Kimberly Nixon

Microglial activation is not equivalent to neuroinflammation in alcohol-induced neurodegeneration: The importance of microglia phenotype.

Excessive alcohol intake, a defining characteristic of an alcohol use disorder (AUD), results in neurodegeneration in the hippocampus and entorhinal cortex that has been linked to a variety of cognitive deficits. Neuroinflammation is thought to be a factor in alcohol-induced neurodegeneration, and microglia activation is a key but not sole component of an inflammatory response. These experiments investigate the effects of ethanol exposure in a well-accepted model of an AUD on both microglial activation and blood brain barrier disruption (BBB) in order to understand their relationship to classical definitions of inflammation and alcohol-induced neurodegeneration. Following a four-day binge ethanol paradigm, rat hippocampal and entorhinal cortex tissue was examined using three distinct approaches to determine microglia phenotype and BBB disruption: immunohistochemistry, autoradiography, and ELISA. After ethanol exposure, there was an increase in [(3)H]-PK-11195 binding and OX-42 immunoreactivity indicative of microglial activation; however, microglia were not fully activated since both OX-6 and ED-1 immunoreactive microglia were absent. This data was supported by functional evidence as there was no increase in the proinflammatory cytokines IL-6 or TNF-α, but a 26% increase in the anti-inflammatory cytokine, IL-10, and a 38% increase in the growth factor, TGF-β, seven days after exposure. Furthermore, there was no evidence of a disruption of the BBB. These data suggest that the four-day binge model of an AUD, which produces neurodegeneration in corticolimbic regions, does not elicit classical neuroinflammation but instead produces partially activated microglia. Partial activation of microglia following binge ethanol exposure suggest that microglia in this model have beneficial or homeostatic roles rather than directly contributing to neurodegeneration and are a consequence of alcohol-induced-damage instead of the source of damage.

Alcohol inhibition of neurogenesis: A mechanism of hippocampal neurodegeneration in an adolescent alcohol abuse model

Adolescents diagnosed with an alcohol use disorder show neurodegeneration in the hippocampus, a region important for learning, memory, and mood regulation. This study examines a potential mechanism by which excessive alcohol intake, characteristic of an alcohol use disorder, produces neurodegeneration. As hippocampal neural stem cells underlie ongoing neurogenesis, a phenomenon that contributes to hippocampal structure and function, we investigated aspects of cell death and cell birth in an adolescent rat model of an alcohol use disorder. Immunohistochemistry of various markers along with Bromo‐deoxy‐Uridine (BrdU) injections were used to examine different aspects of neurogenesis. After 4 days of binge alcohol exposure, neurogenesis was decreased by 33 and 28% at 0 and 2 days after the last dose according to doublecortin expression. To determine whether this decrease in neurogenesis was due to effects on neural stem cell proliferation, quantification of BrdU‐labeled cells revealed a 21% decrease in the dentate gyrus of alcohol‐exposed brains. Cell survival and phenotype of BrdU‐labeled cells were assessed 28 days after alcohol exposure and revealed a significant, 50% decrease in the number of surviving cells in the alcohol‐exposed group. Reduced survival was supported by significant increases in the number of pyknotic‐, FluoroJade B positive‐, and TUNEL‐positive cells. However, so few cells were TUNEL‐positive that cell death is likely necrotic in this model. Although alcohol decreased the number of newborn cells, it did not affect the percentage of cells that matured into neurons (differentiation). Thus, our data support that in a model of an adolescent alcohol use disorder, neurogenesis is impaired by two mechanisms: alcohol‐inhibition of neural stem cell proliferation and alcohol effects on new cell survival. Remarkably, alcohol inhibition of neurogenesis may outweigh the few dying cells per section, which implies that alcohol inhibition of neurogenesis contributes to hippocampal neurodegeneration in alcohol use disorders.

