Neuroimmune Activation and Microglia Reactivity in Female Rats Following Alcohol Dependence.

The rates of alcohol use disorder among women are growing, yet little is known about how the female brain is affected by alcohol. The neuroimmune system, and specifically microglia, have been implicated in mediating alcohol neurotoxicity, but most preclinical studies have focused on males. Further, few studies have considered changes to the microglial phenotype when examining the effects of ethanol on brain structure and function. Therefore, we quantified microglial reactivity in female rats using a binge model of alcohol dependence, assessed through morphological and phenotypic marker expression, coupled with regional cytokine levels. In a time- and region-dependent manner, alcohol altered the microglial number and morphology, including the soma and process area, and the overall complexity within the corticolimbic regions examined, but no significant increases in the proinflammatory markers MHCII or CD68 were observed. The majority of cytokine and growth factor levels examined were similarly unchanged. However, the expression of the proinflammatory cytokine TNFα was increased, and the anti-inflammatory IL-10, decreased. Thus, female rats showed subtle differences in neuroimmune reactivity compared to past work in males, consistent with reports of enhanced neuroimmune responses in females across the literature. These data suggest that specific neuroimmune reactions in females may impact their susceptibility to alcohol neurotoxicity and other neurodegenerative events with microglial contributions.

Adolescent Intermittent Ethanol Drives Modest Neuroinflammation but Does Not Escalate Drinking in Male Rats.

During adolescence, the brain is highly susceptible to alcohol-induced damage and subsequent neuroimmune responses, effects which may enhance development of an alcohol use disorder (AUD). Neuroimmune reactions are implicated in adolescent alcohol exposure escalating adulthood drinking. Therefore, we investigated whether intermittent alcohol exposure in male, adolescent rats (AIE) escalated adult drinking via two-bottle choice (2BC). We also examined the influence of housing environment across three groups: standard (group-housed with enrichment during 2BC), impoverished (group-housed without enrichment during 2BC), or isolation (single-housed without bedding or enrichment throughout). In the standard group immediately after AIE/saline and after 2BC, we also examined the expression of microglial marker, Iba1, reactive astrocyte marker, vimentin, and neuronal cell death dye, FluoroJade B (FJB). We did not observe an escalation of adulthood drinking following AIE, regardless of housing condition. Further, only a modest neuroimmune response occurred after AIE in the standard group: no significant microglial reactivity or neuronal cell death was apparent using this model, although some astrocyte reactivity was detected in adolescence following AIE that resolved by adulthood. These data suggest that the lack of neuroimmune response in adolescence in this model may underlie the lack of escalation of alcohol drinking, which could not be modified through isolation stress.

Sex differences in microglia function in aged rats underlie vulnerability to cognitive decline.

Aging is associated with a significant shift in immune system reactivity ("inflammaging"), as basal inflammation increases but protective responses to infection are compromised. The immune system exhibits considerable sex differences, which may influence the process of inflammaging, including immune cell activation and behavioral consequences of immune signaling (i.e., impaired memory). Here, we test the hypothesis that sex differences in immune aging may mediate sex differences in cognitive decline. Aged male and female rats received peripheral immune stimulation using lipopolysaccharide (LPS), then molecular, cellular, and behavioral outcomes were assessed. We observed that LPS-treated aged male rats showed cognitive impairment and increased neuroinflammatory responses relative to adult males. In contrast, aged female rats did not display these aging-related deficits. Using transcriptomic and flow cytometry analyses, we further observed significant age- and sex- dependent changes in immune cell populations in the brain parenchyma and meninges, indicating a broad shift in the neuroinflammatory environment that may potentiate these behavioral effects. Ovariectomized aged female rats were also resistant to inflammation-induced memory deficits, indicating that ovarian hormones are not required for the attenuated neuroinflammation in aged females. Overall, our results indicate that males have amplified inflammatory priming with age, which contributes to age-associated cognitive decline. Our findings highlight sexual dimorphism in mechanisms of aging, and suggest that sex is a crucial consideration for identifying therapies for aging and neuroinflammation.

Astrocyte Reactivity and Neurodegeneration in the Female Rat Brain Following Alcohol Dependence.

