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Drug addiction is a pervasive worldwide problem characterized by compulsive drug use that continues despite negative consequences and treatment attempts. Historically, the biological basis of drug addiction has focused principally on neuronal activity. However, despite their pivotal role in the underlying pathology of drug addiction, neurons are not the only central nervous system (CNS) component involved. The role of additional cell types, especially the CNS immunocompetent microglial cells, in the development of tolerance and related neuroplastic changes during drug taking, addiction, and withdrawal is also emerging. Within this perspective, this chapter reviews the roles of microglial cells in several aspects of drug addiction and its behavioural consequences, including reward, tolerance, dependence, and withdrawal. The cellular and molecular mechanisms which are particularly recruited will be emphasized. Lastly, we will also summarize the development of pharmacological modulators of microglial activation that offer novel treatment strategies and highlight the need to better understand the roles of microglia in the context of drug addiction. ; Xiaohui Wang, Thomas A. Cochran, Mark R. Hutchinson, Hang Yin, and Linda R. Watkins

BACKGROUND AND PURPOSEEmerging evidence implicates a role for toll‐like receptor 4 (TLR4) in the CNS effects of alcohol. The aim of the current study was to determine whether TLR4–MyD88‐dependent signalling is involved in the acute behavioural actions of alcohol and if alcohol can activate TLR4‐downstream MAPK and NF‐κB pathways.EXPERIMENTAL APPROACHThe TLR4 pathway was evaluated using the TLR4 antagonist (+)‐naloxone (µ‐opioid receptor‐inactive isomer) and mice with null mutations in theTLR4andMyD88genes. Sedation and motor impairment induced by a single dose of alcohol were assessed by loss of righting reflex (LORR) and rotarod tests, separately. The phosphorylation of JNK, ERK and p38, and levels of IκBα were measured to determine the effects of acute alcohol exposure on MAPK and NF‐κB signalling.KEY RESULTSAfter a single dose of alcohol, both pharmacological inhibition of TLR4 signalling with (+)‐naloxone and genetic deficiency of TLR4 or MyD88 significantly (P< 0.0001) reduced the duration of LORR by 45–78% and significantly decreased motor impairment recovery time to 62–88% of controls. These behavioural actions were not due to changes in the peripheral or central alcohol pharmacokinetics. IκBα levels responded to alcohol by 30 min in mixed hippocampal cell samples, from wild‐type mice, but not in cells from TLR4‐ or MyD88‐deficient mice.CONCLUSIONS AND IMPLICATIONSThese data provide new evidence that TLR4–MyD88 signalling is involved in the acute behavioural actions of alcohol in mice.LINKED ARTICLEThis article is commented on by Pandey, pp. 1316–1318 of this issue. To view this commentary visithttp://dx.doi.org/10.1111/j.1476‐5381.2011.01695.x

Adolescents frequently engage in risky behaviours such as binge drinking. Binge drinking, in turn, perturbs neurodevelopment reinforcing reward seeking behaviour in adulthood. Current animal models are limited in their portrayal of this behaviour and the assessment of neuroimmune involvement (specifically the role of Toll-like receptor 4 (TLR4)). Therefore, the aims of this project were to develop a more relevant animal model of adolescent alcohol exposure and to characterise its effects on TLR4 signalling and alcohol-related behaviours later life. Balb/c mice received a short (P22-P25), low dose alcohol binge during in early adolescence, and underwent tests to investigate anxiety (elevated plus maze), alcohol seeking (conditioned place preference) and binge drinking behaviour (drinking in the dark) in adulthood. Four doses of alcohol during adolescence increased alcohol-induced conditioned place preference and alcohol intake in adulthood. However, this model did not affect basal elevated plus maze performance. Subsequent analysis of nucleus accumbal mRNA, revealed increased expression of TLR4-related mRNAs in mice who received alcohol during adolescence. To further elucidate the role of TLR4, (+)-Naltrexone, a biased TLR4 antagonist was administered 30 min before or after the adolescent binge paradigm. When tested in adulthood, (+)-Naltrexone treated mice exhibited reduced alcohol intake however, alcohol seeking and anxiety behaviour was unaltered. This study highlights that even a small amount of alcohol, when given during a critical neurodevelopmental period, can potentiate alcohol-related behaviours and TLR4 activation later in life. Interestingly, attenuation of TLR4 before or after adolescent alcohol exposure reduced only binge alcohol intake in adulthood.

