Alcohol 49 (2015) 781e794

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Alcohol

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Interactions between ethanol and the endocannabinoid system at GABAergic on basolateral amygdala principal neurons

Giuseppe Talani a,b, David M. Lovinger a,* a Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA b Institute of Neuroscience, National Research Council, Monserrato, Cagliari, Italy article info abstract

Article history: The basolateral amygdala (BLA) plays crucial roles in stimulus value coding, as well as drug and alcohol Received 14 April 2015 dependence. Ethanol alters synaptic transmission in the BLA, while endocannabinoids (eCBs) produce Received in revised form presynaptic depression at BLA synapses. Recent studies suggest interactions between ethanol and eCBs 11 August 2015 that have important consequences for alcohol drinking behavior. To determine how ethanol and eCBs Accepted 25 August 2015 interact in the BLA, we examined the physiology and pharmacology of GABAergic synapses onto BLA pyramidal neurons in neurons from young rats. Application of ethanol at concentrations relevant to intoxication increased, in both young and adult animals, the frequency of spontaneous and miniature Keywords: Alcohol GABAergic inhibitory postsynaptic currents, indicating a presynaptic site of ethanol action. Ethanol did CB1 not potentiate sIPSCs during inhibition of adenylyl cyclase while still exerting its effect during inhibition of protein kinase A. Activation of type 1 receptors (CB1) in the BLA inhibited GABAergic Inhibition transmission via an apparent presynaptic mechanism, and prevented ethanol potentiation. Surprisingly, Arachidonoyl ethanolamide ethanol potentiation was also prevented by CB1 antagonists/inverse agonists. Brief depolarization of BLA Cyclic AMP pyramidal neurons suppressed GABAergic transmission (depolarization-induced suppression of inhibi- tion [DSI]), an effect previously shown to be mediated by postsynaptic eCB release and presynaptic CB1 activation. A CB1-mediated suppression of GABAergic transmission was also produced by combined afferent stimulation at 0.1 Hz (LFS), and postsynaptic loading with the eCB arachidonoyl ethanolamide (AEA). Both DSI and LFS-induced synaptic depression were prevented by ethanol. Our findings indicate antagonistic interactions between ethanol and eCB/CB1 modulation at GABAergic BLA synapses that may contribute to eCB roles in ethanol seeking and drinking. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction related behaviors (Läck, Christian, Diaz, & McCool, 2009; Läck, Diaz, Chappell, DuBois, & McCool, 2007). Acute ethanol exposure The basolateral amygdala (BLA) plays key roles in value coding, alters BLA neuron excitability and increases GABAergic synaptic effect, and actions of abused drugs, including conditioned place transmission onto BLA projecting neurons, including increasing preference for ethanol and other substances (Brown & Fibiger, 1993; GABA release at synapses made by local interneurons (Silberman, Davis, Rainnie, & Cassell, 1994; Everitt, Morris, O’Brien, & Robbins, Ariwodola, & Weiner, 2009; Silberman, Shi, Brunso-Bechtold, & 1991; Gremel & Cunningham, 2009; Hiroi & White, 1991; LeDoux, Weiner, 2008; Zhu & Lovinger, 2006). GABAergic transmission at 1993, 2000; Olmstead & Franklin, 1997; Sarter & Markowitsch, interneuron synapses onto BLA projection neurons is also altered 1985; See, Fuchs, Ledford, & McLaughlin, 2003; Stuber et al., after chronic ethanol exposure (Diaz, Christian, Anderson, & 2011). The BLA is also implicated in ethanol withdrawal-induced McCool, 2011). It is important to elucidate ethanol actions in BLA increases in anxiety, as well as other withdrawal/abstinence- to gain a better understanding of the basis of cellular and circuit- level effects of this drug. Presynaptic, Gi/o-interacting G-protein-coupled receptors Conflict of interest: The authors declare no conflict of interest. (GPCRs) inhibit neurotransmitter release and prevent ethanol ef- * Corresponding author. Laboratory for Integrative Neuroscience, National Insti- fects at GABAergic synapses in several brain regions (Ariwodola & tute on Alcohol Abuse and Alcoholism, 5625 Fishers Lane, Room TS-11, Bethesda, MD 20892, USA. Tel.: þ1 301 443 2445; fax: þ1 301 480 8035. Weiner, 2004; Kang-Park, Kieffer, Roberts, Siggins, & Moore, E-mail address: [email protected] (D.M. Lovinger). 2007; Kelm, Criswell, & Breese, 2008; Offermanns, 2003; Roberto http://dx.doi.org/10.1016/j.alcohol.2015.08.006 0741-8329/Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 782 G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794 et al., 2010; Silberman et al., 2009; Theile, Morikawa, Gonzales, & indicate a general antagonistic effect between ethanol and canna- Morrisett, 2008; Wan, Berton, Madamba, Francesconi, & Siggins, binoidergic transmission that has strong implications for acute 1996; Zhu & Lovinger, 2006), suggesting that endogenous neuro- intoxication. transmitter actions limit ethanol-induced increases in release. A possible point of convergence for the ethanol and Gi/o-GPCRs is Materials and methods adenylyl cyclase (AC). This cyclic AMP-generating enzyme can be activated by ethanol (Tabakoff, Nelson, Yoshimura, Hellevuo, & Brain slice preparation Hoffman, 2001) and inhibited by the GPCRs of interest. Deter- mining which neurotransmitters produce these ethanol All experiments were approved by the NIAAA Animal Care and potentiation-limiting effects at which synapses will help us to un- Use Committee. Postnatal day 15e19 (young) or day 50e60 (adult) derstand acute drug actions under different physiological Sprague Dawley rats were anesthetized by halothane inhalation. conditions. After trans-cardial perfusion with ice-cold modified artificial cere- Cannabinoid type 1 (CB1) receptors are predominantly presyn- brospinal fluid (aCSF) containing (in mM): 194 sucrose, 30 NaCl, 4.5 aptic and decrease neurotransmitter release (Freund, Katona, & KCl, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 glucose (pH 7.4 with Piomelli, 2003; López-Moreno, González-Cuevas, Moreno, & 95% O2/5% CO2), they