increased in these animals, but the functional maturation of synapses declined. These results indicate that CX3CL1/CX3CR1 axis works as a “find-me” pathway for microglia to locate unnecessary synapses on neurons during CNS development (Wu et al., 2015) (Fig. 2C).

Complement proteins can also regulate the synaptic elimination. Complement proteins belong to the complement system of the innate immune system, which was originally discovered to facilitate immune cells to clean pathogens and damaged cells in the periphery (Sarma and Ward, 2011). In the developing CNS and retina, the complement protein C1q is widely expressed on developing synapses (Bialas and Stevens, 2013; Schafer et al., 2012; Stevens et al., 2007). Genetic depletion of this protein was found to impair synaptic refinement and neural development in mice (Bialas and Stevens, 2013; Ma et al., 2013; Stevens et al., 2007). Similar phenomena were also observed in the mice lacking the expression of complement protein C3 (Schafer et al., 2012; Stevens et al., 2007). These results indicate that complement proteins serve as “eat-me” signals for unwanted synapses (Fig. 2C). Microglia recognize these “eat-me” signals via the complement receptor 3 (CR3, also named CD11b) on the surface, as the knockout of CR3 significantly decreases the phagocytic capacity of microglia to engulf synaptic inputs (Schafer et al., 2012).

5.2. Astrocytes

Like microglia, astrocytes are also active participants in modulating neuronal transmission and plasticity (Haydon et al., 2009) although relatively little is known regarding the role of astrocyte-associated immune signaling in synaptic regulation mediating opioid reward. Recently, glucocorticoid receptors (GRs) expressed by astrocytes were identified in regulating synaptic transmission that influences the rewarding effect of opioids (Skupio et al., 2019). In this study, the authors found that selective knockdown of astrocytic GR in NAc significantly enhanced sensitivity to morphine reward in mice. Further investigation showed that GR knockdown significantly inhibited dexamethasone-induced glucose uptake and lactate release in astrocytes (Skupio et al., 2019). As previous studies have demonstrated that lactate can potentiate neuronal excitability (Sada et al., 2015; Suzuki et al., 2011; Yang et al., 2014), the astrocytic release of lactate after GR activation may mediate the modulatory effect on morphine reward. Consistent with this idea, a recent study has shown that systemic administration of lactate significantly suppressed morphine-induced place preference in astrocytic GR-knockdown mice (Skupio et al., 2019). Notably, this astroglial GR-dependent regulation only affects the behavioral responses to morphine but not cocaine, suggesting astrocytic GR-dependent signaling as a specific regulatory mechanism for opioid reward (Skupio et al., 2019). Overall, current studies indicate that GR-dependent signaling in astrocytes can modulate opioid reward processing via the regulation of astroglial metabolism.

Previously we discussed the complement component C1q works as an “eat-me” signal and guides microglia to engulf unwanted synapses. Transforming growth factor-β (TGFβ) secreted from astrocytes also participates in this process. Studies using retinal ganglion cells as a neuron model show that the expression of C1q is dependent on the astrocyte-secreted TGFβ (Bialas and Stevens, 2013; Stevens et al., 2007). TGFβ induces C1q expression in retinal ganglion cells by binding to its corresponding receptor TGFBRII since genetic and pharmacological ablation of TGFBRII significantly inhibits TGFβ-induced C1q expression (Bialas and Stevens, 2013) (Fig. 2C). Importantly, the expression of TGFBRII on retinal ganglion cells is time-dependent, which achieves the highest level at postnatal day 5 and sharply decreased by day 15. Taken together, these studies implicate TGFβ signaling regulates complement- and microglia-mediated synaptic elimination.

6. Clinical implications of glial cell modulation in opioid abuse

The opioid epidemic has been difficult to quell due to the lack of novel therapeutics. Knowing that glial cell activity significantly influences rewarding effects of opioids, development of agents which prevent or treat opioid abuse by targeting glial activation is an intriguing option. Currently, several existing medications with the potentials to suppress glial cell activity have been examined (Table 1).

Table 1.

Effects of glial modulators on opioid use disorders in clinical trials.

