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Rationale: Relapse into substance use is often triggered by exposure to drug-related environmental cues. The magnitude of drug seeking depends on the duration of abstinence, a phenomenon known as the incubation of drug craving. Clinical and preclinical research shows that the insular cortex is involved in substance use disorders and cue-induced drug seeking. However, the role of the insula on memory retrieval and motivational integration for cue-elicited drug seeking remains to be determined. Objectives: We investigated the role of the anterior insular cortex (aIC) and its glutamatergic projection to amygdala nuclei (aIC-AMY) on the expression of conditioned place preference (CPP) during early and late abstinence. Methods: Male adult C57BL/6J mice underwent amphetamine-induced CPP, and their preference was tested following 1 or 14 days of abstinence. aIC and aIC-AMY functional role in CPP expression was assessed at both abstinence periods by employing optogenetic silencing and behavioral pharmacology. Results: Compared to a single day, an exacerbated preference for the amphetamine-paired context was observed after 14 days of abstinence. Photoinhibition of either aIC or aIC-AMY projection reduced CPP expression following late but not early abstinence. Similarly, the antagonism of aIC NMDA receptors reduced CPP expression after 14 days of abstinence but not 1 day. Conclusions: These results suggest that aIC and its glutamatergic output to amygdala nuclei constitute critical neurobiological substrates mediating enhanced motivational cue reactivity during the incubation of amphetamine craving rather than contextual memory recall. Moreover, cortical NMDA receptor signaling may become sensitized during abstinence, ultimately modulating disproportioned drug seeking. [ABSTRACT FROM AUTHOR]

• Gut microbiota dysbiosis and intestinal barrier damage during the incubation of methamphetamine (METH) craving. • Depletion of gut bacteria via antibiotic cocktails increased LPS levels, induced a marked change in the microglial morphology in the nucleus accumbens core and prevented the incubation of METH craving. • Klebsiella oxytoca or exogenous LPS administration decreased METH craving after prolonged withdrawal. • Gut-derived LPS accumulation in the nucleus accumbens core promoted the activation of microglia and decreased transcription of dopamine receptor. The microbiota-gut-brain axis plays a critical role in the pathophysiology of neuropsychiatric disorders, and the compositions of gut microbiota are altered by addictive drugs. However, the role of gut microbiota in the incubation of methamphetamine (METH) craving remains poorly understood. 16S rRNA gene sequencing was performed to assess the richness and diversity of gut microbiota in METH self-administration model. Hematoxylin and eosin staining was performed to evaluate the integrity of intestinal barrier. Immunofluorescence and three-dimensional reconstruction were performed to assess the morphologic changes of microglia. Serum levels of lipopolysaccharide (LPS) were determined using the rat enzyme-linked immunosorbent assay kits. Quantitative real-time PCR was performed to assess transcript levels of dopamine receptor, glutamate ionotropic AMPA receptor 3 and brain-derived neurotrophic factor. METH self-administration induced gut microbiota dysbiosis, intestinal barrier damage and microglia activation in the nucleus accumbens core (NAcc), which was partially recovered after prolonged withdrawal. Microbiota depletion via antibiotic treatment increased LPS levels and induced a marked change in the microglial morphology in the NAcc, as indicated by the decreases in the lengths and numbers of microglial branches. Depleting the gut microbiota also prevented the incubation of METH craving and increased the population of Klebsiella oxytoca. Furthermore, Klebsiella oxytoca treatment or exogenous administration of the gram-negative bacterial cell wall component LPS increased serum and central LPS levels, induced microglial morphological changes and reduced the dopamine receptor transcription in the NAcc. Both treatments and NAcc microinjections of gut-derived bacterial LPS significantly decreased METH craving after prolonged withdrawal. These data suggest that LPS from gut gram-negative bacteria may enter circulating blood, activate microglia in the brain and consequently decrease METH craving after withdrawal, which may have important implications for novel strategies to prevent METH addiction and relapse. [ABSTRACT FROM AUTHOR]

