Acute ethanol treatment prevents endocannabinoid-mediated long-lasting disinhibition of striatal output

Neuropharmacology 58 (2010) 799e805

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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Acute ethanol treatment prevents endocannabinoid-mediated long-lasting disinhibition of striatal output Rhona B.C. Clarke, Louise Adermark* Addiction Biology Unit, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, University of Gothenburg, Box 410, 405 30 Gothenburg, Sweden

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 August 2009 Received in revised form 16 November 2009 Accepted 10 December 2009

Recent research has suggested that the neuronal circuit adaptations elicited by drugs of abuse share common features with traditional learning models, and that drugs of abuse cause long-term changes in behavior by altering synaptic function and plasticity. Especially, the endocannabinoid (eCB) system appears to be involved in the neuronal circuitry regulating ethanol (EtOH) preference in rodent. The aim of this study was to evaluate if acute EtOH exposure could modulate eCB-mediated plasticity in the dorsolateral striatum. Our data show that EtOH (20e50 mM) prevents eCB-mediated long-lasting disinhibition (DLL) of striatal output induced by a single stimulation train delivered at 5 Hz for 60 s, and reduces long-term depression (LTD) induced by low-frequency stimulation at inhibitory synapses. Acute EtOH-treatment also prevents DLL induced by the L-type calcium channel activator 2,5-dimethyl4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methylester (FPL64176; 500 nM), or by the cannabinoid 1 receptor (CB1R) agonist WIN55,212-2 (300 nM), indicating that EtOH affects eCB-signaling at a stage that is downstream from eCB production and release. Importantly, high-frequency stimulation, or a higher concentration of WIN55,212-2 (1 mM), induces EtOH-insensitive depression of striatal output, suggesting that EtOH affects CB1R-mediated signaling in a synapse-specific manner. Maintaining the balance between excitation and inhibition is vital for neuronal networks, and EtOH-mediated modulation of eCB-signaling might thus affect the stability and the fine-tuning of neuronal circuits in the striatum. Our data suggest that changes in eCB-signaling could be involved in the physiological response to acute alcohol intoxication. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Alcohol Basal ganglia Medium spiny neurons Synaptic plasticity LTD

1. Introduction Long-term potentiation (LTP) and long-term depression (LTD) appear to be necessary for behavioral adaptations of motor behavior to changing reward-position contingencies in the striatum (Balleine et al., 2007; Lovinger et al., 2003). Striatal LTD is mediated by postsynaptically released endocannabinoids (eCBs) that act on cannabinoid 1 receptors (CB1R) to initiate a short or long-term depression in synaptic strength at excitatory and/or inhibitory synapses (Adermark and Lovinger, 2007b; Gerdeman et al., 2002; Hillard and Campbell, 1997). The crucial molecular switch for eCB production is activation of L-type calcium channels and the subsequent postsynaptic [Ca2þ]i (Adermark and Lovinger, 2007a), but eCB release and LTD-formation are also regulated by neuronal firing (Adermark and Lovinger, 2007b, 2009). The level of neuronal activity required for both eCB release and LTD induction is Abbreviations: EtOH, ethanol; eCB, endocannabinoids; CB1R, cannabinoid 1 receptor; MSN, medium spiny neuron. * Corresponding author. Tel.: þ46 31 786 3975. 0028-3908/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2009.12.006

greater at glutamatergic synapses than at GABAergic synapses, probably due to the fact that CB1Rs are more densely expressed on GABAergic synapses (Uchigashima et al., 2007). GABAergic neurotransmission is also more sensitive to low concentrations of the CB1R-agonist WIN55,212-2 (Adermark and Lovinger, 2009). Taken together, GABAergic synapses onto striatal medium spiny neurons (MSNs) are more susceptible to eCB modulation in comparison to glutamatergic synapses onto the same neurons, and LTD can be induced at lower frequencies of afferent input than those needed to induce LTD at glutamatergic synapses (Adermark and Lovinger, 2009). Thus, the properties of LTD at these synapses generate a unique situation in which the frequency of glutamatergic synaptic input onto MSNs controls whether LTD will have net disinhibitory effect (i.e. depression of inhibitory GABAergic synaptic input), or a net inhibitory effect (depression of excitatory glutamatergic synapses). In other words, if a low stimulation protocol is given the level of eCBs will only be sufficient to depress neurotransmission at inhibitory GABAergic synapses, thus enhancing population spike amplitude and leading to a long-lasting disinhibition (DLL) of striatal output (Adermark and Lovinger, 2009).

