Impact of Elevated Brain IL-6 in Transgenic Mice on the Behavioral and Neurochemical Consequences of Chronic Alcohol Exposure.

Alcohol consumption activates the neuroimmune system of the brain, a system in which brain astrocytes and microglia play dominant roles. These glial cells normally produce low levels of neuroimmune factors, which are important signaling factors and regulators of brain function. Alcohol activation of the neuroimmune system is known to dysregulate the production of neuroimmune factors, such as the cytokine IL-6, thereby changing the neuroimmune status of the brain, which could impact the actions of alcohol. The consequences of neuroimmune-alcohol interactions are not fully known. In the current studies we investigated this issue in transgenic (TG) mice with altered neuroimmune status relative to IL-6. The TG mice express elevated levels of astrocyte-produced IL-6, a condition known to occur with alcohol exposure. Standard behavioral tests of alcohol drinking and negative affect/emotionality were carried out in homozygous and heterozygous TG mice and control mice to assess the impact of neuroimmune status on the actions of chronic intermittent alcohol (ethanol) (CIE) exposure on these behaviors. The expressions of signal transduction and synaptic proteins were also assessed by Western blot to identify the impact of alcohol-neuroimmune interactions on brain neurochemistry. The results from these studies show that neuroimmune status with respect to IL-6 significantly impacts the effects of alcohol on multiple levels.

Neuroimmune interactions with binge alcohol drinking in the cerebellum of IL-6 transgenic mice.

The neuroimmune system of the brain, which is comprised primarily of astrocytes and microglia, regulates a variety of homeostatic mechanisms that underlie normal brain function. Numerous conditions, including alcohol consumption, can disrupt this regulatory process by altering brain levels of neuroimmune factors. Alcohol and neuroimmune factors, such as proinflammatory cytokines IL-6 and TNF-alpha, act at similar targets in the brain, including excitatory and inhibitory synaptic transmission. Thus, alcohol-induced production of IL-6 and/or TNF-alpha could be important contributing factors to the effects of alcohol on the brain. Recent studies indicate that IL-6 plays a role in alcohol drinking and the effects of alcohol on the brain activity following the cessation of alcohol consumption (post-alcohol period), however information on these topics is limited. Here we used homozygous and heterozygous female and male transgenic mice with increased astrocyte expression of IL-6 to examined further the interactions between alcohol and IL-6 with respect to voluntary alcohol drinking, brain activity during the post-alcohol period, IL-6 signal transduction, and expression of synaptic proteins. Wildtype littermates (WT) served as controls. The transgenic mice model brain neuroimmune status with respect to IL-6 in subjects with a history of persistent alcohol use. Results showed a genotype dependent reduction in voluntary alcohol consumption in the Drinking in the Dark protocol and in frequency-dependent relationships between brain activity in EEG recordings during the post-alcohol period and alcohol consumption. IL-6, TNF-alpha, IL-6 signal transduction partners pSTAT3 and c/EBP beta, and synaptic proteins were shown to play a role in these genotypic effects.

Alcohol alters IL-6 Signal Transduction in the CNS of Transgenic Mice with Increased Astrocyte Expression of IL-6.

Neuroimmune factors, including the cytokine interleukin-6 (IL-6), are important chemical regulators of central nervous system (CNS) function under both physiological and pathological conditions. Elevated expression of IL-6 occurs in the CNS in a variety of disorders associated with altered CNS function, including excessive alcohol use. Alcohol-induced production of IL-6 has been reported for several CNS regions including the cerebellum. Cerebellar actions of alcohol occur through a variety of mechanisms, but alcohol-induced changes in signal transduction, transcription, and translation are known to play important roles. IL-6 is an activator of signal transduction that regulates gene expression. Thus, alcohol-induced IL-6 production could contribute to cerebellar effects of alcohol by altering gene expression, especially under conditions of chronic alcohol abuse, where IL-6 levels could be habitually elevated. To gain an understanding of the effects of alcohol on IL-6 signal transduction, we studied activation/expression of IL-6 signal transduction partners STAT3 (Signal Transducer and Activator of Transcription), CCAAT-enhancer binding protein (C/EBP) beta, and p42/p44 mitogen-activated protein kinase (MAPK) at the protein level. Cerebella of transgenic mice that express elevated levels of astrocyte produced IL-6 in the CNS were studied. Results show that the both IL-6 and chronic intermittent alcohol exposure/withdrawal affect IL-6 signal transduction partners and that the actions of IL-6 and alcohol interact to alter activation/expression of IL-6 signal transduction partners. The alcohol/IL-6 interactions may contribute to cerebellar actions of alcohol, whereas the effects of IL-6 alone may have relevance to cerebellar changes occurring in CNS disorders associated with elevated levels of IL-6.

