The Interoceptive Antireward Pathway and Gut Dysbiosis in Addiction

The concept of antireward as a motivator for drug addiction was first proposed in 2005 by Koob and Le Moal [1]. This model conjectures that addictive behaviors, most often the use of substances of abuse, have two positively reinforcing motivators: the stimulation of reward and the inhibition of antireward (Figure1) [2]. Furthermore, opponent-process functionality inherent in biological systems predicts an absence of reward and a stimulation of antireward in absence of the addictive behavior; or abstinence from the xenobiotic in the case of substance dependence (ie withdrawal) [3]. That is, there are also two negative reinforcement motivators for drug-seeking and taking. Biological evidence for a reward pathway in addiction is well-established. Mesolimbic dopamine is stimulated by substance use leading to the activation of pleasure centers, and this pathway is, in fact, depressed in substance abstinence leading to anhedonia [4,5]. The biological evidence for an antireward pathway has been accumulating since Koob and Le Moal’s original article suggesting signaling in the extended amygdale. 

Figure 1: Schematic of Opponent-Process Model in Addiction. (A) Alcohol and/or opioid exposure has two effects: Stimulate reward, via the mesolimbic dopamine pathway, and inhibit antireward. These effects motivate substance use via positive reinforcement (B) Alcohol and/or opioid withdrawal has two effects. Inhibit reward, by inhibiting the mesolimbic dopamine pathway (not shown), and stimulate antireward. This review proposes that amygdala neuroinflammation mediated primarily by astrocytes is an endpoint in antireward stimulation in alcohol and opioid withdrawal, though this hypothesis warrants further testing. These effects, whatever the mechanism, motivate substance dependence via negative reinforcement. Originally published in O’Sullivan et al. 2021. 

They to Koob and Le Moal proposed corticotrophin-releasing factor (CRF) signaling, in addition to norepinephrine (NE) and dynorphin, in the amygdale as substrates of antireward. The extended nucleus of the amygdala is an ostensible location to generate antireward motivations because of its proven function in threat detection, fear, and negative emotion [6]. Additionally, CRF as a substrate is compelling because CRF increases in the amygdala during withdrawal from alcohol [7], cocaine [8], nicotine [9], and cannabinoids [10]. Moreover, CRF antagonism in the amygdala decreases the anxiety-like behavior characteristic of alcohol and opioid withdrawal and also substance intake [11-15]. 

Our group has built on this work. We recently proposed a modified antireward model suggesting that antireward signaling in the amygdala, in both alcohol and opioid withdrawal, involves neuro inflammation mediated by astrocytes [2,16]. We further suggest that gut dysbiosis may contribute to this phenomenon via vagal afferents, gut peptide hormones, or additional blood-borne signaling (Figure 2). This hypothesis is supported by recent studies out of our lab suggesting neuro inflammation in the Central Nucleus of the Amygdala (CeA) and nucleus tractus solitarius (NTS) in opioid and alcohol withdrawal, respectively [16,17]. Additionally, gut dysbiosis was observed 24 hours following acute naltrexone-precipitated opioid withdrawal (Figure 3). These findings in context with other studies demonstrating gut dysbiosis in alcohol and opioid use and withdrawal [18], the effect of astrocyte-mediated neuro inflammation in the amygdala on anxiety-like behavior [19], and the emerging role of neuro inflammation in drug addiction and treatment [20-25], led us to a novel and proposal: The interceptive circuit connecting peripheral organs, especially the gut, to the CeA via the NTS contributes to antireward in both alcohol and opioid withdrawal by inciting neuro inflammation.

Figure 2: Interoceptive Vagal Circuit and Visceral-Emotional Neuraxis. (A) Interoceptive vagal afferents relay the state of the gut, which is highly influenced by gut microflora, and other peripheral organs to the Nucleus Tractus Solitarius (NTS). This information is subsequently relayed to the amygdala and influences emotional states.(B)A simplified cartoon representation showing the integrative roles of the Nucleus Tractus Solitarius (NTS) and the Central Nucleus of the Amygdala (CeA) in emotion, stress, and autonomic regulation. Two neuronal subtypes, GLP-1 and NE neurons, are highlighted. Many anatomical and functional connections are omitted for clarity (NE, Norepinephrine; GLP-1, Glucagon-Like Peptide 1; GABA, γ-Aminobutyric Acid; HPA axis, Hypothalamic-Pituitary-Adrenal axis; CRF, Corticotropin Releasing Hormone).Originally published in O’Sullivan et al. 2021.

Figure 3: Relative abundance of gut microflora from rat cohort 2. Barplots display relative abundance of bacterial species (-ΔΔC_t values). #p<0.1, ^*p<0.05, ^**p<0.008, ^***p=0.0009; two-way ANOVA n=4 animals for each treatment. Originally published in O’Sullivan et al. 2019. 