Adolescent binge alcohol exposure induces long-lasting partial activation of microglia

Accumulating evidence indicates that the adolescent hippocampus is highly susceptible to alcohol-induced structural damage and behavioral deficits. Microglia are vitally important brain constituents needed to support and maintain proper neural function; however, alcohols effects on microglia have only recently gained attention. The microglial response to alcohol during adolescence has yet to be studied; therefore, we examined hippocampal microglial activation in an adolescence binge alcohol exposure model. Adolescent male Sprague-Dawley rats were administered ethanol 3 times/day for 4 days and were sacrificed 2, 7, and 30 days later. Bromo-deoxy-Uridine was injected 2 days after ethanol exposure to label dividing cells. Microglia morphology was scored using the microglia marker Iba-1, while the extent of microglial activation was examined with ED-1, major histocompatibility complex-II (MHC-II), and tumor necrosis factor (TNF)-α expression. Ethanol induced significant morphological change in hippocampal microglia, consistent with activation. In addition, ethanol increased the number of BrdU+ cells throughout all regions of the hippocampus 2 days after the last dose. Confocal microscopy showed that the proliferating BrdU+ cells in each region were Iba-1+ microglia. Importantly, newly born microglia survived and retained their morphological characteristics 30 days after ethanol exposure. Ethanol did not alter hippocampal ED-1, MHC-II, or TNF-α expression, suggesting that a single period of binge ethanol exposure does not induce a full microglial-driven neuroinflammatory response. These results establish that ethanol triggers partial microglial activation in the adolescent hippocampus that persists through early adulthood, suggesting that alcohol exposure during this unique developmental time period has long-lasting consequences.

Distinct cell proliferation events during abstinence after alcohol dependence: microglia proliferation precedes neurogenesis

Excessive alcohol intake characteristic of Alcohol Use Disorders (AUDs) produces neurodegeneration that may recover with abstinence. The mechanism of regeneration is unclear, however neurogenesis from neural stem/progenitor cells is a feasible mechanism of structural plasticity. Therefore, a timecourse of cell proliferation was examined in a rat model of an AUD and showed a striking burst in cell proliferation at 2 days of abstinence preceding the previously reported neurogenic proliferation at 7 days. New cells at 2 days, assessed by bromo-deoxy-uridine incorporation and endogenous markers, were observed throughout hippocampus and cortex. Although the majority of these new cells did not become neurons, neurogenesis was not altered at this specific time point. These new cells expressed a microglia-specific marker, Iba-1, and survived at least 2 months. This first report of microglia proliferation in a model of an AUD suggests that microgliosis could contribute to volume recovery in non-neurogenic regions during abstinence.

Adolescence as a critical window for developing an alcohol use disorder: current findings in neuroscience.

Purpose of review Alcohol consumption during adolescence greatly increases the likelihood that an alcohol use disorder will develop later in life. Elucidating how alcohol impacts the adolescent brain is paramount to understanding how alcohol use disorders arise. This review focuses on recent work addressing alcohols unique effect on the adolescent brain. Recent findings The unique and dynamic state of the developing adolescent brain is discussed with an emphasis on the developmentally distinct effect of alcohol on the dopaminergic reward system and corticolimbic structure and function. Reward neurocircuitry undergoes significant developmental shifts during adolescence, making it particularly sensitive to alcohol in ways that could promote excessive consumption. In addition, developing corticolimbic systems, including the prefrontal cortex and hippocampus, exhibit enhanced vulnerability to alcohol-induced damage. Disruption of white matter integrity, neurotoxicity and inhibition of adult neurogenesis may underlie alcohol-mediated cognitive dysfunction and lead to decreased behavioral control over consumption. Summary In adolescents, alcohol interacts extensively with reward neurocircuitry and corticolimbic structure and function in ways that promote maladaptive behaviors that lead to addiction. Future work is needed to further understand the mechanisms involved in these interactions. Therapeutic strategies that restore proper reward neurochemistry or reverse alcohol-induced neurodegeneration could prove useful in preventing emergence of alcohol use disorders.

Exercise neuroprotection in a rat model of binge alcohol consumption.