Altered brain activity during withdrawal from chronic alcohol is associated with changes in IL-6 signal transduction and GABAergic mechanisms in transgenic mice with increased astrocyte expression of IL-6.

Interleukin-6 (IL-6) is an important neuroimmune factor that is increased in the brain by alcohol exposure/withdrawal and is thought to play a role in the actions of alcohol on the brain. To gain insight into IL-6/alcohol/withdrawal interactions and how these interactions affect the brain, we are studying the effects of chronic binge alcohol exposure on transgenic mice that express elevated levels of IL-6 in the brain due to increased astrocyte expression (IL-6 tg) and their non-transgenic (non-tg) littermate controls. IL-6/alcohol/withdrawal interactions were identified by genotypic differences in spontaneous brain activity in electroencephalogram (EEG) recordings from the mice, and by Western blot analysis of protein activation or expression in hippocampus obtained from the mice after the final alcohol withdrawal period. Results from EEG studies showed frequency dependent genotypic differences in brain activity during withdrawal. For EEG frequencies that were affected by alcohol exposure/withdrawal in both genotypes, the nature of the effect was similar, but differed across withdrawal cycles. Differences between IL-6 tg and non-tg mice were also observed in Western blot studies of the activated form of STAT3 (phosphoSTAT3), a signal transduction partner of IL-6, and subunits of GABAA receptors (GABAAR). Regression analysis revealed that pSTAT3 played a more prominent role during withdrawal in the IL-6 tg mice than in the non-tg mice, and that the role of GABAAR alpha-5 and GABAAR alpha-1 in brain activity varied across genotype and withdrawal. Taken together, our results suggest that IL-6 can significantly impact mechanisms involved in alcohol withdrawal.

Pharmacological Targeting the REV-ERBs in Sleep/Wake Regulation.

The circadian clock maintains appropriate timing for a wide range of behaviors and physiological processes. Circadian behaviors such as sleep and wakefulness are intrinsically dependent on the precise oscillation of the endogenous molecular machinery that regulates the circadian clock. The identical core clock machinery regulates myriad endocrine and metabolic functions providing a link between sleep and metabolic health. The REV-ERBs (REV-ERBα and REV-ERBß) are nuclear receptors that are key regulators of the molecular clock and have been successfully targeted using small molecule ligands. Recent studies in mice suggest that REV-ERB-specific synthetic agonists modulate metabolic activity as well as alter sleep architecture, inducing wakefulness during the light period. Therefore, these small molecules represent unique tools to extensively study REV-ERB regulation of sleep and wakefulness. In these studies, our aim was to further investigate the therapeutic potential of targeting the REV-ERBs for regulation of sleep by characterizing efficacy, and optimal dosing time of the REV-ERB agonist SR9009 using electroencephalographic (EEG) recordings. Applying different experimental paradigms in mice, our studies establish that SR9009 does not lose efficacy when administered more than once a day, nor does tolerance develop when administered once a day over a three-day dosing regimen. Moreover, through use of a time response paradigm, we determined that although there is an optimal time for administration of SR9009 in terms of maximal efficacy, there is a 12-hour window in which SR9009 elicited a response. Our studies indicate that the REV-ERBs are potential therapeutic targets for treating sleep problems as those encountered as a consequence of shift work or jet lag.

Transgenic mice with increased astrocyte expression of IL-6 show altered effects of acute ethanol on synaptic function.