were then decapitated, and their brains Navarro, 2008; Mackie, 2008; Matsuda, Bonner, & Lolait, 1993; rapidly transferred into ice-cold modified aCSF. Coronal sections Wilson & Nicoll, 2001). CB1 expression is robust in the BLA, and (300 mm thickness) were cut in ice-cold modified aCSF using a Leica strongly localized to axon terminals of CCK-expressing GABAergic VT1200S vibratome (Leica Microsystem Nussloch GmbH, Heidel- interneurons (Katona et al., 2001). Endocannabinoids (eCBs), berg, Germany). Slices were transferred immediately to a nylon net endogenous lipid metabolites, activate CB1 receptors (Alger, 2009). submerged in normal aCSF containing (in mM): 124 NaCl, 4.5 KCl, 2 Arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2- CaCl2, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 D-glucose, with AG) are the best characterized eCBs (Heifets & Castillo, 2009). Re- osmolarity set to 320 mOsm (pH 7.4), at 33 C for at least 30 min. ceptor activation by eCBs usually involves “retrograde” After an additional 1-h incubation at room temperature, hemi- postsynaptic-to-presynaptic signaling that produces either short- slices were transferred to a recording chamber, submerged in or long-term synaptic depression (Alger, 2009; Alger et al., 1996; normal aCSF with a constant flow of w2 ml/min. For all experi- Heifets & Castillo, 2009; Lovinger, 2007, 2008; Ohno-Shosaku, ments, the temperature of the bath was maintained at w31 C Maejima, & Kano, 2001; Wilson & Nicoll, 2001), both of which during any given experiment. have been observed at GABAergic synapses onto BLA principal neurons (Marsicano et al., 2002; Patel, Kingsley, Mackie, Marnett, & Neuronal isolation Winder, 2009; Szabo & Schlicker, 2005; Zhu & Lovinger, 2005). The eCB-mediated reduction of GABA release enhances excitatory Mechanical isolation of BLA neurons was performed as previ- output from BLA (Perra, Pillolla, Luchicchi, & Pistis, 2008). ously described (Jun, Cuzon Carlson, Ikeda, & Lovinger, 2011; Zhu & Past studies have described interactions between eCB/CB1 Lovinger, 2005). Briefly, coronal brain slices containing BLA were signaling and acute ethanol effects, in which CB1 activation de- transferred to a 35-mm diameter culture dish containing (in mM) presses GABAergic transmission and prevents ethanol potentiation 150 NaCl, 5 KCl, 10 HEPES, 1 MgCl2, 2.5 CaCl2,10D-glucose, pH of GABA release (Kelm et al., 2008; Roberto et al., 2010). Signaling by adjusted to 7.4 using NaOH, osmolarity set to w300 mOsm with eCBs is also implicated in behavioral actions of ethanol, including sucrose. Using a piezoelectric bimorph (Burleigh Instruments, NY), intake and preference, as well as conditioned place preference the tip of a flame-sealed micropipette was vibrated laterally at (Alén et al., 2009; Arnone et al., 1997; Basavarajappa, Ninan, & 30 Hz as it descended through the BLA. The slice was then removed Arancio, 2008; Basavarajappa, Yalamanchili, Cravatt, Cooper, & from the dish, leaving the isolated neurons. Hungund, 2006; Blednov, Cravatt, Boehm, Walker, & Harris, 2007; Cippitelli et al., 2005, 2007; Colombo et al., 2007; Colombo, Serra, Whole-cell voltage-clamp recording Vacca, Carai, & Gessa, 2005; Economidou et al., 2006; Freedland, Sharpe, Samson, & Porrino, 2001; Houchi et al., 2005; Hungund & In brain slices, whole-cell recordings were performed from BLA Basavarajappa, 2000, 2004; Manzanares, Ortiz, Oliva, Pérez-Rial, & principal neurons. Recording pipettes were pulled from borosilicate Palomo, 2005; Pava et al., 2012; Thanos, Dimitrakakis, Rice, glass on a Flaming Brown micropipette puller (Novato, CA). Pipette Gifford, & Volkow, 2005; Vinod, Sanguino, Yalamanchili, resistance ranged from 2.5 to 4.5 MU, when filled with a CsCl-based Manzanares, & Hungund, 2008; Vinod, Yalamanchili, et al., 2008). internal solution containing (in mM): 150 CsCl, 10 HEPES, 5 lido- Chronic ethanol treatment produces tolerance to the behavioral caine N-ethyl bromide (QX314), 2 MgCl2, 3 Mg-ATP, 0.3 Na-GTP, and effects of (Pava et al., 2012). This evidence suggests 0.2 BAPTA-4K, pH adjusted to 7.2 with CsOH, and osmolarity set to important eCB roles in regulation of the CNS effects of ethanol. 298 mOsm with sucrose. To exclude the presence of glutamatergic In the present study, we examine interactions between ethanol synaptic responses, 50 mM AP5 and 5 mM NBQX were added to the and eCB/CB1 signaling at GABAergic synapses in rat BLA. In addi- solution to block NMDA and AMPA receptor-mediated currents, tion, studies have indicated that adolescent rats are less sensitive respectively. Cells were voltage-clamped at 60 mV. We analyzed than adults are to several ethanol actions in both electrophysio- only recordings with series resistance values ranging from 9 to logical and behavioral experiments (Little, Kuhn, Wilson, & 25 MU without compensation, and if this value changed by more Swartzwelder, 1996; Silveri & Spear, 1998; Van Skike et al., 2010). than 20% during the course of an experiment, the cell was Administration of low to moderate ethanol doses induces social discarded. facilitation in young animals, while the same doses reduce social Synaptic currents were recorded with an Axopatch 700A behavior in adults (Varlinskaya & Spear, 2002). The different amplifier (Axon Instruments, Foster City, CA), filtered at 2 kHz, and response to ethanol in adolescents may play an important role in digitized at 5 kHz. Spontaneous IPSC amplitude and frequency were the development of ethanol-related problems (Spear & Varlinskaya, measured using peak and event detection software in pCLAMP9.2 2005). For this reason, we evaluated, in a separate set of adult an- and analyzed by Minianalysis software (Synaptosoft, Decatur, GA, imals, some of the effects observed in young rats regarding the USA). After initiation of the whole-cell recording, stable baseline interaction between ethanol and the eCB system. Our findings responses were observed within w10 min. After 2e3 additional G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794 783 minutes of baseline recording, we started drug perfusion or the DSI Zhu & Lovinger, 2006). Consistent with previous