Innate immune factors modulate ethanol interaction with GABAergic transmission in mouse central amygdala
Introduction
Recent evidence points to a role for neuroimmune mechanisms, and particularly the innate immunity mediated by the toll-like receptors (TLRs), in ethanol effects and drinking (for review see (Crews et al., 2011, Harris and Blednov, 2012). TLR4 plays an especially critical role in innate immunity and this pathway is activated by both exogenous [e.g., LPS, exhibiting a microbe-/pathogen-associated molecular pattern (MAMP/PAMP)] and endogenous signals or ligands [e.g., high mobility group box 1, heat shock proteins, nucleic acids and fibronectin, exhibiting damage-associated molecular patterns (DAMPs)] (Piccinini and Midwood, 2010). Activation of TLR4 triggers transcriptional activation of pro-interleukin-1β and TNFα and initiation of the innate immune response (Hanamsagar et al., 2012, Kielian, 2009).
Evidence supporting a key role of TLR4 in alcohol effects and drinking includes the following: 1) the alcohol-induced activation of glia, induction of inflammatory mediators, apoptosis, and behavioral and anxiety impairments seen in WT mice are not found in TLR4 deficient mice (Alfonso-Loeches and Guerri, 2011, Alfonso-Loeches et al., 2010, Alfonso-Loeches et al., 2012, Blanco and Guerri, 2007, Blanco et al., 2005, Fernandez-Lizarbe et al., 2013, Fernandez-Lizarbe et al., 2009, Pascual et al., 2011, Valles et al., 2004, Wu et al., 2012); 2) TLR4 is involved in excessive alcohol drinking in preclinical models (Blednov et al., 2011a, Blednov et al., 2012, Liu et al., 2011, Mulligan et al., 2006); 3) alcohol releases endogenous ligands for TLR4 in brain (Crews et al., 2013, He and Crews, 2008, Vetreno and Crews, 2012); and 4) LPS leaks from the gut in human alcoholics to activate pro-inflammatory signaling that may contribute to neuroinflammation and neurodegeneration (Qin et al., 2007). Examination of the TLR4 inflammatory pathways suggests possible approaches to study ethanol effects and drinking. Notably, knockout of the accessory protein CD14, that plays a critical role in LPS activation of TLR4, reduces ethanol preference and blocks the LPS-induced increase in ethanol drinking seen in wild type (WT) mice (Blednov et al., 2011a, Blednov et al., 2012). The reduction of TLR4 expression in the central nucleus of the amygdala (CeA) modulates ethanol binge drinking in a mouse model via a GABAA receptor effect (Liu et al., 2011). Moreover, it has been shown that TLR4–MyD88 signaling is involved in the acute behavioral actions of alcohol, as both pharmacological inhibition of TLR4 signaling with (+)-naloxone, a TLR4-MD-2 complex inhibitor and genetic deficiency of TLR4 or MyD88 significantly reduced the sedation and motor impairment induced by a single dose of alcohol in mice (Wu et al., 2012). Ethyl glucuronide, an ethanol metabolite, causes TLR4-dependent pain allodynia that can be blocked by (+)-naloxone (Lewis et al., 2013). Naloxone has two stereotactic isoforms, (+)-naloxone and (−)-naloxone, that are both potent TLR4 signaling inhibitors (Hutchinson et al., 2008). Whereas (+)-naloxone is selective for TLR4, (−)-naloxone also acts on opioid receptors (Hutchinson et al., 2008). Because another opioid antagonist, naltrexone, is now used in the treatment of alcohol addiction (Jarosz et al., 2013, Thorsell, 2013), there has been an effort to evaluate the therapeutic potential of TLR4-MD-2 complex specific (+)-naloxone and (+)-naltrexone.
The CeA, a major component of the extended amygdala (Heimer and Alheid, 1991), is a brain region known to be critically involved in anxiety and fear-conditioning, as well as in alcohol and drug dependence (Davis and Shi, 1999, Koob and Volkow, 2010, Rosen, 2004). In alcohol dependence, the CeA participates in the learning of stimulus-reward responses and mediation of alcohol’s motivational effects, self-administration, and stress-induced reinstatement of drinking (Koob, 1998, Koob and Volkow, 2010). The great majority of CeA neurons are GABAergic, and the GABAergic system is a key player in ethanol effects in the CeA (Nie et al., 2004, Nie et al., 2009, Roberto et al., 2008, Roberto et al., 2003, Roberto et al., 2004a, Siggins et al., 2005).
