Antagonising TLR4-TRIF signalling before or after a low-dose alcohol binge during adolescence prevents alcohol drinking but not seeking behaviour in adulthood
Introduction
Adolescence is a unique neurodevelopmental period characterized by an increased sensitivity towards rewarding stimuli and an attenuated sensitivity to aversive stimuli (Spear, 2011). This phenotype causes adolescence to engage in risk-taking behaviors such as unprotected sex, reckless driving and binge drinking (Johnston et al., 2015, Hingson et al., 2003, Hingson et al., 2009). Binge drinking in turn profoundly perturbs neurodevelopment causing a retention of adolescent-like phenotypes such as reward-sensitivity, into adulthood (the “locked-in” hypothesis) (Crews et al., 2016, Doremus-Fitzwater and Spear, 2016). Consequently, individuals that consume alcohol during adolescence are more likely to develop problems associated with alcohol use in adulthood (see Spear, 2011 for review). This finding is reinforced by the link between age of first use and alcohol dependence later in life (DeWit et al., 2000). Crucially, these phenomenon are readily translatable to rodents (Spear, 2011). Adolescent rodents exposed to alcohol exhibit potentiated alcohol-reward behaviors in adulthood as inferred by increased conditioned place preference, self-administration or two bottle choice drinking (Pandey et al., 2015, Alaux-Cantin et al., 2013, Maldonado et al., 2008, Rodd-Henricks et al., 2002). However, the magnitude of this potentiation is variable owing to differences in sex, genetic background, age and the model of adolescent alcohol exposure (Strong et al., 2010, Walker and Ehlers, 2009, Blizard et al., 2004, Siciliano and Smith, 2001). The model of alcohol exposure is a particularly important variable. To reach high blood ethanol concentrations (BECs) researchers often use methods that bypass the natural route of administration (for example, Gass et al., 2014, Gilpin et al., 2012). This in turn, influences the molecular and behavioral responses towards alcohol (Osterndorff-Kahanek et al., 2013, Osterndorff-Kahanek et al., 2015, Gilpin et al., 2012) and consequently, it is unclear how much these models reflect the human condition (Ward et al., 2014).
Despite different exposure methodologies rodent studies have identified multiple mechanisms underlying adolescent alcohol-induced reward sensitivities in adulthood with particular emphasis placed upon the molecular and cellular alterations within the nucleus accumbens, hippocampus and amygdala (Spear and Swartzwelder, 2014). For example, adolescent alcohol exposure reduces the expression of plasticity-related genes (BDNF, ARC and CREB), negative regulators of dopaminergic function (dopamine D2 receptor and GABA receptors) and alters dopaminergic firing and tone within these regions in adulthood (Sakharkar et al., 2016, Pandey et al., 2015, Philpot et al., 2009, Pascual et al., 2009, Pietrzykowski et al., 2008). These alterations enhance an individuals sensitivity towards dopamine-inducing experiences such as alcohol use, and reduced the ability to alter learnt behavior (Vetreno et al., 2015, Alaux-Cantin et al., 2013, Maldonado-Devincci et al., 2010).