Others have speculated on the role of gut dysbiosis in substance dependence [18,26,27]. The importance of the gut-brain connection in health and disease and how gut microflora influence this connection is a growing area of investigation [28]. We have built on these advances by connecting this work to the antireward model of addiction. The neuro anatomy of the proposed interoceptive antireward pathway suggests that vagal afferents responding to gut dysbiosis transmit this information via synapses in the NTS which has strong bidirectional connections to the CeA (Figure 2) [29,30].  We conjecture this circuit influences CeA activity by glial-neuronal paracrine signaling or cytokines and chemokines, in addition to transmitters such as CRF, NE, and glucagon-peptide-1 (GLP-1).

Our study shows that astrocytes have the most perturbed transcriptome in the CeA 24 hours following acute naltrexone-precipitated opioid withdrawal [16]. This finding in astrocytes is consistent with the work of others that has yet to be published. The substrate in our dataset that emerged most prominently, however, was Tumor necrosis factor alpha (TNF-α). We found the transcript of this gene to be significantly up regulated in the withdrawal conditionin all three cell types collected-neurons, microglia, and astrocytes. TNF-α up regulation was confirmed with Western blot and immune fluorescence. We speculate that the signal for this elevation of TNF-α may originate in the gut where opioid receptors are numerous. And, that this neuro inflammation lowers the threshold of firing of neurons in the CeA resulting in hyperactivity that contributes to the negative emotion that characterizes substance withdrawal; That is, antireward stimulation [31,32]. 

This hypothesis is further supported by functional magnetic resonance imaging (fMRI) studies. Increased activity in the amygdala is observed during opioid craving in recently detoxed opioid-dependent patients [33]. Activity in the amygdala also increased upon anticipation of opioid use and subsequently decreased upon methadone administration [34]. Similarly, fMRI studies found that alcohol inhibited amygdala activity in heavy drinkers while hyperactive amygdala-orbitofrontal circuits in adolescents predicted future alcohol abuse [35,36]. These imaging studies suggest that, like the mesolimbic reward pathway, amygdale antireward functionality is involved in dependence of multiple substances. The contribution of gut dysbiosis via vagal afferents or other routes to these observations is not yet well understood. However, clinical treatments that target this pathway are likely to demonstrate efficacy for multiple substances of abuse. 

One emerging treatment is the anti-neuro inflammatory ibudilast. This small molecule has multiple mechanisms of action including as a phosphodiesterase inhibitor and macrophage migration inhibitory factor (Mif) inhibitor [37]. In clinical trials, ibudilast has demonstrated efficacy in decreasing opioid cravings and withdrawal symptoms while increasing the analgesic effects of opioids in patients with dependence [23-25]. Additionally, ibudilast demonstrated efficacy in reducing reward in methamphetamine infusions [22,38]. This effect may have been due to reductions in peripheral inflammation implicating the interoceptive antireward pathway in ibudilast’s efficacy [39]. Ibudilast is currently in clinical trials for alcohol dependence as well [21]. The ability of this anti-inflammatory molecule to treat multiple forms of substance dependence is consistent with the antireward interoceptive pathway model. 

The emergence of peripheral nerve stimulation for the treatment of opioid withdrawal adds further evidence to this model. The Food & Drug Administration (FDA) recently approved transcutaneous auricular neuro stimulation with the BRIDGE device to treat opioid withdrawal symptoms [40]. The proposed mechanism of action of this device is that stimulation of peripheral cranial nerves, which input into the NTS, subsequently stimulates the amygdala where “extracellular recordings from single cells in the rat amygdala before and during neuro stimulation with the BRIDGE device showed a 65% reduction in the baseline firing of neurons in the central nucleus of the amygdala” [41]. The effectiveness of the BRIDGE device in treating acute opioid withdrawal symptoms demonstrates that peripheral nerves influence an antireward center whose primary endpoint is the CeA. And, that targeting this pathway has potent effects on decreasing the physical and emotional pain that occurs in opioid withdrawal which may decrease drug-seeking and taking by decreasing negative reinforcement (Figure 1). 

The evidence linking gut microflora dysbiosis to antireward, however, remains minimal. Studies have shown the importance of gut microflora in behavior and disease broadly, but their stimulation of antireward in acute withdrawal and protracted substance abstinence remains hypothetical [28,42]. Recent studies employing sub diaphragmatic vagotomy suggest vagal afferents do influence self-administration behavior [43], but the direct effects of microbes on this circuit and their sequalae influencing antireward in the amygdala remains an unknown frontier for further investigation.

This work was support by NIH HLB U01 HL133360 awarded to James S. Schwaber, Ph. D. and Rajanikanth Vadigepalli, Ph. D. and T32 AA-007463 awarded to Jan B. Hoek, Ph. D.