studies protocol. For evoked IPSC recordings, the concentric bipolar stim- (Silberman et al., 2009; Zhu & Lovinger, 2006), application of ulating electrode was placed approximately 200 mm medial to the 80 mM ethanol induced a significant increase in sIPSC amplitude recording site. This would certainly stimulate the inhibitory affer- and frequency that developed within 3e4 min of the onset of ents arising from local interneurons, but stimulation of afferents ethanol application (Amp: 32 12% increase; Freq: 56 12% in- from paracapsular cells cannot be ruled out. For DSI experiments, crease; p < 0.05, paired t test), (Fig. 1AeC). The potentiation after a recording period of approximately 3 min, the membrane reversed within 5 min after cessation of ethanol application. potential was stepped from 60 to 0 mV for 5 s under voltage Potentiation of sIPSC frequency by ethanol was concentration- clamp. The magnitude of DSI was calculated as the sIPSC amplitude dependent (F[4,58] ¼ 3.89, p ¼ 0.007), without any significant and frequency after the depolarization divided by the average concentration-dependence of the change in event amplitude, during a 3-min pre-depolarization period. In a different set of ex- where only the higher concentrations were significant (p < 0.05, periments, AEA (50 mM) was loaded in the patch electrode to in- paired t test) (Fig. 1D and E). In another set of neurons from young crease the pool of endocannabinoid available for release during DSI. rats, we examined action potential-independent miniature IPSCs The low-frequency stimulation (LFS) protocol consisted of 0.1-Hz (mIPSCs) in the presence of the voltage-dependent sodium chan- stimulation for 3 min using a concentric bipolar electrode (FHC, nel blocker TTX (1 mM) (basal amplitude 45.3 6.2 pA; basal Bowdoin, ME). Stimulation intensity was set to induce an eIPSC frequency 4 0.9 Hz; n ¼ 11). When ethanol (80 mM) was with an amplitude w50% of the maximal response. perfused into the slice it increased mIPSC frequency by 41 18% In some experiments examining ethanol effects on DSI, the DSI (p < 0.05, paired t test vs. control) without any significant change protocol was repeated three times in each neuron, once during the in amplitude (15 8.5%), (Fig. 1F and G). baseline period, once in the presence of ethanol, and once after washout. In other experiments the DSI protocol was run only once Effect of adenylyl cyclase and PKA inhibitors on ethanol potentiation per cell, and SR or ethanol were present throughout the recording. of sIPSCs In the experiments involving ethanol wash-on and washout, we applied the DSI protocol after 3 min of stable baseline and Previous studies evaluated the adenylyl cyclase (AC) and protein continued to record for an additional 3 min after DSI. We then kinase A (PKA) effect on ethanol potentiation of GABA release (Kelm applied ethanol to the slice for a total of 5 min. Three minutes into et al., 2008; Roberto et al., 2010). Ethanol can activate AC (Luthin & the ethanol application, if the response was stable, we applied the Tabakoff, 1984; Rabin & Molinoff, 1981), and we thus examined DSI protocol again. Two minutes after this second DSI protocol, we effects of AC activation and inhibition, as well as PKA inhibition, on began the washout of ethanol. After a total of 4 min of washout, we GABAergic sIPSCs and ethanol potentiation in BLA slices from young again applied the DSI protocol, and recorded for a further 3 min. animals. As previously described in hippocampus (Chevaleyre, Whole-cell voltage-clamp recordings from isolated neurons were Heifets, Kaeser, Südhof, & Castillo, 2007) and prefrontal cortex performed on the stage of an inverted Nikon T200 microscope as (Chiu, Puente, Grandes, & Castillo, 2010), a 30-min pre-incubation previously described (Zhu & Lovinger, 2006), using micropipettes with the 10 mM AC inhibitor dideoxy- (DDA) or the PKA with resistances of 2e4MU filled with the CsCl-based solution. inhibitor H-89 (10 mM) decreased basal sIPSC frequency without Solutions containing ethanol and other compounds were applied affecting amplitude (Fig. 2AeC). Bath application of the AC activator via local perfusion using an array of fused square-tipped glass tubes, forskolin (10 mM) potentiated sIPSC frequency. The forskolin effect with lateral movement driven by the Faststep stepper motor system was prevented by previous incubation of slices in 10 mMDDA (Warner Instruments, Hamden, CT). Recordings were performed in (Fig. 2D and E). The increases in sIPSC amplitude and frequency the presence of 5 mM NBQX and 25 mM AP5 to block fast gluta- induced by 80 mM ethanol were reduced in slices incubated with matergic transmission, allowing for measurement of spontaneous DDA (Fig. 2F), as previously reported (Kelm et al., 2008). However, GABAA receptor-mediated IPSCs. in slices treated with H-89, ethanol was still able to induce an in- crease in sIPSC frequency that was not distinguishable from the Drugs effect of ethanol alone (Fig. 2F). A significant difference among the three treatment groups in ethanol effects on sIPSC amplitude was The following drugs were used: ethanol (Pharma corporation), observed (one-way ANOVA, F[2,14] ¼ 8.28, p ¼ 0.0042), with post WIN 55,212, SR161417A, AEA, 2AG, H-89, BSE, and DDA (Sigma- hoc Tukey multiple comparisons indicating significant differences eAldrich, San Diego, CA), NBQX, D-AP5, TTX (Tocris, Ellisville, MO). only between the ethanol alone and DDA þ ethanol treatments. Analysis of the sIPSC frequency data indicated a significant effect of Statistical analysis treatment (F[2,14] ¼ 7.004, p ¼ 0.0078), with post hoc comparisons indicating significant differences only between the ethanol alone Statistical comparisons of pooled data were performed by t test, versus DDA þ ethanol group. One-sample t tests indicated that or one-way ANOVA followed by the Tukey or Neuman-Keuls post ethanol produced significant increases in sIPSC frequency in all hoc tests, or repeated-measures two-way ANOVA. In all cases, a p groups, and thus DDA did not completely eliminate ethanol value of <0.05 was considered statistically significant. potentiation of GABAergic transmission.

