The current study shows that inhibition of acute alcohol-induced pro-inflammation through the use of mice with a genetic deficiency in TLR4 or MyD88, or treatment with the TLR4 antagonist (+)-naloxone, was successful in attenuating acute alcohol-induced sedation and motor dysfunction in mice, as measured by duration of LORR and rotarod performance, respectively. These behavioural actions were unlikely to result from changes in the peripheral or central pharmacokinetics of alcohol. In addition, we demonstrated, at the cellular level, that IκBα protein levels are elevated in response to 30 min of alcohol exposure in mixed hippocampal cells from WT mice, but not in those from Tlr4–/– or Myd88–/– mice. However, acute alcohol exposure did not alter p38, JNK and ERK phosphorylation in vitro or ex vivo. These results provide a mechanistic hypothesis underlying the behavioural observations. Together, these findings suggest that alcohol is able to induce rapid modification of pro-inflammatory mediator signalling within the brain through the TLR4–MyD88 pathway and subsequently alter animal motor behaviour.
Acute alcohol exposure activates the TLR4–MyD88–NF-κB signalling pathway in the brain
Although brain TLR4 signalling, including MAPK and NF-κB pathways, has been demonstrated to be activated in vitro after acute alcohol exposure (Blanco et al., 2005; Fernandez-Lizarbe et al., 2009), as well as in vivo and ex vivo with chronic models (Valles et al., 2004; Alfonso-Loeches et al., 2010; Liu et al., 2011; Pascual et al., 2011), it is still not known whether this effect mechanistically contributes to the acute behavioural effects induced by alcohol. In this study, we have gone one step further by demonstrating that such signalling can occur after even one dose of alcohol. Importantly, our data indicated that the TLR4 signalling in vivo occurs rapidly, as the robust difference between the WT and null mutant groups started 20 min after alcohol administration in rotarod tests and after about 30 min in LORR tests.
To further explore the link between our behavioural findings and TLR4–MyD88 signalling, we analysed a number of cell signalling proteins that could be up-regulated by TLR4 signalling in the cerebellum and hippocampus. The cerebellum was chosen as it is generally considered to control motor activity (Valenzuela et al., 2010) in the brain regions influenced by alcohol (Vilpoux et al., 2009), and we assessed the modification of motor function by alcohol in this study. The hippocampus was investigated since hippocampal microglial activation was induced by adolescent binge alcohol exposure in rats (McClain et al., 2011). As attenuation of microglia, the prime component of the brain's immune system (Streit et al., 2004), inhibited acute alcohol-induced sedation in mice (Wu et al., 2011), the activation of TLR4–MyD88–NFκB signalling may occur in microglia.
Thus, due to the rapid activation of TLR4 signalling by alcohol suggested from the behavioural data, we assessed the phosphorylation of p38, JNK and ERK in MAPK pathway ex vivo in hippocampal or cerebellum tissue as well as in mixed hippocampal cells in vitro following alcohol exposure in an attempt to delineate the mechanism responsible. However, we found that acute alcohol exposure did not affect either p38, JNK or ERK phosphorylation, which differs from previous reports using chronic alcohol treatment ex vivo (Valles et al., 2004; Alfonso-Loeches et al., 2010) and fetal microglial or astrocyte cultures in vitro (Blanco et al., 2005; Fernandez-Lizarbe et al., 2008). This implies that non-MAPK signalling cascades, such as phosphoinositide 3 kinase (PI3K)/AKT pathways (Hua et al., 2007), may be involved in the acute alcohol-induced signalling downstream from TLR4. Recently, it was found that acute alcohol challenge induced a robust AKT phosphorylation in mouse striatum (Bjork et al., 2010), further highlighting the involvement of the non-MAPK pathways. It is possible that the disparity between our findings and those from previous studies may be related to different phenotypes between adult and neonatal glia (Beauvillain et al., 2008). Nonetheless, it is important to note that the concentration of alcohol (50 mM) used in all of the in vitro experiments is based on the maximum serum (85–100 mM) and brain (30–35 mM) alcohol concentrations observed in our pharmacokinetic study, which also show maximal activity in activating immune signalling in glial cells (Blanco et al., 2005; Fernandez-Lizarbe et al., 2008).
Furthermore, IκBα protein levels were determined in vitro in mixed hippocampal cells from WT, Tlr4–/– and Myd88–/– mice. Our previous study demonstrated that alcohol-induced cellular IκBα protein levels changed in a time-dependent manner with an increase at 15 and 30 min, and a decrease at 45 and 60 min following alcohol exposure in WT mouse mixed hippocampal cells (Wu et al., 2011). The time point of 30 min was chosen to match the behavioural response we observed, and we hypothesized that the increased IκBα protein levels following 30 min of alcohol exposure might be as a result of NF-κB activation leading to IκBα protein stabilization, free IκBα from nuclear NF-κB, or increased transcription of IκBα mRNA (Scott et al., 1993; Ferreiro and Komives, 2010). In this study, we have shown that the elevated cellular IκBα protein levels by alcohol in WT cells were not observed in cells from Tlr4–/– or Myd88–/– mice. As IκBα is the main inhibitory protein of NF-κB (Sun et al., 1993), these results imply that acute alcohol exposure induces a modification to the NF-κB cascade following activation of TLR4–MyD88 signalling. In addition, the elevated pro-inflammatory cytokine levels, such as TNF-α, IL-1β and IL-6, in the brains of WT mice seen after chronic alcohol treatment (Alfonso-Loeches et al., 2010), may also be due to alcohol-induced TLR4–NF-κB activation.