Knocking out TLR4 or CD14 reduced ethanol drinking and ethanol-related behavior in rodents (Alfonso-Loeches et al., 2010, Blednov et al., 2011a, Blednov et al., 2012, Pascual et al., 2011). Importantly, decreased TLR4 expression in CeA but not in basolateral amygdala (BLA) reduced ethanol binge drinking, indicating a critical role of the TLR4 system in the CeA for ethanol drinking (Liu et al., 2011). Although TLR4 receptors are expressed primarily by microglia in the CNS (Lehnardt et al., 2002, Chakravarty and Herkenham, 2005, Pascual et al., 2012), several studies also have shown neuronal expression of TLR4 (Acosta and Davies, 2008, Okun et al., 2011, Rolls et al., 2007, Tu et al., 2011). In the CeA, TLR4 receptors appear to be expressed in neurons, and their expression is regulated by the α2 GABAA subunit (Liu et al., 2011). Overall, these findings indicate that both TLR4 and the GABAergic system, and their cellular interactions in the CeA, may play an important role in ethanol drinking. However, little is known about the cellular aspects of TLR4 activation on neurophysiology and GABAergic transmission or on ethanol-induced potentiation of GABAergic transmission in the CeA (Bajo et al., 2008, Cruz et al., 2011, Roberto et al., 2012, Roberto et al., 2003, Roberto et al., 2004b). Therefore, in the present study we explored these issues using electrophysiological methods in CeA slices from CD14 KO mice, with exogenous administration of LPS and the TLR4 antagonist (+)-naloxone to activate and inhibit TLR4, respectively. We report that acute ethanol effects on GABAergic transmission in the CeA involve, or are mimicked by, components of the innate immune system such as TLR4 and CD14.
Section snippets
Slice Preparation
We prepared in vitro brain slices (300 and 400 μm thick for whole-cell and sharp electrode recordings, respectively) containing CeA as previously described (Bajo et al., 2008, Bajo et al., 2011) from male (20–30 weeks old; 25–31 g) C57Bl/6J mice (Jackson Laboratory and the rodent breeding colony of The Scripps Research Institute) and from male CD14 KO mice (provided by Drs. Blednov and Harris of The University of Texas at Austin; see (Blednov et al., 2011a)). For more detailed information on the
Results
We recorded from a total of 181 mouse CeA neurons from control (WT; n = 99) and null mutant (CD14 KO; n = 82) mice and found no significant differences in passive membrane properties or basal GABAergic transmission (Student’s t-test; see Table S1 in SI). For example, neither the mean amplitudes of GABAA-IPSPs evoked by a half-maximal stimulation intensity, nor paired-pulse facilitation, were significantly different between WT and CD14 KO mice (Table S1 in SI). Thus, expression of CD14 does not
Discussion
To our knowledge this is the first report of the effects of LPS on GABAergic transmission in the CeA and identifies a role for CD14 and TLR4 in acute ethanol effects on this transmission responses. Our data indicate that moderate (44 mM) but not high ethanol concentrations (100 mM) require CD14 for augmentation of GABAergic neurotransmission. In addition, our findings with (+)-naloxone suggest that the TLR4-MD-2 complex may be involved in the early acute effects of 44 mM ethanol, but may not be
Funding and disclosure
Support for this study was provided by NIH/NIAAA Grants U01-AA013498, U01-AA013520, and U01-AA013517 (as part of the INIA West consortium). A portion of this work was supported by the Intramural Research Programs of the National Institute on Drug Abuse and the National Institute on Alcohol Abuse and Alcoholism. The authors declare no conflict of interest. (+)-naloxone was provided by Dr. George Koob and synthesized by Drs. Kenner Rice from the NIH/NIAA and Edward Roberts from The Scripps
Acknowledgements
We thank Drs. George Koob, Floyd Bloom and Cecilia Borghese for their critical reviews and comments on the manuscript, Dr. Logrip for her help with statistical analyses, and Dr. George Koob for the gift of (+)-naloxone. The Scripps Research Institute’s manuscript number for this paper is 24053.
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