Results CB1R interactions with ethanol-induced increases in GABA release

Effect of ethanol on sIPSCs recorded from BLA principal neurons of We next determined whether CB1 receptor activation alters young rats synaptic transmission and ethanol potentiation at GABAergic ter- minals in BLA principal neurons recorded from brain slices made GABAergic sIPSCs occur with reliable frequency and amplitude from young animals, as has been observed at other synapses (Ampl. 56.3 6 pA; Freq. 8.4 0.8 Hz; n ¼ 73) in pyramidal (Roberto et al., 2010). The CB1 agonist WIN 55,212 (WIN) (5 mM) neurons examined in BLA brain slices (Fig. 1) from young rats, as significantly decreased sIPSC frequency (35.7 7% below baseline, previously reported (Diaz, Chappell, Christian, Anderson, & p < 0.01), but not amplitude (6.7 4.4) (Fig. 3A and B). Once sIPSC McCool, 2011; Diaz, Christian, et al., 2011; Silberman et al., 2008; frequency had reached a stable level in the presence of WIN, we 784 G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794

Fig. 1. Ethanol increases GABAergic transmission onto BLA principal neurons. A and B) Graphs showing the effect of 5-min 80 mM ethanol perfusion on both sIPSC amplitude (A) and frequency (B). C) Representative current traces obtained from a single neuron before, during, and 5 min after ethanol perfusion (scale bar 100 pA, 10 s). D and E) Bar graph showing the average ethanol effect on sIPSCs at different concentrations (10, 25, 50, 80, and 150 mM). The extent of the ethanol effect was calculated during the 2 min in which the drug showed its maximal effect. Data are expressed as mean SEM (n ¼ 5, 9, 11, 27, and 11 cells, respectively). F) Bar graph showing 80-mM ethanol effects on amplitude and frequency of TTX-insensitive sIPSCs (mIPSCs) (n ¼ 11 cells) (*p < 0.05 vs. baseline, paired t test). G) Representative traces of mIPSCs recorded from a single neuron before, during, and after ethanol slice perfusion (scale bar 50 pA, 5 s). applied 80 mM ethanol in the continued presence of CB1 agonist. We next evaluated the effect of ethanol in the presence of the No significant change in sIPSC frequency or amplitude was CB1 antagonist/inverse agonist SR141716A (SR) (1 mM) in the slice observed during ethanol (80 and even 150 mM) application in the preparation. The compound was applied for 3 min prior to 5-min presence of WIN (Fig. 3C and D) (p > 0.05, t test vs. baseline). co-application of ethanol (80 mM), and SR did not alter sIPSC G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794 785

Fig. 2. Roles of adenylyl cyclase and protein kinase A in ethanol potentiation of sIPSCs. A and B) Bar graphs showing the effect of 30-min incubation of slices with the PKA inhibitor H-89 (10 mM) or the AC inhibitor DDA (10 mM). Neither compound altered sIPSC amplitude (A), but both produced a decrease in sIPSC frequency (B) (n ¼ 54, 11, 21) (*p < 0.05 vs. aCSF unpaired t test. C) Representative traces obtained from different neurons incubated in normal aCSF, or after 30 min of H-89 or DDA incubation. Scale bar ¼ 50 pA, 10 s. D and E) Forskolin (20 mM) increased sIPSC frequency, and this effect was dramatically decreased in slices incubated for 30 min with DDA. Representative current traces in D show forskolin effects on sIPSCs in the absence (top) and presence (bottom) of DDA. Scale bar ¼ 50 pA, 10 s. Bar graphs in E show the forskolin and forskolin þ DDA effects on sIPSC amplitude and frequency (n ¼ 5, 3) (*p < 0.05 vs. baseline paired t test; #p < 0.05 vs. forskolin, unpaired t test). F) The AC inhibitor DDA decreases ethanol potentiation of the amplitude and frequency of sIPSCs recorded from BLA-projecting neurons, while the PKA inhibitor H-89 did not significantly alter ethanol actions (n ¼ 27, 7, 5) (*p < 0.05 vs. baseline, paired t test). amplitude or frequency, but prevented the increase induced by baseline, t test), consistent with our previous observations (Zhu & ethanol (Fig. 3EeG) (p < 0.05 vs. baseline, t test). The increase in Lovinger, 2005). In a separate set of neurons we applied SR sIPSC frequency induced by 80 mM ethanol was intact in the (1 mM) throughout the recording. Similar to what was observed by presence of DMSO (dimethyl sulfoxide, 0.01%), the carrier used to Zhu and Lovinger (2005), SR perfusion induced an increase in sIPSC dilute SR (Amp: 27.6 4.8%, Freq: 56.9 6.5% increase; data not frequency of 52 20% above baseline in 58% (4 of 7) of cells tested. shown). Another CB1 antagonist/inverse agonist, AM251 (1 mM) However, no potentiation was observed in the remaining cells. In also prevented ethanol (80 mM) potentiation of sIPSC frequency the presence of SR, ethanol 80 mM failed to increase the frequency and amplitude (amp ¼ 0.57 1.3%, paired t test, p > 0.05, and amplitude of sIPSCs (Fig. 4BeD). Thus, the ability of the CB1 freq ¼ 2.3 4.6%, paired t test, p > 0.05, n ¼ 11). However, when a antagonist/inverse agonist to prevent potentiation by ethanol is not high concentration of ethanol (150 mM) was used, SR did not dependent on intact circuitry or the slice milieu, and is likely due to reduce ethanol potentiation of sIPSC frequency (paired t test, a more direct effect on ethanol-sensitive axon terminals. p < 0.05, t ¼ 3.58, df ¼ 6; unpaired t test ethanol þ vehicle vs. ethanol þ SR, p ¼ 0.92, t ¼ 0.098, df ¼ 19) (Fig. 3H). Ethanol reduces DSI at synapses on BLA principal neurons In mechanically isolated BLA principal neurons from young an- imals, we observed sIPSCs, and application of 80-mM ethanol To determine if ethanol alters eCB actions at BLA GABAergic increased sIPSC amplitude and frequency (Fig. 4A, C, D) (p < 0.05 vs. synapses, we first examined depolarization-induced suppression of 786 G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794