Recent evidence suggests that alcohol use disorder (AUD) may manifest itself differently in women compared to men. Women experience AUDs on an accelerated timeline and may have certain regional vulnerabilities. In male rats, neuronal cell death and astrocyte reactivity are noted following induction of alcohol dependence in an animal model of an AUD. However, the regional and temporal patterns of neurodegeneration and astrocyte reactivity have yet to be fully examined in females using this model. Therefore, adult female rats were exposed to a 4-day binge model of alcohol dependence followed by different periods of abstinence. Histological markers for FluoroJade B, a label of degenerating neurons, and vimentin, a marker for reactive astrocytes, were utilized. The expression of these markers in cortical and limbic regions was quantified immediately after their last dose (e.g., T0), or 2, 7, and 14 days later. Significant neuronal cell death was noted in the entorhinal cortex and the hippocampus, similar to previous reports in males, but also in several cortical regions not previously observed. Vimentin immunoreactivity was noted in the same regions as previously reported, in addition to three novel regions. Vimentin immunoreactivity also occurred at earlier and later time points in some cortical and hippocampal regions. These data suggest that both neuronal cell death and astrocyte reactivity could be more widespread in females compared to males. Therefore, this study provides a framework for specific regions and time points which should be examined in future studies of alcohol-induced damage that include female rats.

Chronic intermittent ethanol exposure during adolescence produces sex- and age-dependent changes in anxiety and cognition without changes in microglia reactivity late in life.

Binge-like ethanol exposure during adolescence has been shown to produce long lasting effects in animal models including anxiety-like behavior that can last into young adulthood and impairments in cognition that can last throughout most of the lifespan. However, little research has investigated if binge-like ethanol exposure during adolescence produces persistent anxiety-like behavior and concomitantly impairs cognition late in life. Furthermore, few studies have investigated such behavioral effects in both female and male rats over the lifespan. Finally, it is yet to be determined if binge-like ethanol exposure during adolescence alters microglia activation in relevant brain regions late in life. In the present study female and male adolescent rats were exposed to either 3.0 or 5.0 g/kg ethanol, or water control, in a chronic intermittent pattern before being tested in the elevated plus maze and open field task over the next ∼18 months. Animals were then trained in a spatial reference task via the Morris water maze before having their behavioral flexibility tested. Finally, brains were removed, sectioned and presumptive microglia activation determined using autoradiography for [3H]PK11195 binding. Males, but not females, displayed an anxiety-like phenotype initially following the chronic intermittent ethanol exposure paradigm which resolved in adulthood. Further, males but not females had altered spatial reference learning and impaired behavioral flexibility late in life. Conversely, [3H]PK11195 binding was significantly elevated in females compared to males late in life and the level of microglia activation interacted as a function of sex and brain regions, but there was no long-term outcome related to adolescent alcohol exposure. These data further confirm that binge-like ethanol exposure during adolescence produces alterations in behavior that can last throughout the lifespan. In addition, the data suggest that microglia activation late in life is not exacerbated by prior binge-like ethanol exposure during adolescence but the expression is sex- and brain region-dependent across the lifespan.

The neuroimmune system - Where aging and excess alcohol intersect.

As the percentage of the global population over age 65 grows, and with it a subpopulation of individuals with alcohol use disorder (AUD), understanding the effect of alcohol on the aged brain is of utmost importance. Neuroinflammation is implicated in both natural aging as well as alcohol use, and its role in alterations to brain morphology and function may be exacerbated in aging individuals who drink alcohol to excess. The neuroimmune response to alcohol in aging is complex. The few studies investigating this issue have reported heightened basal activity and either hypo- or hyper-reactivity to an alcohol challenge. This review of preclinical research will first introduce key players of the immune system, then explore changes in neuroimmune function with aging or alcohol alone, with discussion of vulnerable brain regions, changes in cytokines, and varied reactions of microglia and astrocytes. We will then consider different levels of alcohol exposure, relevant animal models of AUD, and neuroimmune activation by alcohol across the lifespan. By identifying key findings, challenges, and targets for future research, we hope to bring more attention and resources to this underexplored area of inquiry.

Effects of voluntary adolescent intermittent alcohol exposure and social isolation on adult alcohol intake in male rats.