BACKGROUND Excessive alcohol intake produces structural and functional deficits in corticolimbic pathways that are thought to underlie cognitive deficits in the alcohol use disorders (AUDs). Animal models of binge alcohol administration support the direct link of high levels of alcohol consumption and neurotoxicity in the hippocampus and surrounding cortex. In contrast, voluntary wheel running enhances hippocampal neurogenesis and generally promotes the health of neurons. METHODS We investigated whether voluntary exercise prior to binge alcohol exposure could protect against alcohol-induced cell loss. Female Long-Evans rats exercised voluntarily for 14 days before undergoing 4 days of binge alcohol consumption. Brains were harvested immediately after the last dose of alcohol and examined for various histological markers of neurodegeneration, including both cell death (FluoroJade B) and cell birth (Ki67) markers. RESULTS Rats that exercised prior to binge exposure were significantly less behaviorally intoxicated, which was not a result of enhanced hepatic metabolism. Rats that exercised prior to binge alcohol consumption had reduced loss of dentate gyrus granule cells and fewer FluoroJade B positive cells in the dentate gyrus and associated entorhinal-perirhinal cortex compared to nonexercisers. However, exercise did not protect against cell death in the piriform cortex nor protect against alcohol-induced decreases in cell proliferation, evidenced by a similar alcohol-induced reduction in Ki67 labeled cells between exercise and sedentary rats. CONCLUSIONS We conclude that exercise can reduce behavioral sensitivity to ethanol intoxication and protect vulnerable brain areas from alcohol-induced cell death. Exercise neuroprotection of alcohol-induced brain damage has important implications in understanding the neurobiology of the AUDs as well as in developing novel treatment strategies.

Long-term suppression of forebrain neurogenesis and loss of neuronal progenitor cells following prolonged alcohol dependence in rats

Alcohol dependence leads to persistent neuroadaptations, potentially related to structural plasticity. Previous work has shown that hippocampal neurogenesis is modulated by alcohol, but effects of chronic alcohol on neurogenesis in the forebrain subventricular zone (SVZ) have not been reported. Effects in this region may be relevant for the impairments in olfactory discrimination present in alcoholism. Here, we examined the effects of prolonged alcohol dependence on neurogenesis. Rats were sacrificed directly after 7 wk of intermittent alcohol vapour exposure, or 3, 7 or 21 d into abstinence. Proliferation was assessed using BrdU and Ki67 immunoreactivity, newly differentiated neurons (neurogenesis) as doublecortin-immunoreactivity (DCX-IR), and neural stem cells using the SOX2 marker. In the dentate gyrus, chronic dependence resulted in a pattern similar to that previously reported for acute alcohol exposure: proliferation and neurogenesis were suppressed by the end of exposure, rebounded on day 3 of abstinence, and returned to control levels by days 7 and 21. In the SVZ, proliferation was also suppressed at the end of alcohol exposure, followed by a proliferation burst 3 d into abstinence. However, in this area, there was a trend for reduced proliferation on days 7 and 21 of abstinence, and this was accompanied by significant suppression of DCX-IR, indicating a long-term suppression of forebrain neurogenesis. Finally, a decrease in the SOX2 stem cell marker was detected at days 7 and 21, suggesting long-term reduction of the SVZ stem cell pool. While suppression of hippocampal neurogenesis by alcohol dependence is transient, the suppression in the forebrain SVZ appears long-lasting.

Similar withdrawal severity in adolescents and adults in a rat model of alcohol dependence