Increasing evidence demonstrates induction of proinflammatory Toll-like receptor (TLR) 2 and TLR4 signaling by morphine and, TLR4 signaling by alcohol; thus indicating a common site of drug action and a potential novel innate immune-dependent hypothesis for opioid and alcohol drug interactions. Hence, the current study aimed to assess the role of TLR2, TLR4, MyD88 (as a critical TLR-signaling participant), NF-κB, Interleukin-1β (IL-1β; as a downstream proinflammatory effector molecule) and the μ opioid receptor (MOR; as a classical site for morphine action) in acute alcohol-induced sedation (4.5g/kg) and alcohol (2.5g/kg) interaction with morphine (5mg/kg) by assessing the loss of righting reflex (LORR) as a measure of sedation. Wild-type male Balb/c mice and matched genetically-deficient TLR2, TLR4, and MyD88 strains were utilized, together with pharmacological manipulation of MOR, NF-κB, TLR4 and Interleukin-1β. Alcohol induced significant LORR in wild-type mice; this was halved by MyD88 and TLR4 deficiency, and surprisingly nearly completely eliminated by TLR2 deficiency. In contrast, the interaction between morphine and alcohol was found to be MOR-, NF-κB-, TLR2- and MyD88-dependent, but did not involve TLR4 or Interleukin-1β. Morphine-alcohol interactions caused acute elevations in microglial cell counts and NF-κB-p65 positive cells in the motor cortex in concordance with wild-type and TLR2 deficient mouse behavioral data, implicating neuroimmunopharmacological signaling as a pivotal mechanism in this clinically problematic drug-drug interaction.

We have previously observed that the non-opioid morphine metabolite, morphine-3-glucuronide, enhances pain via a toll-like receptor 4 (TLR4) dependent mechanism. The present studies were undertaken to determine whether TLR4-dependent pain enhancement generalizes to other classes of glucuronide metabolites. In silico modeling predicted that glucuronic acid alone and ethyl glucuronide, a minor but long-lasting ethanol metabolite, would dock to the same MD-2 portion of the TLR4 receptor complex previously characterized as the docking site for morphine-3-glucuronide. Glucuronic acid, ethyl glucuronide and ethanol all caused an increase in TLR4-dependent reporter protein expression in a cell line transfected with TLR4 and associated co-signaling molecules. Glucuronic acid-, ethyl glucuronide-, and ethanol-induced increases in TLR4 signaling were blocked by the TLR4 antagonists LPS-RS and (+)-naloxone. Glucuronic acid and ethyl glucuronide both caused allodynia following intrathecal injection in rats, which was blocked by intrathecal co-administration of the TLR4 antagonist LPS-RS. The finding that ethyl glucuronide can cause TLR4-dependent pain could have implications for human conditions such as hangover headache and alcohol withdrawal hyperalgesia, as well as suggesting that other classes of glucuronide metabolites could have similar effects.

Microglia and astrocytes, the immunocom -petent cells within the CNS, contribute to the acute and chronic effects of alcohol use; with research over the past decade making it increasingly clear that ethanol induces a neuroinflammatory response. Both acute and chronic ethanol consumption lead to an increase in cytokine production experimen -tally, including, TNF- α, IL-1 β and IL-6

Acute stress has been shown to facilitate the rewarding effects of a number of commonly abused drugs, although the stressor typically must be administered either immediately before or during drug administration and often in the same environment. We have previously reported that a single session of an uncontrollable (inescapable tailshock, IS), but not controllable (escapable tailshock, ES), stressor can enhance the conditioned place preference (CPP) response to morphine, even when stressor and drug administration are separated temporally and spatially. However, this persistent, trans-situational enhancement did not occur to amphetamine CPP.The following experiments were conducted to determine whether the long-term effects of IS on drug reward are specific to opioids.Adult, male Sprague-Dawley rats were exposed to a single session of IS or remained in their home cages (HC). Twenty-four hours later, using an unbiased procedure, CPP conditioning was conducted with either oxycodone (0, 2, or 5 mg/kg, sc), cocaine (0, 1, 5, or 10 mg/kg, ip), or ethanol (0.3, 1, or 2 g/kg, ip). Another group of rats were exposed to IS, ES, or HC treatment and conditioned with oxycodone (5 mg/kg, sc) 24 h later.IS enhanced the subsequent CPP response to oxycodone, but not cocaine or ethanol. This enhancement was dependent on the controllability of the stressor, as ES did not affect oxycodone CPP.These results indicate that the long-term, trans-situational enhancing effect of uncontrollable stress on drug reward is specific to opioids.

Circadian rhythm affects drug-induced reward behaviour and the innate immune system. Peaks in reward-associated behaviour and immune responses typically occur during the active (dark) phase of rodents. While the role of the immune system, specifically, Toll-like receptor 4 (TLR4, an innate immune receptor) in drug-induced reward is becoming increasingly appreciated, it is unclear whether its effects vary according to light-cycle. Therefore, the aim of this study was to characterise the effects of the phase of the light-cycle and the state of the innate immune system on alcohol reward behaviour and subsequently determine whether the efficacy of targeting the immune component of drug reward depends upon the light-cycle. This study demonstrates that mice exhibit greater alcohol-induced conditioned place preference and alcohol two-bottle choice preference during the dark cycle. This effect overlapped with elevations in reward-, thirst- and immune-related genes. Administration of (+)-Naltrexone, a TLR4 antagonist, reduced immune-related gene mRNA expression and alcohol preference with its effects most pronounced during the dark cycle. However, (+)-Naltrexone, like other TLR4 antagonists exhibited off-target side effects, with a significant reduction in overall saccharin intake - an effect likely attributable to a reduction in tyrosine hydroxylase (Th) mRNA expression levels. Collectively, the study highlights a link between a time-of-day dependent influence of TLR4 on natural and alcohol reward-like behaviour in mice.