Glial modulator	Mechanism of action	Participant	Measurement	Effect	references
Pioglitazone	PPARγ agonist	Non-dependent prescription opioid abuser	Positive subjective effects	N.C.	(Jones et al., 2016)
			Negative subjective effects	N.C.
		Heroin-dependent participants	Reinforcing effect	N.C.	(Jones et al., 2018)
			Positive subjective effects	N.C.
			Negative subjective effects (except for anxiety)	N.C.
			Drug-induced anxiety	↓
			Drug craving	↓
Ibudilast	Non-selective Phosphodiesterase inhibitor	Heroin-dependent participants	Withdrawal symptoms	↓	(Cooper et al., 2016)
		Opioid-dependent participants	Positive subjective effects	N.C.	(Cooper et al., 2017)
		Opioid-dependent participants	Positive subjective effects	↓	(Metz et al., 2017)
			Reinforcing effect	↓
			Drug craving	↓
Minocycline	Not identified	Opioid-dependent participants	Drug craving	N.C.	(Arout et al., 2018)
			Withdrawal symptoms	N.C.
Cannabidiol	Not identified	Opioid-abstinent participants with opioid use disorders	Drug craving	↓	(Hurd et al., 2015)
			Drug cue-induced anxiety	↓
		Opioid-abstinent participants with opioid use disorders	Drug craving	↓	(Hurd et al., 2019)
			Drug cue-induced anxiety	↓

Abbreviations: N.C., no significant change identified.

Pioglitazone, a peroxisome proliferator-activated receptor-γ (PPARγ) agonist, is one of the first glial modulators examined for its ability to treat opioid abuse. PPARγ is a nuclear receptor expressed by both microglia and astrocytes (Warden et al., 2016). As the activation of this receptor significantly suppress the production of pro-inflammatory cytokines from glial cells, the PPARγ agonists are widely considered as promising medication to treat neuroinflammatory disorders (Kielian and Drew, 2003). Recently, a study has examined the ability of the PPARγ agonist pioglitazone to alter the abuse liability of oxycodone in opioid abusers (Jones et al., 2016). In this study, 17 non-dependent, prescription opioid abusers were recruited and maintained on pioglitazone (0, 15, and 45 mg per day) for 2–3 weeks in ascending order. At the end of each maintenance period, the positive subjective effects of oral oxycodone (0, 10, 20 mg, cumulative dose = 30 mg) were examined. The results show that pioglitazone did not alter the subjective ratings of drug ‘liking’, ‘high’ or ‘good drug effect’ associated with oxycodone administration, indicating that pioglitazone may not be able to reduce the abuse liability of oxycodone. As pioglitazone has been shown to effectively, suppress opioid reward in preclinical studies (de Guglielmo et al., 2017, 2015), it is unexpected to see the inability of pioglitazone to reduce abuse potential in humans. However, as the participants in this study are non-dependent opioid users, their responses may be different from opioid-dependent abusers. According to previous studies, chronic opioid users may have a higher level of glial activation (Hutchinson et al., 2007; Song and Zhao, 2001), which may enhance the therapeutic effect of pioglitazone. Therefore, another clinical trial was conducted to test the ability of pioglitazone to reduce opioid reward in opioid-dependent participants (Jones et al., 2018). In this study, 30 heroin-dependent volunteers were randomized to receive either pioglitazone (45 mg) or placebo for 3 weeks. On test days, participants performed a verbal choice self-administration procedure to receive either heroin or money. This paradigm allows researchers to evaluate the influence of pioglitazone on the reinforcing and positive subjective effects of heroin. However, no significant difference was found in these effects.

The failure of pioglitazone in treating opioid abuse may not extend to other glial modulators. Ibudilast, a non-selective phosphodiesterase inhibitor, was the second glial agent evaluated clinically for its effectiveness in altering the subjective and reinforcing effects of oxycodone (Metz et al., 2017). In the study, 11 opioid-dependent volunteers were recruited and maintained on active ibudilast (50 mg, b.i.d.) or placebo after in-patient detoxification with morphine. The subjective and reinforcing effects of oxycodone (0 mg, 15 mg and 30 mg/70 kg, p.o.) were then tested under the maintenance treatment. Excitingly, the results indicate that ibudilast produced a mild but significant reduction in the subjective ratings of drug liking for the low dose of oxycodone (15 mg) and the reinforcing effects were also significantly reduced under the treatment of ibudilast. This investigation shows the promise that ibudilast can be an effective medication to inhibit opioid abuse potential. However, inconsistent results were reported by another clinical trial of ibudilast (Cooper et al., 2017). Thirty-one opioid-dependent volunteers participated, stabilizing on morphine treatment (30 mg, p.o., q.i.d.) for two weeks and, at the same time, they all received placebo during the first week of this two-week period and then randomly assigned to receive ibudilast (20 and 40 mg, p.o., b.i.d.) or placebo during the second week. On the testing days (days 4 and 11), the subjective effects of oxycodone (0, 25, 50 mg/70 kg) were tested. The results show that ibudilast does not change the subjective ratings of ‘high’, ‘good effect’ and ‘I would pay’ associated with oxycodone administration, suggesting ibudilast may not affect the rewarding effect of opioids.