Fig. 3. CB1 agonists and antagonists modulate ethanol effects on sIPSCs. A and B) sIPSC amplitude (A) and frequency (B) plotted as a function of time during an experiment in which the CB1 agonist WIN 55,212 (5 mM) was applied to BLA slices prior to application of 80 mM ethanol. The agonist reduced sIPSC frequency and abolished potentiation by ethanol. C, D) Bar graphs showing the average ethanol effect on sIPSC amplitude and frequency at two different concentrations (80 mM, C; and 150 mM, D) in the presence and absence of WIN. Data were collected from 5 to 11 cells (*p < 0.05 vs. baseline, paired t test). E, F) Graphs showing the time course of 80 mM ethanol effects on sIPSC amplitude and frequency, respectively, in the presence and absence of the CB1 antagonist SR (1 mM). G and H) Bar graph showing the average ethanol effect at two different concentrations (80 and 150 mM) in the presence and absence of the CB1 antagonist/inverse agonist. Data were collected from 8 to 12 cells (*p < 0.05 vs. baseline, paired t test). G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794 787

ethanol had no effect. In the pre-ethanol baseline condition, post- synaptic depolarization produced significant sIPSC frequency depression (Fig. 5B and C) (p < 0.05 vs. baseline, t test). As shown in Fig. 5B and C, application of different concentrations of ethanol reduced the depolarization-induced decrease in sIPSC frequency (repeated-measures two-way ANOVA, significant treatment effect F [1,30] ¼ 23.1, p < 0.05), with no significant effect of ethanol con- centration or significant interaction. No significant decrease in sIPSC frequency was observed following depolarization in the presence of the 50 or 80 mM concentrations (Fig. 5C). This decrease in DSI was reversed when ethanol was removed from the prepa- ration (Fig. 5C), indicating that the loss of sIPSC depression was not due to a time-dependent loss of DSI. The depolarization protocol did not alter sIPSC amplitude in the absence or presence of ethanol (Fig. 5D) (p > 0.05, two-way ANOVA). To try to enhance DSI magnitude, and perhaps bypass ethanol effects on eCB synthesis, we filled the patch electrode with AEA (10 mM) or 2-AG (10 mM) and applied the depolarization protocol in these eCB-loaded cells. The magnitude and time course of DSI were unchanged in neurons filled with AEA relative to neurons filled with the standard internal solution containing CsCl (Fig. 5B, E, F). We obtained the same result filling the recording electrode with 2- AG (decrease in sIPSC freq. following DSI ¼ 36.8 6.6 below baseline). Bath application of the CB1 antagonist/inverse agonist SR (1 mM) prevented DSI in AEA-loaded cells (Fig. 5E and F). Bath application of 80 mM ethanol prevented DSI in the AEA-loaded cells, as observed in the non-loaded neurons (Fig. 5E and F), (F [3,45] ¼ 11.43, p < 0.05). This finding suggests that the effects of ethanol are not via inhibition of eCB synthesis.

Effect of ethanol and CB1 agonist in adult rats

The effects of ethanol and CB1 agonists on GABAergic synapses might be dependent on the developmental stage of animals tested, as previous studies have indicated such changes over development in other brain regions and in behavioral tests (Little et al., 1996; Silveri & Spear, 1998; Van Skike et al., 2010). As observed in young rats, GABAergic sIPSCs occur with reliable frequency and amplitude in BLA principal neurons from adults (Ampl. 38.86 3.14 pA; Freq. 3.31 0.38 Hz; n ¼ 26). Application of 80 mM ethanol induced a significant increase in sIPSC amplitude and fre- quency recorded in BLA slices from adult animals, similar to what was observed in young rats (Amp: 27.8 3.38% increase; Freq: 37.8 9.6% increase; p < 0.05 one-sample t test vs. baseline) (Fig. 6AeD). As previously observed, the potentiation reversed within 5 min after cessation of ethanol application. To evaluate the interaction between ethanol and eCB, we first examined ethanol actions in the presence of the CB1 agonist. As observed in young rats, WIN 55,212 (5 mM) decreased sIPSC frequency (65.4 6.2% Fig. 4. Ethanol potentiation is prevented by a CB1 antagonist/inverse agonist in below baseline, p < 0.001) (Fig. 7A) without any change in sIPSC vibrodissociated BLA projection neurons. A and B) Representative traces obtained from single neurons before and during ethanol application using fast perfusion in the amplitude (data not shown). After sIPSC frequency had reached a absence and presence of the CB1 antagonist SR (1 mM) (scale bar 200 pA, 60 s). C) Time stable level in the presence of WIN, we applied 80-mM ethanol in course of the effect of 80 mM ethanol on sIPSC frequency in the presence and absence the continued presence of CB1 agonist. Even in adult animals, no of the CB1 antagonist SR (n ¼ 12 cells). D) Bar graph showing the average ethanol effect significant change in sIPSC frequency was observed during ethanol on sIPSC amplitude and frequency in the presence and absence of the CB1 antagonist/ application in the presence of WIN (Fig. 7A and B) (p < 0.05 vs. inverse agonist (*p < 0.05 vs. baseline, paired t test). baseline, t test). To determine whether ethanol alters eCB actions at GABAergic synapses in neurons from adult animals, we used the DSI inhibition (DSI) in slices from young rats. Depolarization of the protocol. DSI induced a decrease in sIPSC frequency (40.61 6.42% pyramidal neuron from 60 to 0 mV for 5 s induced a fast-onset, below baseline, p < 0.01 paired t test, n ¼ 4) (Fig. 7C) during the first transient (15e20 s post-depolarization duration) decrease in 15 s after cessation of the depolarization step. Application of 80- sIPSC frequency (25.7 2.7% below baseline, p < 0.01 paired t test, mM ethanol significantly reduced the magnitude of the decrease n ¼ 45) (Fig. 5A and B), as observed in previous studies (Patel et al., in sIPSC frequency induced by the DSI protocol, and the effect of DSI 2009; Zhu & Lovinger, 2006). Application of 50 or 80 mM ethanol recovered completely after a 5e10-min ethanol washout (Fig. 7C for 3 min increased sIPSC frequency as previously observed (% and D) (p < 0.05 vs. baseline, t test), indicating that the loss of sIPSC increase ¼ 28 5.1 at 50 mM, 33 13.6 at 80 mM), while 25 mM depression was not due to a time-dependent loss of DSI. 788 G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794