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Recent research has suggested that the eCB system could be involved in the neuronal circuitry regulating EtOH preference in rodent. Mice lacking the CB1R and rats treated with CB1R antagonists show reduced alcohol consumption and motivation to consume alcohol (Lallemand and De Witte, 2006; Vinod et al., 2008b). EtOH self-administration is also reduced after treatment with the eCB anandamide transport blocker AM404, and after manipulation of the eCB-degrading enzyme fatty acid amide hydrolase (FAAH), further supporting a role for the eCB system in reward mechanisms and addictive behavior (Cippitelli et al., 2007; Vinod et al., 2008a). Studies from other brain regions suggest that eCB-signaling is enhanced by acute EtOH exposure (Basavarajappa et al., 2008; Perra et al., 2008; Yin et al., 2007), and high-frequencyinduced plasticity in the dorsomedial striatum has been shown to be shifted from LTP to LTD in EtOH-treated slices, suggesting that EtOH could modulate learning of all instrumental behaviors under its influence (Yin et al., 2007). The aim of this study was to evaluate if acute EtOH exposure would modulate eCB-signaling in the dorsolateral striatum, a brain region vital for habit formation and involved in the motivation to procure the drug in addicted humans (Volkow et al., 2007; Yin et al., 2008). We hypothesized that EtOH would facilitate eCB-signaling, and enhance LTD-formation induced by lower stimulation frequencies. 2. Materials and methods 2.1. Brain slice preparation Experiments were carried out in accordance with the guidelines laid down by the Swedish Council regarding the care and use of animals for experimental procedures and were approved by the Local Ethics Committee of University of Gothenburg (42-07, 2007-02-28). Striatal slices were prepared from 18 to 25-dayold Wistar rats (Charles River, Germany). A subset of slices was also prepared from male Wistar rats weighing 270e350 g (Taconic, Denmark). The animals were deeply anesthetized with Isofluran Baxter (Baxter medical AB, Kista, Sweden) and decapitated. The brains were quickly removed and placed in ice-cold modified artificial cerebrospinal fluid (aCSF) containing (in mM); 194 sucrose, 30 NaCl, 4.5 KCl, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4 and 10 D-glucose, saturated with oxygen. After a 5 min equilibration period the brain tissue was attached with histoacryl (Aesculap & CO. KG, Tuttlingen, Germany) to a Teflon pad submerged into ice-cold modified aCSF and sectioned coronally in 400 mm thick slices with a vibrating tissue sectioning system (Campden Instruments Ltd., Loughborough, England). Brain slices were allowed to equilibrate for at least 1 h at room temperature in normal aCSF containing (in mM); 124 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.2 NaH2PO4 and 10 D-glucose, continuously bubbled with a mixture of 95% O2/5% CO2 gas. 2.2. Striatal field potential recordings One hemisphere of a slice was transferred to a recording chamber and perfused at a constant rate of 2.6 ml/min with pre-warmed aCSF kept at 30  C. Stimulation was delivered as 0.1 ms negative constant current pulses via a monopolar tungsten electrode placed at the border of the dorsolateral striatum and the overlying white matter (World Precision Instruments, FL, USA, type TM33B), activated at a frequency of 0.05 Hz. Stimulus intensity was set to yield an evoked PS amplitude approximately half the size of the maximal evoked response. The half-maximal responses ranged from 0.3 to 1.3 mV, and were evoked with stimuli of 0.03e0.05 mA in intensity, and 0.1 ms duration. Long-lasting disinhibition (DLL) was induced by low/ moderate frequency stimulation (one 5 Hz train delivered for 1 min, 60 s-5 Hz), by treatment with the L-type calcium channel activator 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methylester (FPL64176, FPL), or by the CB1R-agonist WIN55,212-2 (300 nM) (Adermark and Lovinger, 2009; Adermark et al., 2009). CB1R-mediated depression of PS amplitude was induced by highfrequency stimulation (HFS) (four trains of 100 pulses delivered at 100 Hz) paired with an increase in stimulation amplitude to 0.08 mA, and by treatment with 1 mM WIN55,212-2. Slices were perfused with EtOH for at least 20 min before DLL or LTD induction, and continuously throughout the experiments. In a subset of slices eCBsignaling and/or DLL was prevented with the CB1R antagonist AM251 (2 mM), or bicuculline (20 mM). AM251 or bicuculline was applied at least 20 min before and continuously throughout the experiments. We also evaluated the role of group 1 mGluRs in FPL-DLL. The mGlu1 receptor antagonist 7-(Hydroxyimino)cyclopropa[b] chromen-1a-carboxylate ethyl ester (CPCCOEt, 40 mM), and the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP, 40 mM) were applied at least 20 min before FPL and continuously throughout the experiment. We also confirmed EtOH effects on DLL on slices from adult Wistar rats. Signals were