Relapse to drug use is one of the major challenges in treating substance use disorders. Exposure to drug-related cues and contexts triggers drug craving, which drives cocaine seeking, and increases the probability of relapse. Clinical and animal studies have shown a progressive intensification of cocaine seeking and craving that develops over the course of abstinence, a phenomenon commonly referred to as incubation of cocaine craving. Although the neurobiology underlying incubation of cocaine craving has been examined – particularly within the context of glutamate plasticity– the extent to which increased cocaine craving engenders mesolimbic dopamine (DA) changes has received relatively little attention. To assess whether incubation of cocaine craving is associated with alterations in DA terminal neurotransmission in the nucleus accumbens core (NAc), we used ex vivo fast scan cyclic voltammetry in female and male rats to assess DA dynamics following short access, long access, or intermittent access to cocaine self-administration followed by 28 days of abstinence. Results indicated that both long access and intermittent access to cocaine produced robust incubation of cocaine craving, which was associated with increases in cocaine potency. In addition, intermittent access self-administration also produced a robust increase in DA uptake rate at baseline. In contrast, short access to cocaine did not engender incubation of cocaine craving, nor produce changes in DA neurotransmission. Together these observations indicate that incubation of cocaine craving coincides with changes in DA transmission, suggesting that underlying changes in mesolimbic DA signaling may contribute to the progressive intensification of drug craving that occurs across periods of abstinence.

People suffering from opioid use disorder (OUD) exhibit cognitive dysfunctions. Here, we investigated potential changes in the expression of glutamate receptors in rat hippocampi at 2 h and 31 days after the last session of oxycodone self-administration (SA). RNA extracted from the hippocampus was used in quantitative polymerase chain reaction analyses. Rats, given long-access (9 h per day) to oxycodone (LgA), took significantly more drug than rats exposed to short-access (3 h per day) (ShA). In addition, LgA rats could be further divided into higher oxycodone taking (LgA-H) or lower oxycodone taking (LgA-L) groups, based on a cut-off of 50 infusions per day. LgA rats, but not ShA, rats exhibited incubation of oxycodone craving. In addition, LgA rats showed increased mRNA expression of GluA1-3 and GluN2a-c subunits as well as Grm3, Grm5, Grm6, and Grm8 subtypes of glutamate receptors after 31 days but not after 2 h of stopping the SA experiment. Changes in GluA1-3, Grm6, and Grm8 mRNA levels also correlated with increased lever pressing (incubation) after long periods of withdrawal from oxycodone. More studies are needed to elucidate the molecular mechanisms involved in altering the expression of these receptors during withdrawal from oxycodone and/or incubation of drug seeking.

People suffering from opioid use disorder (OUD) exhibit cognitive dysfunctions. Here, we investigated potential changes in the expression of glutamate receptors in rat hippocampi at 2 h and 31 days after the last session of oxycodone self-administration (SA). RNA extracted from the hippocampus was used in quantitative polymerase chain reaction analyses. Rats, given long-access (9 h per day) to oxycodone (LgA), took significantly more drug than rats exposed to short-access (3 h per day) (ShA). In addition, LgA rats could be further divided into higher oxycodone taking (LgA-H) or lower oxycodone taking (LgA-L) groups, based on a cut-off of 50 infusions per day. LgA rats, but not ShA, rats exhibited incubation of oxycodone craving. In addition, LgA rats showed increased mRNA expression of GluA1-3 and GluN2a-c subunits as well as Grm3 , Grm5 , Grm6 , and Grm8 subtypes of glutamate receptors after 31 days but not after 2 h of stopping the SA experiment. Changes in GluA1-3, Grm6, and Grm8 mRNA levels also correlated with increased lever pressing (incubation) after long periods of withdrawal from oxycodone. More studies are needed to elucidate the molecular mechanisms involved in altering the expression of these receptors during withdrawal from oxycodone and/or incubation of drug seeking. [ABSTRACT FROM AUTHOR]