Initiating alcohol use in adolescence significantly increases the likelihood of developing adult alcohol use disorder (AUD). However, it has been difficult to replicate adolescent alcohol exposure leading to increased adult alcohol intake across differing preclinical models. In the present study, differentially housed male rats (group vs. single cages) were used to determine the effects of voluntary intermittent exposure of saccharin-sweetened ethanol during adolescence on adult intake of unsweetened 20% ethanol. Adolescent male rats were assigned to group- or isolated-housing conditions and underwent an intermittent 2-bottle choice in adolescence (water only or water vs. 0.2% saccharin/20% ethanol), and again in adulthood (water vs. 20% ethanol). Intermittent 2-bottle choice sessions lasted for 24 h, and occurred three days per week, for five weeks. Rats were moved from group or isolated housing to single-housing cages for 2-bottle choice tests and returned to their original housing condition on off days. During adolescence, rats raised in isolated-housing conditions consumed significantly more sweetened ethanol than rats raised in group-housing conditions, an effect that was enhanced across repeated exposures. In adulthood, rats raised in isolated-housing conditions and exposed to sweetened ethanol during adolescence also consumed significantly higher levels of unsweetened 20% ethanol compared to group-housed rats. The effect was most pronounced over the first five re-exposure sessions. Housing conditions alone had little effect on adult ethanol intake. These preclinical results suggest that social isolation stress, combined with adolescent ethanol exposure, may play a key role in adult AUD risk.

Effects of adolescent alcohol exposure via oral gavage on adult alcohol drinking and co-use of alcohol and nicotine in Sprague Dawley rats.

BACKGROUND: Preclinical models simulating adolescent substance use leading to increased vulnerability for substance use disorders in adulthood are needed. Here, we utilized a model of alcohol and nicotine co-use to assess adult addiction vulnerability following adolescent alcohol exposure. METHODS: In Experiment 1, adolescent (PND30) male and female Sprague-Dawley rats received 25% ethanol (EtOH) or a control solution via oral gavage every 8 h, for 2 days. In young adulthood, animals were tested with a 2-bottle choice between H20% and 15% EtOH or 0.2% saccharin/15% EtOH, followed by co-use of oral Sacc/EtOH and operant-based i.v. nicotine (0.03 mg/kg/infusion) self-administration. In Experiment 2, adolescents received control gavage, EtOH gavage, or no-gavage, and were tested in young adulthood in a 2-bottle choice between H20% and 15% EtOH, Sacc/EtOH, or 0.2% saccharin. RESULTS: In Experiment 1, the adolescent EtOH gavage reduced adult EtOH consumption in the 2-bottle choice, but not during the co-use phase. During co-use, Sacc/EtOH served as an economic substitute for nicotine. In Experiment 2, the control gavage increased adult EtOH drinking relative to the no-gavage control group, an effect that was mitigated in the EtOH gavage group. In both experiments, treatment group differences in EtOH consumption were largely driven by males. CONCLUSIONS: EtOH administration via oral gavage in adolescence decreased EtOH consumption in adulthood without affecting EtOH and nicotine co-use. Inclusion of a no-gavage control in Experiment 2 revealed that the gavage procedure increased adult EtOH intake and that including EtOH in the gavage buffered against the effect.

Neuron-Derived Extracellular Vesicles Modulate Microglia Activation and Function.

Microglia act as the immune cells of the central nervous system (CNS). They play an important role in maintaining brain homeostasis but also in mediating neuroimmune responses to insult. The interactions between neurons and microglia represent a key process for neuroimmune regulation and subsequent effects on CNS integrity. However, the molecular mechanisms of neuron-glia communication in regulating microglia function are not fully understood. One recently described means of this intercellular communication is via nano-sized extracellular vesicles (EVs) that transfer a large diversity of molecules between neurons and microglia, such as proteins, lipids, and nucleic acids. To determine the effects of neuron-derived EVs (NDEVs) on microglia, NDEVs were isolated from the culture supernatant of rat cortical neurons. When NDEVs were added to primary cultured rat microglia, we found significantly improved microglia viability via inhibition of apoptosis. Additionally, application of NDEVs to cultured microglia also inhibited the expression of activation surface markers on microglia. Furthermore, NDEVs reduced the LPS-induced proinflammatory response in microglia according to reduced gene expression of proinflammatory cytokines (TNF-α, IL-6, MCP-1) and iNOS, but increased expression of the anti-inflammatory cytokine, IL-10. These findings support that neurons critically regulate microglia activity and control inflammation via EV-mediated neuron-glia communication. (Supported by R21AA025563 and R01AA025591).