amplified by a custom-made amplifier, filtered at 3 kHz, digitized and transferred to a PC clone computer for on-line and off-line analysis.

2.3. Whole-cell patch clamp recordings Electrophysiological recordings were performed as previously described (Adermark and Lovinger, 2007a). Internal solutions consisted of (in mM); 120 CsMeSO3, 5 NaCl, 10 TEA-Cl, 10 HEPES, 5 QX-314, 1.1 EGTA, 4 Mg-ATP, 0.3 Na-GTP, for experiments examining EPSCs, and 150 CsCl, 10 HEPES, 2 MgCl2, 0.3 Na-GTP, 3 Mg-ATP, 0.2 BAPTA, for experiments examining IPSCs. For EPSC recordings 50 mM 5 D, L-2-Amino-5 phosphonovaleric acid (AP-5) and 50 mM picrotoxin were added to the aCSF. For IPSC measurements, 5 mM 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f] quinoxaline-7-sulfonamide disodium salt (NBQX) and 50 mM AP-5 were added to the aCSF. Currents were measured in conventional ruptured-patch whole-cell mode in MSNs voltage clamped at 50 mV, which resembles the characteristic depolarized ‘upstate’ as previously described by Surmeier and co-workers (Surmeier and Kitai, 1997). Baseline synaptic currents were evoked by a paired stimuli (50 ms interpulse interval) delivered every 20 s through a bipolar electrode placed in the border of the overlaying white matter. Stimulus parameters were adjusted to elicit baseline EPSC or IPSC amplitudes between 200 and 400 pA eCB-signaling was induced by a single train delivered at 1 Hz for 1 min (60 pulses) (60 s-1 Hz). Experiments were performed at 29e32  C, and were discontinued if the series resistance varied by more than 20% or increased over 30 MU.

2.4. Data analysis Data was analyzed with Clampex 10.1 (Molecular Devices, Foster City, CA), and graphs were assembled in GraphPad Prism (GraphPad Software, Inc., San Diego, CA) and Photoshop. Data is presented in text as mean values compared to baseline with 95% confidence interval (CI). Time course figures are plotted as mean amplitude compared to baseline, with standard error of the mean (SEM). Two-tailed paired t-test was used for statistical analysis unless anything else is stated.

