Behavioural and cellular basis of the vulnerability to develop compulsive heroin seeking habits

Addiction is a chronic relapsing disorder for which there is no effective treatment. This may reflect our lack of understanding of the psychological and neural mechanisms that support the transition, in vulnerable individuals, from recreational drug use to compulsive drug seeking habits. Over the last decade clinical and preclinical studies have begun to shed light on the psychological and neural basis of the individual vulnerability to cocaine addiction, but despite the epidemic in opiates addiction in the USA and incremental opioid drug abuse and addiction in the UK, heroin addiction has hitherto been under-investigated. Using a novel preclinical model of compulsive heroin seeking behaviour in which some rats self-administering heroin persist in responding under a second-order schedule of reinforcement despite punishment (Chapter 3), the experiments in this thesis investigated the psychological, behavioural, neural and cellular mechanisms involved in the vulnerability to develop compulsive heroin seeking. Chapter 4 aimed to identify behavioural traits, such as anxiety, stress reactivity or decision making, that predict an increased vulnerability to develop compulsive heroin seeking. Chapter 5 aimed to characterise the neural and cellular correlates of heroin seeking habits, and compulsivity. Based on the combination of hotspot analysis, quantitative PCR, RNAscope and western-blot analyses, the data presented demonstrate that compulsive habits are associated with a differential pattern of cellular plasticity within corticostriatal networks, and are preceded by diverse cellular adaptations, especially in the striatum, in vulnerable individuals. Finally, chapter 6 further investigated the cellular specificity of the observed adaptations in experiments that revealed exposure to heroin and cocaine, triggers a downregulation of the dopamine transporter preferentially in astrocytes, and not in neurons as previously thought. The results presented in this thesis offer new insights into the neural and cellular basis of the vulnerability to develop compulsive heroin seeking, a key feature of opioid addiction.Cambridge Commonwealth, European & International Trus

Effective Glucose Uptake by Human Astrocytes Requires Its Sequestration in the Endoplasmic Reticulum by Glucose-6-Phosphatase-β.

After its uptake into the cytosol, intracellular glucose is phosphorylated to glucose-6-phosphate (G6P), trapping it within the cell and preparing it for metabolism. In glucose-exporting tissues, like liver, G6P is transported into the ER, where it is dephosphorylated by G6Pase-α. The glucose is then returned to the cytosol for export [1, 2]. Defects in these pathways cause glycogen storage diseases [1]. G6Pase-β, an isozyme of G6Pase-α, is widely expressed [3, 4]. Its role in cells that do not export glucose is unclear, although mutations in G6Pase-β cause severe and widespread abnormalities [5-7]. Astrocytes, the most abundant cells in the brain, provide metabolic support to neurons, facilitated by astrocytic endfeet that contact blood capillaries or neurons [8-12]. Perivascular endfeet are the main site of glucose uptake by astrocytes [13], but in human brain they may be several millimeters away from the perineuronal processes [14]. We show that cultured human fetal astrocytes express G6Pase-β, but not G6Pase-α. ER-targeted glucose sensors [15, 16] reveal that G6Pase-β allows the ER of human astrocytes to accumulate glucose by importing G6P from the cytosol. Glucose uptake by astrocytes, ATP production, and Ca2+ accumulation by the ER are attenuated after knockdown of G6Pase-β using lentivirus-delivered shRNA and substantially rescued by expression of G6Pase-α. We suggest that G6Pase-β activity allows effective uptake of glucose by astrocytes, and we speculate that it allows the ER to function as an intracellular "highway" delivering glucose from perivascular endfeet to the perisynaptic processes

How Preclinical Models Evolved to Resemble the Diagnostic Criteria of Drug Addiction.