Collectively, the current results demonstrate that both a binge drinking dose (3.5 and 4.5 g·kg−1) and a lower moderate dose (2.0 g·kg−1) of alcohol rapidly activates pro-inflammatory signalling cascades within the brain, which appear to be critical to alcohol-induced sedation and motor impairment through activation of TLR4–MyD88-dependent signalling and NF-κB. The possible mechanisms between this immune activation and behavioural effects of alcohol are discussed below. It has been hypothesized that the acute activation of NF-κB leads to the release of pro-inflammatory cytokines, which in turn could modulate neuronal activity in the brain, although the mechanism by which this modulation occurs is only beginning to be understood (Ren and Dubner, 2008). Interestingly, IL-1β signalling, which was activated by acute alcohol administration in our previous study (Wu et al., 2011), drove excitotoxic motor neuron injury (Prow and Irani, 2008). Furthermore, chemokine (C-X-C motif) ligand 12 (CXCL12) may enhance GABA synaptic activity at 5-HT neurons in rats (Heinisch and Kirby, 2010). Therefore, cytokines and chemokines could alter neuronal receptor functions, and these actions raise the possibility that pro-inflammatory mediators could facilitate the activation of GABAA receptors by acute alcohol exposure (Ikonomidou et al., 2000; Mukherjee et al., 2008). Thus, apart from directly acting on neurons, alcohol could modify neuronal receptor signalling indirectly via immune signalling activation, and subsequently induce sedation and motor behaviours.
Alcohol-induced behavioural changes are protected by (+)-naloxone treatment
Signalling by TLR4 occurs in response to both clinically employed opioid antagonists [(–)-isomers] and their non-opioid receptor (+)-isomers (Hutchinson et al., 2010b). In this study, we showed firstly, that in contrast to WT mice, there is no effect of (+)-naloxone treatment in the LORR test when mice are deficient in TLR4 or MyD88. This is consistent with the specificity of (+)-naloxone for the TLR4–MyD88 signalling cascade. Secondly, (+)-naloxone induced an increase in IκBα protein levels 30 min following the initial (+)-naloxone exposure, indicating that the mechanism of (+)-naloxone action may be related to interference of IκBα protein synthesis or degradation. Thirdly, this alteration in IκBα protein levels by (+)-naloxone was TLR4–MyD88-dependent. To maintain physiological relevance, the (+)-naloxone concentration in our in vitro experiments was equivalent to the blood (–)-naloxone concentrations in a previous rodent pharmacokinetic study (Kleiman-Wexler et al., 1989), as there was a paucity of (+)-naloxone pharmacokinetic data available at the time of this study.
Behavioural changes are not the result of modified alcohol pharmacokinetic profiles in null mutant or (+)-naloxone-treated animals
To confirm that the behavioural changes induced by (+)-naloxone and genetic deficiency of either TLR4 or MyD88 were not simply a result of modifying the peripheral or central pharmacokinetics of alcohol, we measured alcohol concentrations following the dosing regimens used in the LORR tests (3.5 g·kg−1 of alcohol). Overall, neither (+)-naloxone treatment nor the absence of TLR4 or MyD88 altered alcohol concentrations in either serum or brain samples.
Because of the decreased alcohol pharmacodynamic responses and unchanged alcohol pharmacokinetics in TLR4 signalling attenuated groups compared with controls, we expected that mice which awoke earlier in the LORR test would have higher peripheral and brain alcohol concentrations following their awakening. However, there was no significant difference in serum or brain alcohol concentrations between groups at the time of waking from alcohol-induced sedation, which may be due to the shallow slopes of the alcohol concentration-time curves.
TLR4–MyD88 signalling plays a pivotal role in the acute behavioural actions of alcohol
Amongst the acute behavioural effects of alcohol, sedation and motor inco-ordination are probably responsible for traffic accident-related deaths in humans and accompany self-administration of alcohol in mice (Chuck et al., 2006). Thus, our results not only suggests that the initial effects of alcohol are related to TLR4 signalling but also may have important clinical applications in binge drinking-related brain conditions and alcohol dependence, which may culminate in preventing traffic accidents and decreasing the social burden of alcohol abuse.
In conclusion, the current study provides new evidence linking the contribution of TLR4–MyD88-dependent signalling to the behavioural response induced by acute alcohol administration. The consequences of blocking TLR4 signalling that support this theory include inhibition of the influence of alcohol on IκBα protein levels and a reduction in the sedative and motor effects of alcohol. Therefore, novel pharmacological strategies targeting TLR4 signalling, such as (+)-naloxone, may have an important and highly relevant clinical application. The use of TLR4 antagonists would potentially also reduce alcohol-induced peripheral TLR4 signalling in the liver and gut (Szabo and Bala, 2010).