3. Results 3.1. LTD induced by high-frequency stimulation is not modulated by acute EtOH exposure in the dorsolateral striatum Recent studies from different brain regions have indicated that EtOH-treatment promotes eCB-signaling (Basavarajappa et al., 2008; Perra et al., 2008; Yin et al., 2007). We were thus interested to see whether eCB-signaling and LTD-formation in the dorsolateral striatum would be facilitated by acute EtOH-treatment. Highfrequency stimulation (4 trains of 100 pulses delivered at 100 Hz) (HFS) induced eCB-mediated LTD in untreated control slices (HFSLTD), which was blocked by the CB1R antagonist AM251 (2 mM) (PS amplitude at t ¼ 30e35 ¼ 85  11% of baseline, t ¼ 2.75, df ¼ 11, n ¼ 12, p < 0.05; PS amplitude at t ¼ 30e35 in slices treated with AM251 for at least 20 min before, and continuously throughout the experiment ¼ 99  3.85% of baseline, t ¼ 0.64, df ¼ 11, n ¼ 12, p > 0.05) (Fig. 1A). Exposure to 50 mM EtOH for 20 min before HFS, and continuously throughout the experiment, did not affect HFSLTD (PS amplitude at t ¼ 30e35 ¼ 81 11% of baseline, t ¼ 3.60, df ¼ 9, n ¼ 10, p < 0.01; control vs. 50 mM EtOH, t ¼ 0.55, df ¼ 20, p > 0.05). HFS-LTD was also not modulated in slices treated with 50 mM EtOH for 20 min and then subjected to a 30 min washout period before HFS (PS amplitude at t ¼ 30e35 ¼ 80  15% of baseline, n ¼ 9, t ¼ 2.72, df ¼ 8, p < 0.05) (Fig. 1A). Treatment with a higher EtOH concentration (80 mM) was also insufficient to significantly modulate HFS-LTD (PS amplitude at t ¼ 30e35 min ¼ 75  11% of baseline, t ¼ 4.63, df ¼ 14, n ¼ 15, p < 0.001; 80 mM EtOH vs. control, t ¼ 1.20, df ¼ 24, p > 0.05) (Fig. 1B). HFS can induce NMDAR-dependent LTP at corticostriatal synapses and thus modulate net striatal output (Partridge et al., 2000). However, treatment with the NMDAR inhibitor AP-5 did not enhance EtOH-mediated effects on HFS-LTD (PS amplitude at t ¼ 30e35 in AP-5-treated slices ¼ 84  6.9% of baseline, t ¼ 4.70, df ¼ 13, n ¼ 14, p < 0.001; PS amplitude at t ¼ 30e35 in AP5 þ EtOH-treated slices ¼ 76  10% of baseline, t ¼ 4.61, df ¼ 8,

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Fig. 1. EtOH does not enhance HFS-LTD. A) HFS (4 trains of 100 pulses delivered at 100 Hz) induced eCB-mediated LTD in control slices, which was completely blocked by the CB1R antagonist AM251. HFS-LTD was not modulated in slices treated with EtOH (50 mM), or subjected to 20 min EtOH-treatment followed by a 30 min washout period. Example traces show PS at baseline and after HFS in a control slice. B) Treatment with 80 mM EtOH for 20 min before HFS, and continuously throughout the experiment, did not enhance HFS-LTD significantly. Example traces show PS at baseline and after HFS in an EtOH-treated slice. C) EtOH (50 mM) did also not modulate HFS-LTD in slices treated with the NMDAR antagonist AP-5 (50 mM) PS data are mean values compared to baseline with SEM. Calibrations are 0.2 mV and 5 ms.