Fig. 5. Depolarization-induced suppression of inhibition in BLA is prevented by ethanol. A) Representative traces showing that a depolarizing step from 60 mV to 0 mV, applied for 5 s to a single BLA pyramidal neuron, produced a rapid, short-lasting decrease in sIPSC frequency vs. the baseline-recording period, without any significant modification in amplitude (scale bar 50 pA, 5 s). B) Graph showing normalized sIPSC frequency over time before, during, and after depolarization using our standard CsCl-containing intracellular solution (open circles) and in the presence of 80 mM ethanol (closed circles) (n ¼ 28, 11). C and D) Bar graphs show the average normalized sIPSC frequency (C) and amplitude (D) measured over the first 15 s after depolarization in control conditions (ACSF only), in the presence of different concentrations of ethanol (25, 50, 80 mM), and after 5 min of ethanol washout (n ¼ 11 for all groups) (*p < 0.05 vs. baseline, paired t test). E) Time course of normalized sIPSC frequency before, during, and after depolarization in recordings made from BLA projection neurons after loading the patch electrode with AEA (10 mM). Recordings were made either in standard aCSF (white circles), aCSF containing SR141716A (1 mM) (gray circles), or aCSF containing 80 mM ethanol (black triangles). (n ¼ 12, 6, 3 cells). F) Summary graph showing DSI magnitude in all CsCl-filled, AEA-filled, AEA þ SR and AEA þ ethanol cells (n ¼ 28, 12, 6, 3 cells) (*p < 0.05 vs. baseline, paired t test). G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794 789

Fig. 6. Ethanol increases GABAergic transmission onto BLA principal neurons in adult rats (PND 50e60). A and B) Time course plots showing the effect of 5-min 80 mM ethanol perfusion on both sIPSC amplitude (A) and frequency (B). C) Representative current traces obtained from a single neuron before, during, and 5 min after ethanol perfusion (scale bar 100 pA, 10 s). D) Bar graph showing the average effect of 80 mM ethanol on sIPSCs amplitude and frequency. The extent of the ethanol effect was calculated during the 2 min in which the drug showed its maximal effect. Data are expressed as mean SEM (n ¼ 8 cells), (*p < 0.05 vs. baseline, paired t test).

Ethanol prevents eCB-mediated synaptic depression induced Discussion by low-frequency afferent activation The present study indicates that ethanol interacts with the BLA We combined afferent stimulation with postsynaptic loading of eCB/CB1 signaling system in several ways. Strong activation of the AEA in neurons from young animals, in the presence of NBQX CB1 receptor prevents ethanol potentiation of GABA release, (5 mM) and APV (50 mM), to determine whether we could produce consistent with previous studies (Perra et al., 2008; Roberto et al., CB1-dependent synaptic depression while bypassing mechanisms 2010), and consistent with other studies indicating that several involved in eCB production. Afferent stimulation at 0.1 Hz (LFS) for Gi/o-coupled GPCRs prevent presynaptic ethanol actions in BLA and 3 min induced a significant decrease in sIPSC frequency that per- elsewhere (Ariwodola & Weiner, 2004; Kang-Park et al., 2007; Kelm sisted for over 3 min following cessation of stimulation in re- et al., 2008; Roberto et al., 2010; Silberman et al., 2009; Wan et al., cordings from AEA-filled BLA pyramidal neurons (Fig. 8A and B). In 1996; Zhu & Lovinger, 2006). Importantly, we observed that ethanol cells filled only with the CsCl-based pipette solution, LFS did not interactions with eCB/CB1 signaling are not only confined to young alter sIPSC frequency (Fig. 8A and B). The decrease in sIPSC fre- animals but are also present in the adult. Thus, developmental quency observed with LFS plus AEA loading (10 mM) was prevented changes in ethanol sensitivity that have been noted in other brain by SR (Fig. 8C and D), consistent with previous observations in regions and in behavioral tests (Little et al., 1996; Silveri & Spear, striatum that combined synaptic activation and postsynaptic eCB 1998; Van Skike et al., 2010) do not appear to be occurring at BLA loading induce CB1-dependent synaptic depression (Adermark & GABAergic synapses. The opposing ethanol and GPCR actions may Lovinger, 2009; Ronesi & Lovinger, 2005). LFS failed to alter sIPSC involve AC, as ethanol is known to potentiate the function of certain frequency in AEA-loaded neurons in the presence of 80 mM ethanol ACs (Rabin & Molinoff, 1981; Tabakoff et al., 2001), while Gi/oa (Fig. 8E and F). One-way ANOVA indicated significant differences subunits liberated by receptor activation are AC inhibitors (Davis, across conditions (F[3,140] ¼ 26.6, p < 0.05), and post hoc analyses Ronesi, & Lovinger, 2003; Freund et al., 2003; Wartmann, with the Neuman-Keuls Multiple Comparison Test revealed signif- Campbell, Subramanian, Burstein, & Davis, 1995). Our findings icant differences (p < 0.05) in the AEA vs. CsCl alone-loaded com- indicate that AC inhibition interferes with ethanol potentiation of parison, the AEA vs. AEA þ SR comparison, and the AEA vs. GABA release, supporting a role for this signaling system in ethanol AEA þ ethanol comparison, but not in other comparisons. These actions. Interestingly, PKA inhibition did not appear to alter ethanol findings indicate that ethanol inhibits eCB-mediated synaptic actions as effectively as did AC inhibition. This might indicate a role depression and likely does so at a step that occurs after eCB syn- for increased cAMP independent of PKA activation. It is not yet thesis, as the depression we observed only occurred at synapses known how these signaling pathways are recruited in vivo, and how onto eCB-loaded neurons. they may alter ethanol-related behaviors. The ability of CB1 790 G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794