Reactive, Adult Neurogenesis From Increased Neural Progenitor Cell Proliferation Following Alcohol Dependence in Female Rats.

Hippocampal neurodegeneration is a consequence of excessive alcohol drinking in alcohol use disorders (AUDs), however, recent studies suggest that females may be more susceptible to alcohol-induced brain damage. Adult hippocampal neurogenesis is now well accepted to contribute to hippocampal integrity and is known to be affected by alcohol in humans as well as in animal models of AUDs. In male rats, a reactive increase in adult hippocampal neurogenesis has been observed during abstinence from alcohol dependence, a phenomenon that may underlie recovery of hippocampal structure and function. It is unknown whether reactive neurogenesis occurs in females. Therefore, adult female rats were exposed to a 4-day binge model of alcohol dependence followed by 7 or 14 days of abstinence. Immunohistochemistry (IHC) was used to assess neural progenitor cell (NPC) proliferation (BrdU and Ki67), the percentage of increased NPC activation (Sox2+/Ki67+), the number of immature neurons (NeuroD1), and ectopic dentate gyrus granule cells (Prox1). On day seven of abstinence, ethanol-treated females showed a significant increase in BrdU+ and Ki67+ cells in the subgranular zone of the dentate gyrus (SGZ), as well as greater activation of NPCs (Sox2+/Ki67+) into active cycling. At day 14 of abstinence, there was a significant increase in the number of immature neurons (NeuroD1+) though no evidence of ectopic neurogenesis according to either NeuroD1 or Prox1 immunoreactivity. Altogether, these data suggest that alcohol dependence produces similar reactive increases in NPC proliferation and adult neurogenesis. Thus, reactive, adult neurogenesis may be a means of recovery for the hippocampus after alcohol dependence in females.

Primed for addiction: A critical review of the role of microglia in the neurodevelopmental consequences of adolescent alcohol drinking.

Alcohol is one of the most widely used recreational substances worldwide, with drinking frequently initiated during adolescence. The developmental state of the adolescent brain makes it vulnerable to initiating alcohol use, often in high doses, and particularly susceptible to alcohol-induced brain changes. Microglia, the brain parenchymal macrophages, have been implicated in mediating some of these effects, though the role that these cells play in the progression from alcohol drinking to dependence remains unclear. Microglia are uniquely positioned to sense and respond to central nervous system insult, and are now understood to exhibit innate immune memory, or "priming," altering their future functional responses based on prior exposures. In alcohol use disorders (AUDs), the role of microglia is debated. Whereas microglial activation can be pathogenic, contributing to neuroinflammation, tissue damage, and behavioral changes, or protective, it can also engage protective functions, providing support and mediating the resolution of damage. Understanding the role of microglia in adolescent AUDs is complicated by the fact that microglia are thought to be involved in developmental processes such as synaptic refinement and myelination, which underlie the functional maturation of multiple brain systems in adolescence. Thus, the role microglia play in the impact of alcohol use in adolescence is likely multifaceted. Long-term sequelae may be due to a failure to recover from EtOH-induced tissue damage, altered neurodevelopmental trajectories, and/or persistent changes to microglial responsivity and function. Here, we review critically the literature surrounding the effects of alcohol on microglia in models of adolescent alcohol misuse. We attempt to disentangle what is known about microglia from other neuroimmune effectors, to which we apply recent discoveries on the role of microglia in development and plasticity. Considered altogether, these studies challenge assumptions that proinflammatory microglia drive addiction. Alcohol priming microglia and thereby perturbing their homeostatic roles in neurodevelopment, especially during critical periods of plasticity such as adolescence, may have more serious implications for the neuropathogenesis of AUDs in adolescents.

Functional Activation of Newborn Neurons Following Alcohol-Induced Reactive Neurogenesis.