n ¼ 9, p < 0.01; AP-5 vs. AP-5 þ EtOH-treated slices, t ¼ 1.23, df ¼ 21, p > 0.05) (Fig. 1C). 3.2. EtOH prevents long-lasting disinhibition induced by low-frequency stimulation We have previously shown that GABAergic synapses are more sensitive to eCB-signaling, and that stimulation with a low frequency (1e5 Hz) for short time periods (1 min) leads to CB1Rdependent long-lasting disinhibition (DLL) of striatal output caused by reduced synaptic strength at inhibitory synapses (Adermark and Lovinger, 2009). In order to distinguish DLL from LTP we treated slices with the NMDAR blocker AP-5 (50 mM) (Dang et al., 2006). Moderate afferent activation with a train of 300 pulses delivered at 5 Hz (1 min stimulation) (60 s-5 Hz) induced robust eCB-mediated DLL in AP-5-treated slices, which was blocked by AM251 (PS amplitude at t ¼ 30e35 min ¼ 136  15% of baseline, n ¼ 15, t ¼ 4.7, df ¼ 14, p < 0.001; PS amplitude in AM251-treated slices at t ¼ 30e35 min ¼ 103  5.0% of baseline, n ¼ 12, t ¼ 1.02, df ¼ 11, p > 0.05). Interestingly, DLL was also blocked in slices treated with as low as 20 mM EtOH and AP-5 (50 mM) for at least 20 min before stimulation, and continuously throughout the experiment (PS amplitude at t ¼ 30e35 min in EtOH-treated slices ¼ 106  5.8% of baseline, t ¼ 2.04, df ¼ 6, n ¼ 7, p > 0.05 (20 mM EtOH); 105  7.3% of baseline, t ¼ 1.37, df ¼ 8, n ¼ 9, p > 0.05 (50 mM EtOH); AP-5treated vs. AP-5 þ EtOH-treated (20 mM), t ¼ 2.58, df ¼ 20, p < 0.05) (Fig. 2A). This lack of DLL could be connected to reduced LTD-formation at inhibitory synapses, or by facilitated eCBsignaling at excitatory synapses, thus stabilizing striatal output. To determine if EtOH promotes eCB-signaling at excitatory synapses we treated slices with the GABAA receptor antagonist picrotoxin (50 mM) þ EtOH (50 mM) for at least 20 min before stimulation, and continuously throughout the experiment. Striatal output was not affected by moderate frequency stimulation in these slices, indicating that EtOH-treatment impedes eCB-signaling at GABAergic synapses (PS amplitude at t ¼ 30e35 min ¼ 98  6.7% of baseline, t ¼ 0.77, df ¼ 7, n ¼ 8, p > 0.05; control vs. AP-5-treated, t ¼ 1.82, df ¼ 13, p > 0.05) (Fig. 2A). To confirm this EtOH effect on DLL we performed additional recordings in slices from adult Wistar rats. DLL induced by 5 Hz-60 s stimulation was not significantly different in slices from adult rats as compared to slices from juvenile rats (PS amplitude at t ¼ 30e35 ¼ 118  5.7% of baseline, t ¼ 6.32, df ¼ 9, n ¼ 10, p < 0.001; adult vs. juvenile, t ¼ 1.97, df ¼ 20, p > 0.05). Furthermore, treatment with 50 mM for 20 min before and continuously throughout the experiment completely blocked DLL (PS amplitude at