Drug addiction is a complex neuropsychiatric disorder that affects a subset of the individuals who take drugs. It is characterized by maladaptive drug-seeking habits that are maintained despite adverse consequences and intense drug craving. The pathophysiology and etiology of addiction is only partially understood despite extensive research because of the gap between current preclinical models of addiction and the clinical criteria of the disorder. This review presents a brief overview, based on selected methodologies, of how behavioral models have evolved over the last 50 years to the development of recent preclinical models of addiction that more closely mimic diagnostic criteria of addiction. It is hoped that these new models will increase our understanding of the complex neurobiological mechanisms whereby some individuals switch from controlled drug use to compulsive drug-seeking habits and relapse to these maladaptive habits. Additionally, by paving the way to bridge the gap that exists between biobehavioral research on addiction and the human situation, these models may provide new perspectives for the development of novel and effective therapeutic strategies for drug addiction.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.biopsych.2015.01.00

Heroin seeking becomes dependent on dorsal striatal dopaminergic mechanisms and can be decreased by N-acetylcysteine.

The alarming increase in heroin overdoses in the USA is a reminder of the need for efficacious and novel treatments for opiate addiction. This may reflect the relatively poor understanding of the neural basis of heroin, as compared to cocaine, seeking behaviour. While cocaine reinforcement depends on the mesolimbic system, well-established cocaine seeking is dependent on dorsolateral striatum (aDLS) dopamine-dependent mechanisms which are disrupted by N-acetylcysteine, through normalisation of corticostriatal glutamate homeostasis. However, it is unknown whether a functional recruitment of aDLS dopamine-dependent control over instrumental responding also occurs for heroin seeking, even though heroin reinforcement does not depend on the mesolimbic dopamine system. Lister Hooded rats acquired heroin self-administration and were subsequently trained to seek heroin daily over prolonged periods of time under the control of drug-paired cues, as measured under a second-order schedule of reinforcement. At different stages of training, that is, early on and when heroin seeking behaviour was well established, we measured the sensitivity of drug-seeking responses to either bilateral aDLS infusions of the dopamine receptor antagonist α-flupenthixol (5, 10 and 15 μg/side) or systemic administration of N-acetylcysteine (30, 60 and 90 mg/kg). The results demonstrate that control over heroin seeking behaviour devolves to aDLS dopamine-dependent mechanisms after extended training. Further aDLS-dependent well-established, cue-controlled heroin seeking was disrupted by N-acetylcysteine. Comparison with previous data on cocaine suggests that the development of drug seeking habits and the alteration of corticostriatal glutamate homeostasis, which is restored by N-acetylcysteine, are quantitatively similar between heroin and cocaine

The anterior insular cortex in the rat exerts an inhibitory influence over the loss of control of heroin intake and subsequent propensity to relapse.

The anterior insular cortex (AIC) has been implicated in addictive behaviour, including the loss of control over drug intake, craving and the propensity to relapse. Evidence suggests that the influence of the AIC on drug-related behaviours is complex as in rats exposed to extended access to cocaine self-administration, the AIC was shown to exert a state-dependent, bidirectional influence on the development and expression of loss of control over drug intake, facilitating the latter but impairing the former. However, it is unclear whether this influence of the AIC is confined to stimulant drugs that have marked peripheral sympathomimetic and anxiogenic effects or whether it extends to other addictive drugs, such as opiates, that lack overt acute aversive peripheral effects. We investigated in outbred rats the effects of bilateral excitotoxic lesions of AIC induced both prior to or after long-term exposure to extended access heroin self-administration, on the development and maintenance of escalated heroin intake and the subsequent vulnerability to relapse following abstinence. Compared to sham surgeries, pre-exposure AIC lesions had no effect on the development of loss of control over heroin intake, but lesions made after a history of escalated heroin intake potentiated escalation and also enhanced responding at relapse. These data show that the AIC inhibits or limits the loss of control over heroin intake and propensity to relapse, in marked contrast to its influence on the loss of control over cocaine intake.This work, carried out at the Department of Psychology, University of Cambridge, was funded by a Programme Grant from the Medical Research Council to BJE and DB (MR/N02530X/1) and a research grant from the Leverhulme Trust to DB (RPG‐2016‐117). DJ is supported by a BBSRC-DTP/Shionogi joint grant to DB (G101457). Heroin hydrochloride was provided to DB by the NIDA Drug Supply Programme

Pan-striatal reduction in the expression of the astrocytic dopamine transporter precedes the development of dorsolateral striatum dopamine-dependent incentive heroin seeking habits.

