Acute and chronic ethanol exposure differentially regulate CB1 receptor function at glutamatergic synapses in the rat basolateral amygdala
Introduction
Dependence-induce anxiety is a significant risk factor for relapse to alcohol use during withdrawal. Ethanol-induced alterations to a network of brain regions involved in the regulation of emotionality likely contribute to this anxiogenic state. The amygdala is critically involved in fear-learning, stress, and anxiety-related behaviors. Importantly, the lateral/basal amygdala (BLA), which serves as the major input nuclei of the amygdaloid complex, undergoes significant alterations during chronic ethanol (for review see (McCool et al., 2010)). Inhibition of BLA principal neuron activity during withdrawal from ethanol is anxiolytic in rodent dependence models (Läck et al., 2007), and chronic ethanol-induced alterations to BLA synaptic transmission potently contribute to the development and expression of withdrawal-related anxiety (Läck et al., 2007, Diaz et al., 2011, Christian et al., 2012, Christian et al., 2013). Specifically, chronic exposure/withdrawal significantly enhances both pre- and postsynaptic components of excitatory transmission coupled with a specific decrease in feedforward inhibitory transmission. Overall, enhanced glutamatergic function and diminished GABAergic function likely drive a net increase in BLA output (Läck et al., 2007, Diaz et al., 2011, Christian et al., 2012, Christian et al., 2013). The impact of chronic ethanol on the neuromodulators controlling glutamatergic and GABAergic transmission is unknown but is poised to dramatically influence these dependence-related outcomes.
The endogenous cannabinoid (eCB) system acts primarily through the modulation of glutamate and GABA synaptic activity and is richly expressed in areas related to the regulation of emotional behavior, including the BLA (Häring et al., 2012). Importantly, the eCB system has been implicated in both the acute and chronic effects of ethanol as well as in the development and maintenance of alcohol dependence (For review see (Pava and Woodward, 2012, Vinod and Hungund, 2005, Basavarajappa, 2007)). In contrast to traditional neurotransmitters which are released by excitation-secretion from presynaptic terminals, eCB ligands are generated ‘on demand’ within postsynaptic compartments and act as retrograde signals that activate presynaptic cannabinoid receptors. The first eCB to be identified, anandamide (N-arachidonoylethanolamine or AEA), is generated primarily by the cleavage of the membrane-bound precursor, N-arachidonoyl phosphatidylethanolamine (NAPE), via phospholipase D (NAPE-PLD) (for review see (Okamoto et al., 2007)). Following its release into the synapse space, AEA undergoes reuptake and is degraded by fatty acid amide hydrolase (FAAH) (Egertová et al., 2003, Gulyas et al., 2004). The other most well-studied endogenous ligand, 2-arachidonoylglycerol (2-AG), is synthesized from diacylglycerol by a cellular lipase (DAGLα) and subsequently degraded by monoacylglycerol lipase (MAGL) (Ueda et al., 2011). These endogenous ligands interact with two main receptors, cannabinoid receptor 1 and 2 (CB1 and CB2, respectively), although CB1 is generally considered the primary cannabinoid receptor in neuronal signaling. CB1 is a Gi/o protein-coupled receptor which inhibits transmitter release at both BLA glutamatergic and GABAergic synapses through numerous downstream intercellular signaling mechanisms (Azad et al., 2003).