t ¼ 30e35 ¼ 90  6.64% of baseline, t ¼ 2.85, df ¼ 8, n ¼ 7, p < 0.05; control vs. EtOH-treated, t ¼ 6.28, df ¼ 16, p < 0.001) (Fig. 2B). To further address the synapse specificity we studied evoked excitatory and inhibitory postsynaptic currents (EPSCs/IPSCs) in MSNs from the dorsolateral striatum voltage clamped at 50 mV eCB-signaling was induced by a single train delivered at 1 Hz for 1 min (60 pulses) (60 s-1 Hz), a protocol that has previously been shown to be compatible with the 60 s-5 Hz stimulation protocol applied to unclamped cells in field potential recordings (Adermark and Lovinger, 2009). This stimulation protocol induced a small but significant depression of EPSC amplitude (EPSC amplitude at t ¼ 15e20 min ¼ 87  6.1% of baseline, t ¼ 4.4, df ¼ 5, n ¼ 6, p < 0.01) (Fig. 2C), which was not enhanced by EtOH-treatment (EPSC amplitude at t ¼ 15e20 min in EtOH-treated slices ¼ 92  12% of baseline, t ¼ 1.30, df ¼ 6, n ¼ 7, p > 0.05; control vs. EtOH-treated, t ¼ 0.76, df ¼ 11, p > 0.05) (Fig. 2C). At inhibitory synapses the 1 Hz60 s-stimulation induced a robust LTD (IPSC amplitude at t ¼ 15e20 min ¼ 47  12% of baseline, t ¼ 8.38, df ¼ 6, n ¼ 7, p < 0.001), which was significantly reduced in EtOH-treated slices (IPSC amplitude at t ¼ 15e20 min in EtOH-treated slices ¼ 85  11% of baseline, t ¼ 3.26, df ¼ 6, n ¼ 7, p < 0.05; control vs. EtOH-treated slices, t ¼ 4.60, df ¼ 12, p < 0.001) (Fig. 2D), suggesting that acute EtOH impairs low/moderate frequency-induced plasticity at GABAergic synapses in the dorsolateral striatum. 3.3. EtOH modulates eCB-signaling at the presynaptic level In order to determine at what level (postsynaptic (production, mobilization, release) or presynaptic) EtOH affects DLL-formation we treated slices with the L-type calcium channel activator FPL64176 (FPL). This treatment has previously been shown to induce eCB-dependent LTD (FPL-LTD) that is independent on group 1 metabotropic glutamate receptors (mGluRs), dopamine D2 receptors and phospholipase C (PLC) at excitatory synapses (Adermark and Lovinger, 2007a). FPL-LTD at excitatory synapses needs to be combined with paired pulse activation and in the field potential recordings conducted here, we would thus expect FPL-LTD to occur primarily at GABAergic synapses (Adermark et al., 2009). Supporting this hypothesis we found that 10 min treatment with FPL (500 nM) enhanced PS amplitude (PS amplitude at t ¼ 30e35 min ¼ 123  10% of baseline, t ¼ 4.4, df ¼ 6, n ¼ 7, p < 0.01) (Fig. 3A). We could thus use this experimental set up to study eCB-signaling that was independent on presynaptic activation (Adermark et al., 2009). In line with stimulation-induced DLL, FPL-DLL was inhibited in slices treated with 50 mM EtOH (PS amplitude at t ¼ 30e35 min in EtOH-treated slices ¼ 104  8.1% of baseline, t ¼ 1.13, df ¼ 8,

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Fig. 2. EtOH impairs eCB-signaling induced by low/moderate frequency stimulation at inhibitory synapses. A) Stimulation with a 60s train delivered at 5 Hz (60s-5Hz) induced eCBmediated long-lasting disinhibition (DLL) of striatal output in control slices. DLL was inhibited in slices treated with 20 mM EtOH, and in slices treated with 50 mM EtOH and the GABAAR inhibitor picrotoxin (PTX, 50 mM), suggesting that EtOH impairs eCB-signaling at inhibitory synapses. Example traces show PS amplitude at baseline (black) and after FPLtreatment (gray) in a control slice. B) EtOH-sensitive DLL was also induced by 60s-5Hz stimulation in slices from adult rats. Example traces show PS amplitude at baseline (black) and after 60s-5Hz stimulation (gray) in control slices. C) eCB-signaling induced by 60s-5Hz stimulation in field potential recordings has previously been shown to correspond to eCB-signaling induced by a lower stimulation frequency (1eHz) in MSNs clamped at 50 mV (Adermark and Lovinger, 2009). EtOH (50 mM) did not enhance low-frequencyinduced eCB-signaling (60s-1Hz) at excitatory synapses in MSNs clamped at 50 mV. Example traces show evoked EPSCs at baseline (black) and after 60s-1Hz (gray) in an EtOHtreated slice. D) Low-frequency stimulation (60s-1Hz) induced a robust depression of IPSC amplitude, which was significantly depressed in slices treated with EtOH for at least 20 min before low-frequency stimulation, and continuously throughout the experiment. Example traces show evoked IPSCs at baseline (black) and after 60s-1Hz (gray) in an untreated control slice. Calibrations in A and B are 0.2 mV and 5 ms, and 100 pA and 25 ms in C and D. Arrows mark time points for moderate and low-frequency stimulation. PS, EPSC and IPSC data are mean values compared to baseline with SEM.