Abstinence after alcohol dependence leads to structural and functional recovery in many regions of the brain, especially the hippocampus. Significant increases in neural stem cell (NSC) proliferation and subsequent "reactive neurogenesis" coincides with structural recovery in hippocampal dentate gyrus (DG). However, whether these reactively born neurons are integrated appropriately into neural circuits remains unknown. Therefore, adult male rats were exposed to a binge model of alcohol dependence. On day 7 of abstinence, the peak of reactive NSC proliferation, rats were injected with bromodeoxyuridine (BrdU) to label dividing cells. After six weeks, rats underwent Morris Water Maze (MWM) training then were sacrificed ninety minutes after the final training session. Using fluorescent immunohistochemistry for c-Fos (neuronal activation), BrdU, and Neuronal Nuclei (NeuN), we investigated whether neurons born during reactive neurogenesis were incorporated into a newly learned MWM neuronal ensemble. Prior alcohol exposure increased the number of BrdU+ cells and newborn neurons (BrdU+/NeuN+ cells) in the DG versus controls. However, prior ethanol exposure had no significant impact on MWM-induced c-Fos expression. Despite increased BrdU+ neurons, no difference in the number of activated newborn neurons (BrdU+/c-Fos+/NeuN+) was observed. These data suggest that neurons born during alcohol-induced reactive neurogenesis are functionally integrated into hippocampal circuitry.

Microglia Phenotypes Following the Induction of Alcohol Dependence in Adolescent Rats.

BACKGROUND: Activation of the innate immune system may play a role in the development of alcohol use disorders (AUDs), which often originate with adolescent alcohol abuse. A key player in the innate immune system is microglia, the activation of which occurs along a spectrum from proinflammatory, or M1-like, to anti-inflammatory, or M2-like, phenotypes. METHODS: Adolescent, male rats were gavaged with ethanol (EtOH) or isocaloric control diet every 8 hours for 4 days and then sacrificed at 0, 2, 7, and 14 days later. Microglia were isolated from the entorhinal cortex and hippocampus by Percoll gradient centrifugation, labeled with surface antigens for activation, and analyzed by flow cytometry. Polarization states of microglia, defined as CD11b+ CD45low cells, were determined by the expression of M1 surface markers, major histocompatibility complex (MHC) II, CD32, and CD86, and M2 surface marker, CD206 (mannose receptor). Cytokine gene expression was measured by reverse transcriptase polymerase chain reaction. RESULTS: Isolated cells were a highly enriched population (>95% pure) of microglia/macrophages according to CD11b immunoreactivity. EtOH rats showed the most dramatic increases in microglia activation markers CD11b and CD45, and M1 (MHC-II) and M2 (CD206) markers at T2, when additional M1 markers CD86 and CD32 were also increased. Surprisingly, proinflammatory gene expression of CCL2, IL-1ß, IL-6, and TNF-α generally was decreased at all time points in EtOH rats except for IL-6 which was increased at T0 and TNF-α which was not changed at T0 in either region. Simultaneously, BDNF expression was increased at T2 and T7, while IGF1 and TGF-ß gene expression was decreased. Arginase was also increased at T0 in hippocampus, but not changed by alcohol otherwise. CONCLUSIONS: These data show that microglia phenotype after alcohol dependence is not a simple M1 or M2 classification, though more indicators of an anti-inflammatory phenotype were observed. Determining microglia phenotype is critical for understanding their role in the development of AUDs.

Microglia Dystrophy Following Binge-Like Alcohol Exposure in Adolescent and Adult Male Rats.

Microglia are dynamic cells that have roles in neuronal plasticity as well as in recovery responses following neuronal injury. Although many hypothesize that hyperactivation of microglia contributes to alcohol-induced neuropathology, in other neurodegenerative conditions disruption of normal microglial processes also contributes to neuronal loss, particularly as microglia become dystrophic or dysfunctional. Based on the observation of a striking, abnormal morphology in microglia during binge-like ethanol exposure, the present study investigated the impact of excessive ethanol exposure on microglia number and dystrophic morphology in a model of alcohol dependence that includes neurodegeneration in both adult and adolescent rats. Following 2- and 4-day binge ethanol exposure, the number of microglia was decreased in the hippocampus and the perirhinal and entorhinal cortices of both adult and adolescent rats. Furthermore, a significant number of microglia with a dystrophic morphology were observed in ethanol-exposed tissue, accompanied by a significant decrease in brain-derived neurotrophic factor (BDNF) expression in the hippocampus. Together these findings suggest another means by which microglia may contribute to alcohol-induced neurodegeneration, specifically dystrophic microglia and/or loss of microglia may disrupt homeostatic and recovery mechanisms. These results demonstrate that microglia also degenerate with excessive alcohol exposure, which has important implications for understanding the role of microglia-and specifically their contributions to plasticity and neuronal survival-in neurodegenerative disease.