A growing body of evidence has revealed that resident cells of the central nervous system (CNS), and particularly the glial cells, comprise a neuroimmune system that serves a number of functions in the normal CNS and during adverse conditions. Cells of the neuroimmune system regulate CNS functions through the production of signaling factors, referred to as neuroimmune factors. Recent studies show that ethanol can activate cells of the neuroimmune system, resulting in the elevated production of neuroimmune factors, including the cytokine interleukin-6 (IL-6). Here we analyzed the consequences of this CNS action of ethanol using transgenic mice that express elevated levels of IL-6 through increased astrocyte expression (IL-6-tg) to model the increased IL-6 expression that occurs with ethanol use. Results show that increased IL-6 expression induces neuroadaptive changes that alter the effects of ethanol. In hippocampal slices from non-transgenic (non-tg) littermate control mice, synaptically evoked dendritic field excitatory postsynaptic potential (fEPSP) and somatic population spike (PS) at the Schaffer collateral to CA1 pyramidal neuron synapse were reduced by acute ethanol (20 or 60 mM). In contrast, acute ethanol enhanced the fEPSP and PS in hippocampal slices from IL-6 tg mice. Long-term synaptic plasticity of the fEPSP (i.e., LTP) showed the expected dose-dependent reduction by acute ethanol in non-tg hippocampal slices, whereas LTP in the IL-6 tg hippocampal slices was resistant to this depressive effect of acute ethanol. Consistent with altered effects of acute ethanol on synaptic function in the IL-6 tg mice, EEG recordings showed a higher level of CNS activity in the IL-6 tg mice than in the non-tg mice during the period of withdrawal from an acute high dose of ethanol. These results suggest a potential role for neuroadaptive effects of ethanol-induced astrocyte production of IL-6 as a mediator or modulator of the actions of ethanol on the CNS, including persistent changes in CNS function that contribute to cognitive dysfunction and the development of alcohol dependence.

Strain-specific viral distribution and neuropathology of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) is a naturally occurring lentivirus of domestic cats, and is the causative agent of feline AIDS. Similar to human immunodeficiency virus (HIV), the pathogenesis of FIV involves infection of lymphocytes and macrophages, and results in chronic progressive immune system collapse and death. Neuropathologic correlates of FIV infection have not yet been elucidated, and may be relevant to understanding HIV-associated neurologic disease (neuroAIDS). As in HIV, FIV strains have been shown to express differential tendencies towards development of clinical neuroAIDS. To interrogate viral genetic determinants that might contribute to neuropathogenicity, cats were exposed to two well-characterized FIV strains with divergent clinical phenotypes and a chimeric strain as follows: FIV(PPR) (PPR, relatively apathogenic but associated with neurologic manifestations), FIV(C36) (C36, immunopathogenic but without associated neurologic disease), and Pcenv (a chimeric virus consisting of a PPR backbone with substituted C36 env region). A sham inoculum control group was also included. Peripheral nerve conduction velocity, CNS imaging studies, viral loads and hematologic analysis were performed over a 12 month period. At termination of the study (350 days post-inoculation), brain sections were obtained from four anatomic locations known to be involved in human and primate lentiviral neuroAIDS. Histological and immunohistochemical evaluation with seven markers of inflammation revealed that Pcenv infection resulted in mild inflammation of the CNS, microglial activation, neuronal degeneration and apoptosis, while C36 and PPR strains induced minimal neuropathologic changes. Conduction velocity aberrations were noted peripherally in all three groups at 63 weeks post-infection. Pcenv viral load in this study was intermediate to the parental strains (C36 demonstrating the highest viral load and PPR the lowest). These results collectively suggest that (i) 3' C36 genomic elements contribute to viral replication characteristics, and (ii) 5' PPR genomic elements contribute to CNS manifestations. This study illustrates the potential for FIV to provide valuable information about neuroAIDS pathogenesis related to genotype and viral kinetics, as well as to identify strains useful to evaluation of therapeutic intervention.

Biochemical modulation of the sleep-wake cycle: endogenous sleep-inducing factors.

Regulation of the sleep-wake cycle involves diverse brain circuits and molecules. Further complexity has been introduced by the recognition of sleep-promoting factors that accumulate in the brain naturally or during prolonged waking. The variety of sleep-inducing molecules includes peptides, cytokines, and lipids. With regard to the lipids, current evidence indicates the existence of endogenous lipids, called endocannabinoids, that mimic the pharmacological actions of the psychoactive ingredient of marijuana and that are likely to be essential factors in sleep promotion. This Mini-Review presents current knowledge concerning the role of endogenous compounds with sleep-promoting properties.