The emergence of compulsive drug-seeking habits, a hallmark feature of substance use disorder, has been shown to be predicated on the engagement of dorsolateral striatal control over behaviour. This process involves the dopamine-dependent functional coupling of the anterior dorsolateral striatum (aDLS) with the nucleus accumbens core, but the mechanisms by which this coupling occurs have not been fully elucidated. The striatum is tiled by a syncytium of astrocytes that express the dopamine transporter (DAT), the level of which is altered in individuals with heroin use disorder. Astrocytes are therefore uniquely placed functionally to bridge dopamine-dependent mechanisms across the striatum. Here we tested the hypothesis that exposure to heroin influences the expression of DAT in striatal astrocytes across the striatum before the development of DLS-dependent incentive heroin seeking habits. Using Western-blot, qPCR, and RNAscope™, we measured DAT protein and mRNA levels in whole tissue, culture and in situ astrocytes from striatal territories of rats with a well-established cue-controlled heroin seeking habit and rats trained to respond for heroin or food under continuous reinforcement. Incentive heroin seeking habits were associated with a reduction in DAT protein levels in the anterior aDLS that was preceded by a heroin-induced reduction in DAT mRNA and protein in astrocytes across the striatum. Striatal astrocytes were also shown to be susceptible to direct dopamine- and opioid-induced downregulation of DAT expression. These results suggest that astrocytes may critically regulate the striatal dopaminergic adaptations that lead to the development of incentive heroin seeking habits

Pan‐striatal reduction in the expression of the astrocytic dopamine transporter precedes the development of dorsolateral striatum dopamine‐dependent incentive heroin seeking habits

Publication status: PublishedAbstractThe emergence of compulsive drug‐seeking habits, a hallmark feature of substance use disorder, has been shown to be predicated on the engagement of dorsolateral striatal control over behaviour. This process involves the dopamine‐dependent functional coupling of the anterior dorsolateral striatum (aDLS) with the nucleus accumbens core, but the mechanisms by which this coupling occurs have not been fully elucidated. The striatum is tiled by a syncytium of astrocytes that express the dopamine transporter (DAT), the level of which is altered in individuals with heroin use disorder. Astrocytes are therefore uniquely placed functionally to bridge dopamine‐dependent mechanisms across the striatum. Here we tested the hypothesis that exposure to heroin influences the expression of DAT in striatal astrocytes across the striatum before the development of DLS‐dependent incentive heroin seeking habits. Using Western‐blot, qPCR, and RNAscope™, we measured DAT protein and mRNA levels in whole tissue, culture and in situ astrocytes from striatal territories of rats with a well‐established cue‐controlled heroin seeking habit and rats trained to respond for heroin or food under continuous reinforcement. Incentive heroin seeking habits were associated with a reduction in DAT protein levels in the anterior aDLS that was preceded by a heroin‐induced reduction in DAT mRNA and protein in astrocytes across the striatum. Striatal astrocytes were also shown to be susceptible to direct dopamine‐ and opioid‐induced downregulation of DAT expression. These results suggest that astrocytes may critically regulate the striatal dopaminergic adaptations that lead to the development of incentive heroin seeking habits.</jats:p

Negative Urgency Exacerbates Relapse to Cocaine Seeking After Abstinence.

BACKGROUND: The mechanisms through which drug-cue-induced negative affective states are involved in relapse have not been defined. We tested the hypothesis that in individuals having developed a dorsolateral striatum (DLS)-dependent cue-controlled cocaine-seeking habit, the loss of the opportunity to enact the drug-seeking response during abstinence results in an urge to act that exacerbates relapse severity mediated by negative urgency. METHODS: Eighty-seven male Sprague Dawley rats were trained to seek cocaine under the influence of the conditioned reinforcing properties of drug-paired cues or not. We investigated whether the tendency to relapse depended on the aversive state of withdrawal or instead on the loss of opportunity to perform the ingrained drug-seeking response after periods of abstinence. The striatal locus of control over cocaine seeking at baseline and relapse was investigated using in situ hybridization of the cellular activity marker C-fos and assessment of the sensitivity of instrumental drug seeking to dopamine receptor blockade in the dorsomedial striatum-dependent goal-directed and DLS-dependent habit systems. RESULTS: The development of a DLS-dependent cue-controlled cocaine-seeking habit prior to abstinence resulted in a marked increase in drug seeking at relapse, which was not motivated by a cocaine withdrawal state and was no longer dependent on the DLS habit system. Instead, it reflected the emergence of negative urgency caused by the prevention of the performance of the habit during abstinence and underpinned by transient engagement of the goal-directed system. CONCLUSIONS: These results show that ingrained cue-controlled drug-seeking habits increase the pressure to relapse.Leverhulme Trus