Effects of ethanol, naltrexone, nicotine and varenicline in an ethanol and nicotine co-use model in Sprague-Dawley rats.

BACKGROUND: As alcohol and nicotine use disorders are entwined, it may be possible to develop a single medication to treat both. We previously developed a model for ethanol (EtOH) and nicotine co-use in female selectively bred alcohol-preferring (P) rats. To model co-use in a genetically diverse population, we adapted the model to outbred Sprague-Dawley rats of both sexes and assessed the effect of drug pretreatments. METHODS: In phase 1, rats were trained in a 2-bottle choice between water and a sweetened or unsweetened EtOH solution in operant chambers. In phase 2, rats were trained in nicotine self-administration under an increasing fixed ratio (FR) schedule with 2 bottles containing water or saccharin-sweetened EtOH also available. In phase 3, rats were pretreated with EtOH (0.5, 1.5 g/kg), naltrexone (0.3 mg/kg), nicotine (0.2, 0.6 mg/kg), varenicline (3.0 mg/kg) or vehicle before the session. RESULTS: Sweetening the EtOH solution was required to obtain pharmacologically relevant levels of consumption in Phase 1, with males showing increased sweetened EtOH preference compared to females. In Phase 2, increasing the FR requirement for nicotine decreased nicotine infusions, but increased EtOH consumption. In Phase 3, EtOH, naltrexone, and nicotine failed to alter EtOH consumption; however, varenicline decreased both EtOH and nicotine intake. CONCLUSIONS: The co-use model was successfully adapted to Sprague-Dawley rats by adding saccharin to the EtOH solution. In contrast to previous results in P rats, varenicline reduced both EtOH and nicotine intake, indicating it may be a useful monotherapy for co-use in a genetically diverse population.

Recovery of Hippocampal-Dependent Learning Despite Blunting Reactive Adult Neurogenesis After Alcohol Dependence.

BACKGROUND: The excessive alcohol drinking that occurs in alcohol use disorder (AUD) causes neurodegeneration in regions such as the hippocampus, though recovery may occur after a period of abstinence. Mechanisms of recovery are not clear, though reactive neurogenesis has been observed in the hippocampal dentate gyrus following alcohol dependence and correlates to recovery of granule cell number. OBJECTIVE: We investigated the role of neurons born during reactive neurogenesis in the recovery of hippocampal learning behavior after 4-day binge alcohol exposure, a model of an AUD. We hypothesized that reducing reactive neurogenesis would impair functional recovery. METHODS: Adult male rats were subjected to 4-day binge alcohol exposure and two approaches were tested to blunt reactive adult neurogenesis, acute doses of alcohol or the chemotherapy drug, temozolomide (TMZ). RESULTS: Acute 5 g/kg doses of EtOH gavaged T6 and T7 days post binge did not inhibit significantly the number of Bromodeoxyuridine-positive (BrdU+) proliferating cells in EtOH animals receiving 5 g/kg EtOH versus controls. A single cycle of TMZ inhibited reactive proliferation (BrdU+ cells) and neurogenesis (NeuroD+ cells) to that of controls. However, despite this blunting of reactive neurogenesis to basal levels, EtOH-TMZ rats were not impaired in their recovery of acquisition of the Morris water maze (MWM), learning similarly to all other groups 35 days after 4-day binge exposure. CONCLUSIONS: These studies show that TMZ is effective in decreasing reactive proliferation/neurogenesis following 4-day binge EtOH exposure, and baseline levels of adult neurogenesis are sufficient to allow recovery of hippocampal function.

Its complicated: The relationship between alcohol and microglia in the search for novel pharmacotherapeutic targets for alcohol use disorders.