Abstract Background Methamphetamine (Meth) seeking progressively increases after withdrawal (incubation of Meth craving). We previously demonstrated a role of anterior intralaminar nucleus of thalamus (AIT) to dorsomedial striatum (DMS) projections in this incubation. Here, we examined molecular alterations in DMS and AIT neurons activated (identified by neuronal activity marker Fos) during “incubated” Meth‐seeking relapse test after prolonged withdrawal. Methods We trained male rats to self‐administer Meth or saline (control condition) for 10 days (6 hr/day). Using fluorescence‐activated cell sorting, we examined gene expression in Fos‐positive (activated during a 2‐hr relapse test) and Fos‐negative (nonactivated) DMS and AIT neurons. Results In DMS, we found increased mRNA expressions of immediate early genes (IEGs) (Arc, Egr1, Npas4, Fosb), Trkb, glutamate receptors subunits (Gria3, Grin1, Grin2b, Grm1), and epigenetic enzymes (Hdac3, Hdac5, Crebbp) in Fos‐positive neurons, compared with Fos‐negative neurons. In AIT, we found that fewer genes (Egr1, Fosb, TrkB, Grin1, and Hdac5) exhibited increased mRNA expression in Fos‐positive neurons. Unexpectedly, in both brain regions, gene alterations described above also occurred in drug‐naïve saline self‐administration control rats. Conclusions These results demonstrated that transcriptional regulations in Fos‐positive neurons activated during the relapse tests are brain region‐specific but are not uniquely associated with drug exposure during the self‐administration training.

High rates of relapse to drug use during abstinence is a defining feature of human drug addiction. This clinical scenario has been studied at the preclinical level using different animal models in which relapse to drug seeking is assessed after cessation of operant drug self-administration in rodents and monkeys. In our Society for Neuroscience (SFN) session entitled "Circuit and Synaptic Plasticity Mechanisms of Drug Relapse," we will discuss new developments of our understanding of circuits and synaptic plasticity mechanisms of drug relapse from studies combining established and novel animal models with state-of-the-art cellular, electrophysiology, anatomical, chemogenetic, and optogenetic methods. We will also discuss the translational implications of these new developments. In the minireview that introduces our SFN session, we summarize results from our laboratories on behavioral, cellular, and circuit mechanisms of drug relapse within the context of our session. [ABSTRACT FROM AUTHOR]

In humans, cues associated with the rewarding effect of drugs of abuse induce drug craving and activate drug-associated memories after prolonged abstinence. In animal studies with the self-administration (SA) paradigm, responses to drug-associated cues increase within time after extinction, a phenomenon described as incubation of craving. Conditioned place preference (CPP) is widely used to measure the rewarding effect of drugs and the reward memory thereof. However, little is known whether responses to drug associated cues progressively increase after abstinence from the drugs in the CPP paradigm. To test whether the drug-associated cues could increase specific responses over the abstinence period in the CPP paradigm, we employed the high dose morphine-induced CPP paradigms in rats and tree shrews in the present study. We examined the CPP scores and the entrances to side chambers of the CPP apparatus to check whether they would progressively increase in the CPP paradigms. Twenty-one male adult Sprague-Dawley rats and eight adult male tree shrews were used to establish morphine-induced CPP and another ten rats treated with saline were controls for the rat experiments. After morphine conditioning, rats and tree shrews showed significant higher CPP scores at the first or second post tests than at baseline but then the CPP scores in the abstinence period decreased gradually. During the abstinence period, animals with morphine-conditioning experiences entered progressively more times to both side compartments, whereas the number of entrances to side chambers of the saline group in rats had no such significant differences. These findings suggest that progressively increased entrances to the side chambers in the extended abstinence period reflect the incubation of craving in high dose morphine-induced CPP paradigms. Also, our data imply that reward memory and drug craving can be distinguished in the CPP paradigm. [ABSTRACT FROM AUTHOR]

We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we studied the role of dorsolateral striatum (DLS) and dorsomedial striatum (DMS) in this incubation. We trained rats to self-administer palatable food pellets (6 d, 6 h/d) and methamphetamine (12 d, 6 h/d). We then assessed relapse to methamphetamine seeking under extinction conditions after 1 and 21 abstinence days. Between tests, the rats underwent voluntary abstinence (using a discrete choice procedure between methamphetamine and food; 20 trials/d) for 19 d. We used in situ hybridization to measure the colabeling of the activity marker Fos with Drd1 and Drd2 in DMS and DLS after the tests. Based on the in situ hybridization colabeling results, we tested the causal role of DMS D1 and D2 family receptors, and DMS neuronal ensembles in “incubated” methamphetamine seeking, using selective dopamine receptor antagonists (SCH39166 or raclopride) and the Daun02 chemogenetic inactivation procedure, respectively. Methamphetamine seeking was higher after 21 d of voluntary abstinence than after 1 d (incubation of methamphetamine craving). The incubated response was associated with increased Fos expression in DMS but not in DLS; Fos was colabeled with both Drd1 and Drd2. DMS injections of SCH39166 or raclopride selectively decreased methamphetamine seeking after 21 abstinence days. In Fos-lacZ transgenic rats, selective inactivation of relapse test-activated Fos neurons in DMS on abstinence day 18 decreased incubated methamphetamine seeking on day 21. Results demonstrate a role of DMS dopamine D1 and D2 receptors in the incubation of methamphetamine craving after voluntary abstinence and that DMS neuronal ensembles mediate this incubation. [ABSTRACT FROM AUTHOR]