Fig. 7. CB1 agonists modulate ethanol effects on sIPSCs in BLA slices from adult rats. A) sIPSC frequency plotted as a function of time during an experiment in which the CB1 agonist WIN 55,212 (5 mM) was applied to BLA slices prior to application of 80 mM ethanol. The agonist reduced sIPSC frequency and abolished potentiation by ethanol. B) Bar graph showing the effect of ethanol alone as well as ethanol þ WIN in the modulation of sIPSC frequency (*p < 0.05 vs. baseline, paired t test). C) Graph showing normalized sIPSC frequency over time before, during, and after depolarization using our standard CsCl (open circles), in the presence of 80 mM ethanol (closed squares) and after ethanol washout (open triangles). D) Bar graphs show the DSI effect on sIPSC frequency during the first 15 s after DSI, using our standard CsCl, in the presence of ethanol and after ethanol washout (*p < 0.05 vs. baseline, paired t test). agonists to prevent ethanol potentiation of GABAergic transmission antagonist/inverse agonist effect can be overcome if the alcohol has implications for the effect of combined ethanol and synthetic or effect is sufficiently strong. This finding may provide a clue as to phytocannabinoid use. The cannabinoid drugs appear to be able to how prevention of ethanol effects differs when CB1 is occupied by suppress this ethanol action, adding another layer of complexity to different ligands. Our findings are similar to the observation in the interactions of these often co-abused substances that must be hippocampus by Basavarajappa et al. (2008), who showed antago- kept in mind when examining interactions in vivo. nism between SR and ethanol effects on glutamate release as well Surprisingly, CB1 antagonists/inverse agonists also reduced as in BLA by Perra et al. (2008), but differ from those in previous ethanol potentiation in both brain slices and isolated neurons. It is studies that examined effects of GPCR blockade on ethanol poten- odd that both agonists and antagonists/inverse agonists have the tiation of GABA release (Silberman et al., 2009), including a study in same action. Perhaps CB1 receptors are constantly activated by which CB1 antagonist/inverse agonist treatment did not interfere tonic eCB signaling that would produce a tone of AC inhibition. The with ethanol potentiation in central amygdala (Roberto et al., 2010). antagonist/inverse agonist could relieve this tone, and might then It is likely that the interactions between ethanol and eCB/CB1 occlude ethanol potentiation. This scenario seems unlikely, given signaling are more complex in BLA than in other brain regions, as that no increase in sIPSC frequency, indicative of relief of the tone, our findings with DSI- and LFS-induced synaptic depression (dis- was observed during CB1 antagonist/inverse agonist application in cussed below) suggest. BLA slices. Another possibility is that ethanol potentiation involves, While the observation that ethanol interacts with CB1 receptor at least in part, actions on the CB1 receptor or proximal signaling function is not new when considering findings from other brain molecules such as the G protein itself. In this case, strong receptor regions, acute ethanol interactions with synaptic signaling by activation would counteract ethanol actions while receptor occu- endocannabinoids has not been explored. This set of experiments is pancy with an inverse agonist might also render the receptor or G crucial for understanding the full range of effects that ethanol will protein insensitive to other modulators. The SR prevention of have on this neuromodulatory system in the intact nervous system. ethanol actions in the neuron/synaptic bouton preparation sup- Thus, we examined ethanol effects on DSI, and observed a revers- ports the idea that the site of interaction is located at the synaptic ible prevention of the decrease in GABA release normally induced terminal itself. A high ethanol concentration was effective in by postsynaptic depolarization in the DSI protocol. This action may potentiating GABA release in the presence of SR, indicating that the actually override any effects of CB1 activation on ethanol G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794 791