Alcohol use disorder (AUD) is a chronic relapsing disorder with wide-ranging health consequences. Alcohol targets the central nervous system producing neurodegeneration and subsequent cognitive and behavioral deficits, but the mechanisms behind these effects remain unclear. Recently, evidence has been mounting for the role of neuroimmune activation in the pathogenesis of AUDs, but our nascent state of knowledge about the interaction of alcohol with the neuroimmune system supports that the relationship is complicated. As the resident macrophage of the central nervous system, microglia are a central focus. Human and animal research on the interplay between microglia and alcohol in AUDs has proven to be complex, and though early research focused on a pro-inflammatory phenotype of microglia, the anti-inflammatory and homeostatic roles of microglia must be considered. How these new roles for microglia should be incorporated into our thinking about the neuroimmune system in AUDs is discussed in the context of developing novel pharmacotherapies for AUDs.

An improved model of ethanol and nicotine co-use in female P rats: Effects of naltrexone, varenicline, and the selective nicotinic α6ß2* antagonist r-bPiDI.

Background Although pharmacotherapies are available for alcohol (EtOH) or tobacco use disorders individually, it may be possible to develop a single pharmacotherapy to treat heavy drinking tobacco smokers by capitalizing on the commonalities in their mechanisms of action. Methods Female alcohol-preferring (P) rats were trained for EtOH drinking and nicotine self-administration in two phases: (1) EtOH alone (0 vs. 15% EtOH, 2-bottle choice) and (2) concomitant access, during which EtOH access continued with access to nicotine (0.03 mg/kg/infusion, i.v.) using a 2-lever choice procedure (active vs. inactive lever) in which the fixed ratio (FR) requirement was gradually increased to FR30. When stable co-use was obtained, rats were pretreated with varying doses of naltrexone, varenicline, or r-bPiDI, an α6ß2* subtype-selective nicotinic acetylcholine receptor antagonist shown previously to reduce nicotine self-administration. Results While EtOH intake was initially suppressed in phase 2 (co-use), pharmacologically relevant intake for both substances was achieved by raising the "price" of nicotine to FR30. In phase 2, naltrexone decreased EtOH and water consumption but not nicotine intake; in contrast, naltrexone in phase 1 (EtOH only) did not significantly alter EtOH intake. Varenicline and r-bPiDI in phase 2 both decreased nicotine self-administration and inactive lever pressing, but neither altered EtOH or water consumption. Conclusions These results indicate that increasing the "price" of nicotine increases EtOH intake during co-use. Additionally, the efficacy of naltrexone, varenicline, and r-bPiDI was specific to either EtOH or nicotine, with no efficacy for co-use. Nevertheless, future studies on combining these treatments may reveal synergistic efficacy.

Effects of the nicotinic agonist varenicline, nicotinic antagonist r-bPiDI, and DAT inhibitor (R)-modafinil on co-use of ethanol and nicotine in female P rats.

RATIONALE: Co-users of alcohol and nicotine are the largest group of polysubstance users worldwide. Commonalities in mechanisms of action for ethanol (EtOH) and nicotine proposes the possibility of developing a single pharmacotherapeutic to treat co-use. OBJECTIVES: Toward developing a preclinical model of co-use, female alcohol-preferring (P) rats were trained for voluntary EtOH drinking and i.v. nicotine self-administration in three phases: (1) EtOH alone (0 vs. 15%, two-bottle choice), (2) nicotine alone (0.03 mg/kg/infusion, active vs. inactive lever), and (3) concurrent access to both EtOH and nicotine. Using this model, we examined the effects of (1) varenicline, a nicotinic acetylcholine receptor (nAChR) partial agonist with high affinity for the α4ß2* subtype; (2) r-bPiDI, a subtype-selective antagonist at α6ß2* nAChRs; and (3) (R)-modafinil, an atypical inhibitor of the dopamine transporter (DAT). RESULTS: In phases 1 and 2, pharmacologically relevant intake of EtOH and nicotine was achieved. In the concurrent access phase (phase 3), EtOH consumption decreased while nicotine intake increased relative to phases 1 and 2. For drug pretreatments, in the EtOH access phase (phase 1), (R)-modafinil (100 mg/kg) decreased EtOH consumption, with no effect on water consumption. In the concurrent access phase, varenicline (3 mg/kg), r-bPiDI (20 mg/kg), and (R)-modafinil (100 mg/kg) decreased nicotine self-administration but did not alter EtOH consumption, water consumption, or inactive lever pressing. CONCLUSIONS: These results indicate that therapeutics which may be useful for smoking cessation via selective inhibition of α4ß2* or α6ß2* nAChRs, or DAT inhibition, may not be sufficient to treat EtOH and nicotine co-use.