Alcohol use during adolescence leads to increased risk of developing an alcohol use disorder (AUD) during adulthood. Converging evidence suggests that this period of enhanced vulnerability for developing an AUD may be due to the adolescents unique sensitivity and response to alcohol. Adolescent rats have been shown to be less sensitive to alcohol intoxication and withdrawal susceptibility; however, age differences in ethanol pharmacokinetics may underlie these effects. Therefore, this study investigated alcohol intoxication behavior and withdrawal severity using a modified Majchrowicz model of alcohol dependence that has been shown to result in similar blood ethanol concentrations (BECs) despite age differences. Adolescent (postnatal day, PND, 35) and adult rats (PND 70+) received ethanol according to this 4-day binge paradigm and were observed for withdrawal behavior for 17h. As expected, adolescents showed decreased sensitivity to alcohol-induced CNS depression as evidenced by significantly lower intoxication scores. Thus, adolescents received significantly more ethanol each day (12.3+/-0.1g/kg/day) than adults (9.2+/-0.2g/kg/day). Despite greater ethanol dosing in adolescent rats, both adolescent and adult groups had comparable peak BECs (344.5+/-10.2 and 338.5+/-7.8mg/dL, respectively). Strikingly, withdrawal severity was similar quantitatively and qualitatively between adolescent and adult rats. Further, this is the first time that withdrawal behavior has been reported for adolescent rats using this model of alcohol dependence. A second experiment confirmed the similarity in BECs at various time points across the binge. These results demonstrate that after consideration of ethanol pharmacokinetics between adults and adolescents by using a model that produces similar BECs, withdrawal severity is nearly identical. This study, in combination with previous reports on ethanol withdrawal in adolescents and adults, suggests only a BEC-dependent effect of ethanol on withdrawal severity regardless of age.

Adolescent binge alcohol exposure alters hippocampal progenitor cell proliferation in rats: Effects on cell cycle kinetics

Binge alcohol exposure in adolescent rats potently inhibits adult hippocampal neurogenesis by altering neural progenitor cell (NPC) proliferation and survival; however, it is not clear whether alcohol results in an increase or decrease in net proliferation. Thus, the effects of alcohol on hippocampal NPC cell cycle phase distribution and kinetics were assessed in an adolescent rat model of an alcohol use disorder. Cell cycle distribution was measured using a combination of markers (Ki‐67, bromodeoxyuridine incorporation, and phosphohistone H3) to determine the proportion of NPCs within G1, S, and G2/M phases of the cell cycle. Cell cycle kinetics were calculated using a cumulative bromodeoxyuridine injection protocol to determine the effect of alcohol on cell cycle length and S‐phase duration. Binge alcohol exposure reduced the proportion of NPCs in S‐phase, but had no effect on G1 or G2/M phases, indicating that alcohol specifically targets S‐phase of the cell cycle. Cell cycle kinetics studies revealed that alcohol reduced NPC cell cycle duration by 36% and shortened S‐phase by 62%, suggesting that binge alcohol exposure accelerates progression through the cell cycle. This effect would be expected to increase NPC proliferation, which was supported by a slight, but significant increase in the number of Sox‐2+ NPCs residing in the hippocampal subgranular zone following binge alcohol exposure. These studies suggest the mechanism of alcohol inhibition of neurogenesis and also reveal the earliest evidence of the compensatory neurogenesis reaction that has been observed a week after binge alcohol exposure. J. Comp. Neurol. 519:2697–2710, 2011.

Roles of neural stem cells and adult neurogenesis in adolescent alcohol use disorders

This review discusses the contributions of a newly considered form of plasticity, the ongoing production of new neurons from neural stem cells, or adult neurogenesis, within the context of neuropathologies that occur with excessive alcohol intake in the adolescents. Neural stem cells and adult neurogenesis are now thought to contribute to the structural integrity of the hippocampus, a limbic system region involved in learning, memory, behavioral control, and mood. In adolescents with alcohol use disorders (AUDs), the hippocampus appears to be particularly vulnerable to the neurodegenerative effects of alcohol, but the role of neural stem cells and adult neurogenesis in alcoholic neuropathology has only recently been considered. This review encompasses a brief overview of neural stem cells and the processes involved in adult neurogenesis, how neural stem cells are affected by alcohol, and possible differences in the neurogenic niche between adults and adolescents. Specifically, what is known about developmental differences in adult neurogenesis between the adult and adolescent is gleaned from the literature, as well as how alcohol affects this process differently among the age groups. Finally, this review suggests differences that may exist in the neurogenic niche between adults and adolescents and how these differences may contribute to the susceptibility of the adolescent hippocampus to damage. However, many more studies are needed to discern whether these developmental differences contribute to the vulnerability of the adolescent to developing an AUD.