These mixed clinical trials of glial modulators on opioid reward may be unexpected and bring doubt on a promising therapeutic potential for attenuating opioid abuse potential in humans. Yet, the reasons for these failures are not clear including a small sample volume and too few trials. All the clinical studies had participants withdrawn from the experiments, some of which even lost one third of their starting number of participants. This participant withdrawal together with the considerable variability across individuals might make the studies significantly underpowered. Therefore, further investigations with greater sample volumes are needed to confirm the clinical influence of glial modulators on opioid reward. In addition, the dosing parameters of the drugs being tested may not be related to their effects on glial activation. For example, the dosage and duration of pioglitazone being used to suppress the rewarding effects of oxycodone in the Jones’ investigation (Jones et al., 2016) were based on the clinical utility of pioglitazone for treating diabetes while no data suggest if pioglitazone may reduce the levels of inflammatory markers at this dosing parameter. In future clinical investigations, methods of detecting glial activity in human brain, such as glial activation biomarker-based on positron emission tomography (Arlicot et al., 2012; Kumar et al., 2012; Loggia et al., 2015), may be used during the testing period to establish proper dosage of glial targeting agents for attenuation of glial activation.

Although the clinical results of pioglitazone and ibudilast are not very exciting, the therapeutic potential of other glial modulators in suppressing opioid abuse potential should not be ignored. Currently, two other glial modulators, cannabidiol and minocycline, have been tested for their ability to treat opioid use disorders (OUD) (Arout et al., 2018; Hurd et al., 2019, 2015) (Table 1). Cannabidiol was found to effectively attenuate opioid cue-induced craving and anxiety without causing any severe adverse effects in drug-abstinent individuals with OUD (Hurd et al., 2019, 2015). Minocycline was able to improve the cognitive performance in opioid-dependent individuals, even though no effect on pain or other behaviors associated with opioid use disorders was observed (Arout et al., 2018). These studies implicate that both cannabidiol and minocycline are promising candidates for treating opioid abuse potential. Further investigations are required to figure out their actual roles.

7. Conclusion

Despite considerable efforts to explore the neuronal mechanisms underlying opioid reward, very few developed interventions have produced a promising effect on the treatment of opioid abuse. Recent studies finding the involvement of glial neuroimmune signaling in opioid reward have provided a new field of study that could develop potential therapies. In this review, we explore the evidence that opioids can induce the activation of glial cells, and how this ability may substantially contribute to the development and maintenance of opioid reward. Furthermore, we also discuss the neuroimmune signaling pathways that mediate the opioid-induced glial activation, especially TLR4 signaling. The studies introduced here suggest that blocking the interaction of opioids and glia or direct suppressing the activation of glial cells by targeting specific neuroimmune signaling could be promising therapeutics to treat opioid abuse. However, an enhanced understanding of opioid actions on neuroimmune signaling would lead to the development of more successful interventions.

Highlights.

• Molecular mechanisms underlying opioid-induced glial activation.

• Recent studies exploring the effects of glial immune factors on opioid reward.

• Current clinical implications of glial modulators in treating opioid reward.

Abbreviation:

ACM

astrocyte-conditioned medium

BDNF

brain-derived neurotrophic factor

CNS

central nerve system

CPP

conditioned place preference

CR3

complement receptor 3

DDDs

daily defined doses

GFAP

glial fibrillary acidic protein

GPCRs

G protein-coupled receptors

GRs

glucocorticoid receptors

Iba1

ionized calcium binding adaptor molecule 1

IL-1β

Interleukin-1β

LTP

Long-term potentiation

LPS

lipopolysaccharide

M3G

morphine-3-glucuronide

M6G

morphine-6-glucuronide

MAPKs

mitogen-activated protein kinases

MD2

Myeloid differentiation factor 2

MOR

μ opioid receptors

NAc

nucleus accumbens

NLRP3

NOD-, LRR- and pyrin domain-containing protein 3

OUD

opioid use disorders

PPARγ

peroxisome proliferator-activated receptor-γ

TLR4

toll-like receptor 4

TPP

tegmental pedunculopontine nucleus

RMTg

rostromedial tegmental nucleus

VTA

ventral tegmental area