Background Cue-induced methamphetamine craving increases after prolonged forced (experimenter-imposed) abstinence from the drug (incubation of methamphetamine craving). Here, we determined whether this incubation phenomenon would occur under conditions that promote voluntary (self-imposed) abstinence. We also determined the effect of the novel metabotropic glutamate receptor 2 positive allosteric modulator, AZD8529, on incubation of methamphetamine craving after forced or voluntary abstinence. Methods We trained rats to self-administer palatable food (6 sessions) and then to self-administer methamphetamine under two conditions: 12 sessions (9 hours/day) or 50 sessions (3 hours/day). We then assessed cue-induced methamphetamine seeking in extinction tests after 1 or 21 abstinence days. Between tests, the rats underwent either forced abstinence (no access to the food- or drug-paired levers) or voluntary abstinence (achieved via a discrete choice procedure between methamphetamine and palatable food; 20 trials per day) for 19 days. We also determined the effect of subcutaneous injections of AZD8529 (20 and 40 mg/kg) on cue-induced methamphetamine seeking 1 day or 21 days after forced or voluntary abstinence. Results Under both training and abstinence conditions, cue-induced methamphetamine seeking in the extinction tests was higher after 21 abstinence days than after 1 day (incubation of methamphetamine craving). AZD8529 decreased cue-induced methamphetamine seeking on day 21 but not day 1 of forced or voluntary abstinence. Conclusions We introduce a novel animal model to study incubation of drug craving and cue-induced drug seeking after prolonged voluntary abstinence, mimicking the human condition of relapse after successful contingency management treatment. Our data suggest that positive allosteric modulators of metabotropic glutamate receptor 2 should be considered for relapse prevention. [ABSTRACT FROM AUTHOR]

Rationale and objectives: Responding to heroin cues progressively increases after cessation of heroin self-administration (incubation of heroin craving). We investigated whether this incubation is associated with time-dependent changes in brain-derived neurotrophic factor (BDNF) and methyl-CpG binding protein 2 (MeCP2) signaling and mu opioid receptor (MOR) expression in nucleus accumbens (NAc), dorsal striatum (DS), and medial prefrontal cortex (mPFC). We also investigated the effect of the preferential MOR antagonist naloxone on cue-induced heroin seeking during abstinence. Methods: We trained rats to self-administer heroin or saline for 9-10 days and then dissected the NAc, DS, and mPFC at different abstinence days and measured mRNA and protein levels of BDNF, TrkB, and MeCP2, as well as MOR mRNA ( Oprm1). In other groups, we assessed cue-induced heroin seeking in extinction tests after 1, 11, and 30 abstinence days, and naloxone's (0-1.0 mg/kg) effect on extinction responding after 1 and 15 days. Results: Cue-induced heroin seeking progressively increased or incubated during abstinence. This incubation was not associated with changes in BDNF, TrkB, or MeCP2 mRNA or protein levels in NAc, DS, or mPFC; additionally, no molecular changes were observed after extinction tests on day 11. In NAc, but not DS or mPFC, MOR mRNA decreased on abstinence day 1 and returned to basal levels over time. Naloxone significantly decreased cue-induced heroin seeking after 15 abstinence days but not 1 day. Conclusions: Results suggest a role of MOR in incubation of heroin craving. As previous studies implicated NAc BDNF in incubation of cocaine craving, our data suggest that different mechanisms contribute to incubation of heroin versus cocaine craving. [ABSTRACT FROM AUTHOR]