Baclofen decreases compulsive alcohol drinking in rats characterized by reduced levels of GAT‐3 in the central amygdala

Abstract: While most individuals with access to alcohol drink it recreationally, some vulnerable individuals eventually lose control over their intake and progressively develop compulsive alcohol drinking and decreased interest in alternative sources of reinforcement, two key features of addiction. The neural and molecular mechanisms underlying this vulnerability to switch from controlled to compulsive alcohol intake have not been fully elucidated. It has been shown that rats having reduced levels of expression of the gamma‐aminobutyric acid (GABA) transporter, GAT‐3, in the amygdala tend to persist in seeking and drinking alcohol even when adulterated with quinine, suggesting that pharmacological interventions aimed at restoring GABA homeostasis in these individuals may provide a targeted treatment to limit compulsive alcohol drinking. Here, we tested the hypothesis that the GABAB receptor agonist baclofen, which decreases GABA release, specifically reduces compulsive alcohol drinking in vulnerable individuals. In a large cohort of Sprague–Dawley rats allowed to drink alcohol under an intermittent two‐bottle choice procedure, a cluster of individuals was identified that persisted in drinking alcohol despite adulteration with quinine or when an alternative ingestive reinforcer, saccharin, was available. In these rats, which were characterized by decreased GAT‐3 mRNA levels in the central amygdala, acute baclofen administration (1.5 mg/kg, intraperitoneal) resulted in a decrease in compulsive drinking. These results indicate that low GAT‐3 mRNA levels in the central amygdala may represent an endophenotype of vulnerability to develop a compulsive drinking of alcohol that is shown here to be mitigated by baclofen

The development of compulsive coping behavior depends on dorsolateral striatum dopamine-dependent mechanisms.

Humans greatly differ in how they cope with stress, a natural behavior learnt through negative reinforcement. Some individuals engage in displacement activities, others in exercise or comfort eating, and others still in alcohol use. Across species, adjunctive behaviors, such as polydipsic drinking, are used as a form of displacement activity that reduces stress. Some individuals, in particular those that use alcohol to self-medicate, tend to lose control over such coping behaviors, which become excessive and compulsive. However, the psychological and neural mechanisms underlying this individual vulnerability have not been elucidated. Here we tested the hypothesis that the development of compulsive adjunctive behaviors stems from the functional engagement of the dorsolateral striatum (DLS) dopamine-dependent habit system after a prolonged history of adjunctive responding. We measured in longitudinal studies in male Sprague Dawley rats the sensitivity of early established vs compulsive polydipsic water or alcohol drinking to a bilateral infusion into the anterior DLS (aDLS) of the dopamine receptor antagonist α-flupentixol. While most rats acquired a polydipsic drinking response with water, others only did so with alcohol. Whether drinking water or alcohol, the acquisition of this coping response was insensitive to aDLS dopamine receptor blockade. In contrast, after prolonged experience, adjunctive drinking became dependent on aDLS dopamine at a time when it was compulsive in vulnerable individuals. These data suggest that habits may develop out of negative reinforcement and that the engagement of their underlying striatal system is necessary for the manifestation of compulsive adjunctive behaviors.This work, carried out at the department of Psychology of the University of Cambridge, was funded by a UKRI grant to DB (MR/W019647/1), a UKRI grant (MR/N02530X/1) to BJE and DB, a Brain and Behavior Research Foundation NARSAD Young Investigator Grant (RG95599 Grant ID: 27353) to CG, and a Leverhulme Trust Early Career (ECF-2018-713) and Isaac Newton Trust fellowship (18.08(g)) to LMP