Fig. 8. Ethanol antagonized the decreased sIPSC frequency induced by low-frequency afferent stimulation (LFS) in BLA slices. A and B) Low frequency electrical afferent stimulation (0.1 Hz) in BLA induced a decrease in sIPSC frequency only when the patch electrode was loaded with the standard CsCl-based intracellular solution plus the eCB AEA (AEA loaded), but not in the absence of the eCB (CsCl) (n ¼ 3, 9). C and D) The decrease in sIPSC frequency induced by LFS in AEA-loaded neurons is antagonized by SR 1 mM bath application throughout the experiment (n ¼ 7). E and F) The decrease in sIPSC frequency induced by LFS in AEA-loaded neurons is also prevented by bath application of 80 mM ethanol (n ¼ 5) (all scale bars 50 pA, 5 s). potentiation of GABAergic synaptic transmission (i.e., those experimental condition. Application of LFS to activate transmission observed with exogenous agonist application as in Figs. 3, 4 and 7). onto AEA-loaded BLA neurons induced depression of sIPSC fre- It appears that, in the presence of ethanol, the endocannabinoid is quency. This synaptic depression resembles effects of post- either not released or cannot activate CB1 receptors, and this action synaptically loaded eCBs previously observed in striatum and would have the net effect of maintaining strong GABAergic trans- hypothalamus (Adermark & Lovinger, 2009; Gerdeman, Ronesi, & mission under conditions when it would normally be suppressed. Lovinger, 2002; Jo, Chen, Chua, Talmage, & Role, 2005; Ronesi & The combined potentiation of GABAergic transmission and DSI Lovinger, 2005). As observed with DSI, ethanol prevented the LFS/ suppression by ethanol has the potential to suppress BLA output loading-induced synaptic depression. The ability of ethanol to quite strongly. There are several mechanisms through which prevent eCB/CB1-mediated synaptic plasticity at GABAergic BLA ethanol might alter endocannabinoid modulation of GABA release, synapses is likely due to interference with eCB release or presyn- and we explored some of these possibilities with additional ex- aptic signaling. It seems unlikely that interference with eCB syn- periments. Ethanol prevented DSI even when the postsynaptic thesis accounts for these actions, as there is ample AEA available in neuron was loaded with AEA during the experiment, suggesting the LFS/AEA-loading experiment. Given the ethanol potentiation of that increasing the eCB available for release could not overcome the GABA release, the interference with eCB-retrograde signaling by ethanol effect. It must be noted, however, that postsynaptic AEA this drug might be due to increased probability of GABA release that loading did not alter DSI magnitude, and thus we cannot be confi- counteracts the ability of CB1 to inhibit this process in both young dent that this treatment enhances eCB signaling under this and adult rats. However, the experiments examining CB1 agonist 792 G. Talani, D.M. Lovinger / Alcohol 49 (2015) 781e794 interactions with ethanol suggest that the interpretation is not so Becker, 2010; Kliethermes, 2005). Treatments that reduce activa- simple, as agonist-induced synaptic depression is clearly intact tion of BLA projection neurons reduce withdrawal-induced in- during ethanol exposure (Fig. 3B). creases in anxiety (Läck et al., 2007, 2009). Furthermore, CB1 Ethanol blockade of eCB retrograde signaling and antagonist/ receptors regulate affective behavior and responses to stressful inverse agonist prevention of ethanol potentiation may be related. stimuli via mechanisms involving the BLA (Brzózka, Fischer, Falkai, Ethanol may interfere with CB1/G protein signaling when receptors & Havemann-Reinecke, 2011; Martin, Ledent, Parmentier, Maldo- are occupied by a partial agonist like AEA, but this effect might be nado, & Valverde, 2002), and these neural actions are likely to be reduced in the presence of an antagonist/inverse agonist and altered by ethanol. Altering inhibitory input to BLA projection eliminated altogether when a full agonist acts on the receptor. neurons will influence output from this important nucleus, and Regardless of the molecular mechanisms involved, our findings thus it will be interesting to see whether manipulation of BLA indicate complex antagonistic interactions between ethanol and GABAergic inhibition by eCBs or CB1 alters withdrawal-induced eCB/CB1 signaling that may differ when ethanol is the only drug anxiety. ingested or when a CB1 agonist is also present. Consistent with previous studies, we observed that ethanol in- Acknowledgments creases GABAergic synaptic transmission via apparent presynaptic mechanisms in brain slices and the “neuron/synaptic bouton” We acknowledge members of the Section on Synaptic Pharma- preparation from BLA, supporting the idea that ethanol actions take cology, Laboratory for Integrative Neuroscience for helping us with place at the axon terminal itself (Lovinger & Roberto, 2013; Roberto, the research and preparation of this manuscript, with a special Madamba, Moore, Tallent, & Siggins, 2003; Silberman et al., 2009; thanks to Dr. Anton Sheinin for training in the vibrodissociation Zhu & Lovinger, 2006). It is tempting to speculate that this may technique. This work was supported by the Division of Intramural occur mainly at synapses made by local, as opposed to intercalated, Clinical and Biological Research of the National Institute on Alcohol BLA interneurons, given the finding that presynaptic ethanol Abuse and Alcoholism ZIAAA000407. potentiation mainly occurs at local-circuit synapses (Silberman et al., 2009). It is known that CB1 receptors are expressed by CCK- References positive local interneurons in the basal and lateral amygdala, most likely on synaptic terminals (Katona et al., 2001). This study Adermark, L., & Lovinger, D. M. (2009). Frequency-dependent inversion of net and physiological evidence from Geracitano, Kaufmann, Szabo, striatal output by endocannabinoid-dependent plasticity at different synaptic e Ferraguti, and Capogna (2007) indicate that CB1 receptors are not inputs. The Journal of Neuroscience, 29,1375 1380. 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Q., Puente, N., Grandes, P., & Castillo, P. E. (2010). Dopaminergic modulation evidence indicates important roles for BLA GABAergic transmission of endocannabinoid-mediated plasticity at GABAergic synapses in the pre- e in control of anxiety (Bueno, Zangrossi, & Viana, 2005; Diaz, frontal cortex. The Journal of Neuroscience, 30, 7236 7248. Cippitelli, A., Bilbao, A., Gorriti, M. A., Navarro, M., Massi, M., Piomelli, D., et al. Chappell, et al., 2011; Gonzalez, Andrews, & File, 1996; Pesold & (2007). The anandamide transport inhibitor AM404 reduces ethanol self Treit, 1995; Sanders & Shekhar, 1995; Silberman, Ariwodola, administration. The European Journal of Neuroscience, 26, 476e486. Chappell, Yorgason, & Weiner, 2010). Acute ethanol intoxication is Cippitelli, A., Bilbao, A., Hansson, A. C., del Arco, I., Sommer, W., Heilig, M., et al. (2005). 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