Cortistatin promotes and negatively correlates with slow-wave sleep.

Sleep need is characterized by the level of slow-wave activity (SWA) and increases with time spent awake. The molecular nature of this sleep homeostatic process is practically unknown. Here, we show that intracerebroventricular administration of the neuropeptide, cortistatin (CST-14), enhances EEG synchronization by selectively promoting deep slow-wave sleep (SWS) during both the light and dark period in rats. CST-14 also increases the level of slow-wave activity (SWA) within deep SWS during the first two hours following CST-14 administration. Steady-state levels of preprocortistatin mRNA oscillate during the light:dark cycle and are four-fold higher upon total 24-h sleep deprivation, returning progressively to normal levels after eight hours of sleep recovery. Preprocortistatin mRNA is expressed upon sleep deprivation in a particular subset of cortical interneurons that colocalize with c-fos. In contrast, the number of CST-positive cells coexpressing pERK1/2 decreases under sleep deprivation. The capacity of CST-14 to increase SWA, together with preprocortistatin's inverse correlation with time spent in SWS, suggests a potential role in sleep homeostatic processes.

Injection of neuropeptide W into paraventricular nucleus of hypothalamus increases food intake.

Neuropeptide W (NPW) is an endogenous ligand for G protein-coupled receptor 7 (GPR7). There are two forms of the peptide, designated as neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). In the current study we found that intracerebroventricular administration of NPW23 increased c-Fos immunoreactivity (IR) in a variety of brain sites, many of which are involved in the regulation of feeding. In particular, we noted that c-Fos IR levels were increased in hypocretin-expressing neurons in the perifornical region of the lateral hypothalamus (LH). We then studied whether injection of NPW23 into the paraventricular nucleus of the hypothalamus (PVN) and the LH increased food intake over a 24-h time period. Intra-PVN injection of NPW23 at doses ranging from 0.1 to 3 nmol increased feeding for up to 4 h, and doses ranging from 0.3 to 3 nmol increased feeding for up to 24 h. In contrast, only the 3-nmol dose of NPW23 increased feeding after administration into the LH. Together, these data suggest a modulatory role for NPW in the control of food intake.

Profound increase in sensitivity to glutamatergic- but not cholinergic agonist-induced seizures in transgenic mice with astrocyte production of IL-6.

Transgenic mice with glial fibrillary acidic protein (GFAP) promoter driven-astrocyte production of the cytokines interleukin-6 (IL-6) and tumor necrosis factor (TNF) were used to determine whether the pre-existing production of these cytokines in vivo might modulate the sensitivity of neurons to excitotoxic agents. Low doses of kainic acid (5 mg/kg) that produced little or no behavioral or electroencephalogram (EEG) alterations in wild type or glial fibrillary acidic protein (GFAP)-TNF animals induced severe tonic-clonic seizures and death in GFAP-IL6 transgenic mice of 2 or 6 months of age. GFAP-IL6 mice were also significantly more sensitive to N-methyl-D-aspartate (NMDA)- but not pilocarpine-induced seizures. Kainic acid uptake in the brain of the GFAP-IL6 mice was higher in the cerebellum but not in other regions. Kainic acid binding in the brain of GFAP-IL6 mice had a similar distribution and density as wild type controls. In the hippocampus of GFAP-IL6 mice that survived low dose kainic acid, there was no change in the extent of either neurodegeneration or astrocytosis. Immunostaining revealed degenerative changes in gamma aminobutyric acid (GABA)- and parvalbumin-positive neurons in the hippocampus of 2-month-old GFAP-IL6 mice which progressed to the loss of these cells at 6 months of age. Thus, GFAP-IL6 but not GFAP-TNF mice showed markedly enhanced sensitivity to glutamatergic- but not cholinergic-induced seizures and lethality. This may relate, in part, to a compromise of inhibitory interneuron function. Therefore, pre-existing IL-6 production and inflammation in the central nervous system (CNS) not only causes spontaneous neurodegeneration but also synergizes with other neurotoxic insults to induce more severe acute functional neurological impairment.