Recent research has additionally highlighted the importance of the neuroimmune system in contributing to the adverse neurodevelopmental consequences of adolescent alcohol exposure (Crews et al., 2016, Montesinos et al., 2016a, Montesinos et al., 2016b). Particular emphasis has been placed on Toll-like receptor 4 (TLR4), a pattern recognition receptor broadly expressed throughout the central nervous system (Akira and Takeda, 2004, Bsibsi et al., 2002). Following activation, TLR4 signals via the MyD88 or TRIF pathways culminating in the expression of classical pro-inflammatory cytokines and type 1 interferon's respectively (see Akira and Takeda, 2004 for review). Alcohol indirectly activates TLR4 recruiting MyD88 and TRIF in vitro (Crews et al., 2013, Fernandez-Lizarbe et al., 2009). However, whether both pathways are activated in vivo remains to be determined. Alcohol-induced recruitment of these adapters causes a signaling cascade resulting in the translocation of immune-related transcription to the nucleus. This in turn increases the expression of inflammatory proteins from both microglia and astrocytes (Fernandez-Lizarbe et al., 2009, Blanco et al., 2005). Importantly, TLR4−/− mice display reduced levels of cytokines, chemokines and inflammatory transcription factors immediately following adolescent alcohol exposure and later in adulthood compared to wildtype mice (Montesinos et al., 2016a, Montesinos et al., 2016b, Pascual et al., 2016, Kane et al., 2013). This coincides with reduced synaptic and myelin derangements, long-term aberrant synaptic remodelling, decreased histone acetylation at BDNF and FosB (Montesinos et al., 2016a, Montesinos et al., 2016b). Behaviourally, TLR4−/− mice do not exhibit long-term cognitive impairments (Montesinos et al., 2015), display less anxiety-like and drug seeking behaviour in adulthood compared to wildtype following adolescent exposure (Montesinos et al., 2016a, Montesinos et al., 2016b). While the precise neuroanatomical area underlying the long-term actions of adolescent alcohol-induced TLR4 activation remains to be determined, studies using morphine (another TLR4 agonist) have identified the nucleus accumbens as a key substrate (Schwarz and Bilbo, 2013).
However, TLR4 is pivotal to normal neurodevelopmental processes (see Okun et al., 2011 for review), therefore, studies using TLR4−/− animals are inherently confounded. For example, TLR4−/− mice have higher levels of neurons and relatively fewer glia compared to wildtype mice (Rolls et al., 2007). Further, the use of TLR4−/− mice does not enable researchers to investigate the relative contribution of the MyD88 or TRIF pathways in the behavioral and molecular response to alcohol. Lastly, studies investigating the TLR4 often use excessive doses/treatments of alcohol exposure which may exaggerate endpoints. Therefore, the aims of this study were to determine whether a more relevant model of adolescent alcohol exposure alters reward-related behavior and mRNA and the TLR4 pathway later in life and secondly, to determine the whether pharmacologically attenuating TLR4 prevents any alcohol-induced reward alterations later in life. These alterations were assessed using conditioned place preference, drinking in the dark and the elevated plus maze with the transcription of a selection of gene targets relating to reward (dopaminergic, opioidergic, gabaergic and glutamaterigic processes) and alcohol-induced neuroadaptions (BDNF and CREB) within the nucleus accumbens additionally assessed.
Section snippets
Subjects
Pregnant female Balb/c mice (10–15 days into their gestation cycle) were obtained from the University of Adelaide Laboratory Animal Services, Adelaide, SA, Australia. Following their arrival to the animal facility, mice were housed in light/dark (12/12 h, lights on/off at 7am/7pm respectively) and temperature (23 ± 3 °C) controlled rooms. Food and water was available ad libitum.
After the dams had given birth, their offspring developed undisturbed until postnatal (P) day 22 at which point they
Experiment 1: can a short alcohol exposure during adolescence potentiate anxiety and alcohol-seeking behaviour in adulthood?
An important consideration when examining the effects of adolescent alcohol exposure on later life behaviour is the relative rise in blood alcohol following the initial alcohol experience. One hour after the last gavage tail blood was isolated and BEC was quantified. The gavage model produced a dose dependent increase in blood ethanol ranging from 57 to 431mg/100 mL at the lowest (0.5 g/kg) and highest (3.5 g/kg) doses respectively (effect of dose, F(3. 32) = 319.8, p < 0.0001). The precise
Discussion
Adolescence is a vulnerable stage of neurodevelopment, throughout which the brain undergoes substantial reorganisation and maturation. Exposure to drugs of abuse, in particular alcohol, can perturb normal brain development, reinforcing an immature brain state in both rodents and humans (Spear and Swartzwelder, 2014). As adults, these individuals are at risk of developing psychiatric disorders such as addiction and anxiety disorders (Spear and Swartzwelder, 2014). Results from our study
Acknowledgements
This research was support by grants Australian Research Council Research Fellowship (DP110100297). A portion of this work was supported by the NIH Intramural Research Programs of the National Institute on Drug Abuse (NIDA) and the National Institute of Alcohol Abuse and Alcoholism.
Competing financial interests
The authors declare no competing financial interests.
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