The eCB system serves as a point of particular interest as interactions between ethanol and the eCB system have been observed across a variety of paradigms. Behavioral cross-tolerance between ethanol and CB1 agonists has been observed for decades (Newman et al., 1972, Sprague and Craigmill, 1976, Pava et al., 2012) and the eCB system is thought to participate in innate ethanol preference in both rats (Ortiz et al., 2004, Vinod et al., 2012) and mice (Vinod et al., 2008, Hungund and Basavarajappa, 2000). Further, both acute and chronic ethanol exposure alter expression levels of the CB1 receptor as well as the endogenous ligands and their respective metabolic and catabolic enzymes (Ceccarini et al., 2013, Caillé et al., 2007, Ferrer et al., 2007, Rubio et al., 2007, González et al., 2004, Vinod et al., 2006, Basavarajappa et al., 1998a, Basavarajappa et al., 1998b, Basavarajappa and Hungund, 1999a, Basavarajappa and Hungund, 1999b, Basavarajappa et al., 2000, Basavarajappa et al., 2003, Basavarajappa et al., 2008). Similar alterations have been shown in human alcoholics, indicating ethanol-induced changes in this system may be conserved across species (Ceccarini et al., 2014, Vinod et al., 2010). Importantly, modulation of the eCB system in preclinical models can alleviate withdrawal symptoms, including those associated with altered amygdala function such as anxiety-like behaviors (Rubio et al., 2008). Critically, the eCB system is also known to play a role in modulating synaptic plasticity within the BLA (Gunduz-Cinar et al., 2013, Basavarajappa et al., 2014, Shin et al., 2010, Fonseca, 2013, Monory et al., 2015). Ethanol-induced alterations to the relative expression of the endogenous ligands and the function of CB1 receptors are therefore poised to significantly impact the excitatory/inhibitory balance within the BLA.
Within the BLA, CB1 is robustly expressed by CCK + GABAergic interneurons and is also localized to glutamatergic afferent terminals (Marsicano and Lutz, 1999). Although the majority of BLA CB1 receptors are located on GABAergic terminals, CB1 activation within the BLA decreases regional activity (Azad et al., 2003), suggesting the modest population of CB1 receptors localized to glutamatergic terminals exerts a privileged influence over BLA-mediated behaviors. We have previously demonstrated that chronic ethanol induces dramatic up-regulation of presynaptic function at glutamatergic projections arriving to the BLA from the internal capsule/stria terminalis (Christian et al., 2013). These synapses are potently influenced by the eCB system (Shin et al., 2010, Fonseca, 2013). Within the current experiments, we evaluated CB1-modulation of glutamatergic transmission at this BLA input during both acute and chronic ethanol exposure. The impact of chronic ethanol exposure on CB1 modulation of GABAergic synapses within the BLA and on the expression levels of the primary eCB ligands and their respective metabolic and catabolic pathways were also investigated.
Section snippets
Animals
Male Sprague–Dawley rats (Harlan, Indianapolis, IN, USA) weighing 100–150 g on arrival were group housed for the duration of the experiments (n = 138). Animals were given 5–7 days to acclimate to the housing area prior to being placed in experimental groups. Food and water were available ad libitum; and housing conditions were consistent with the NIH Guidelines for the Care and Use of Laboratory Animals (68–74 °F, 30–70% relative humidity). All experimental procedures were reviewed and approved
Acute ethanol inhibits internal capsule BLA afferents
fEPSPs were stimulated at internal capsule inputs and recorded within the BLA in drug naïve control animals. One-way ANOVA detected a significant effect of 80 mM ethanol during the final 2 min of drug application (F(3,32) = 6.477, p < .01). Subsequent Dunnett's Multiple Comparison Test was used to directly compare the different EtOH treatment-groups to the aCSF control group. This analysis determined that compared to continuous aCSF perfusion alone (mean percent change: 1.63 ± 2.39, n = 16), at
Discussion
Ethanol significantly alters the expression and function of endogenous cannabinoid system components during both acute and chronic ethanol exposure across numerous brain regions (for review see (Pava and Woodward, 2012) and (Basavarajappa, 2007)). The present study suggests glutamatergic inputs to the BLA arriving along the internal capsule number among these sites of interaction. Pharmacologically relevant doses of acute ethanol inhibited fEPSPs produced by stimulation of these terminals.
Acknowledgment
This work is funded by National Institutes of Health grants T32 AA007565 (BAM & SLR), F31 MH106192 (RJB), R21 MH103515 (SP), R01 MH100096 (SP), and R01 AA014445 (BAM).
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