n ¼ 9, p > 0.05; control vs. EtOH-treated, t ¼ 2.70, df ¼ 14, p < 0.01). However, FPL-DLL was also inhibited in slices treated with the group 1 mGluR antagonists MPEP (40 mM) and CPCCOEt (40 mM) (PS amplitude at t ¼ 30e35 min ¼ 101  7.94% of baseline, t ¼ 0.27, df ¼ 14, n ¼ 15, p > 0.05), suggesting that the postsynaptic requirements for FPL-induced eCB-signaling differs between inhibitory and excitatory synapses. We then went on to evaluate if EtOH-mediated impairment of DLL occurred at the postsynaptic or presynaptic level, and used the CB1R-agonist WIN55,212-2 as a replacement for the 60 s-5 Hz protocol in order to bypass postsynaptic requirements. Since GABAergic synapses show a higher sensitivity to the CB1R-agonist WIN55,212-2 as compared to glutamatergic synapses (Adermark and Lovinger, 2009), and we treated slices with increasing concentrations of WIN55,212-2 (50e300 nM) to find a threshold at which a robust increase in PS amplitude could be detected (Fig. 3B). Treatment with 300 nM WIN55,212-2 significantly enhanced striatal output (PS amplitude t ¼ 30e35 in slices treated with 300 nM WIN55,212-2 ¼ 121  7.2% of baseline, t ¼ 5.57, df ¼ 6, n ¼ 7, p < 0.01) (Fig. 3C). This enhancement was prevented by the GABAA receptor antagonist bicuculline (20 mM), suggesting that 300 nM WIN55,212-2 primarily depresses inhibitory transmission in this experimental set up (PS amplitude t ¼ 30e35 in bicuculline and WIN55,212-2-treated slices ¼ 104  6.9% of baseline, t ¼ 1.07, df ¼ 7, n ¼ 8, p > 0.05). Treatment with 50 mM EtOH for at least 20 min prior to agonist application, and continuously throughout the experiment, prevented the increase in PS amplitude induced by 300 nM WIN55,212-2 (PS amplitude at t ¼ 30e35 in EtOH-treated slices ¼ 102  5.5% of baseline, t ¼ 0.79, df ¼ 8, n ¼ 9, p > 0.05; WIN

vs. WIN þ EtOH, t ¼ 3.98, df ¼ 14, p < 0.01) (Fig. 3C), indicating that EtOH modulates eCB-signaling at inhibitory synapses at a level that is downstream from eCB mobilization and release. In order to determine whether EtOH interfered with CB1R-activation we treated bicuculline (20 mM) perfused slices with a higher concentration of WIN55,212-2 (1 mM), which should decrease the probability for neurotransmitter release also at excitatory synapses and thus lead to a net depressant effect on PS amplitude. Treatment with 1 mM WIN55,212-2 significantly depressed PS amplitude (PS amplitude at t ¼ 30e35 in bicuculline and WIN55,212-2-treated slices ¼ 79  7.1% of baseline, t ¼ 5.86, df ¼ 20, n ¼ 21, p < 0.001). This depression was not sensitive to EtOH, suggesting that EtOH does not interfere with CB1R activation (PS amplitude at t ¼ 30e35 in slices treated with bicuculline and EtOH for at least 20 min prior to WIN55,212-2 application, and continuously throughout the experiment ¼ 82  7.1% of baseline, t ¼ 5.01, df ¼ 20, n ¼ 21, p < 0.001; control vs. EtOH-treated, t ¼ 0.53, df ¼ 40, p > 0.05) (Fig. 3D). 3.4. EtOH does not induce CB1R-mediated changes in baseline striatal output Based on the high sensitivity to CB1R activation at GABAergic synapses and the fact that previous studies have indicated that eCBsignaling might be enhanced by EtOH (Basavarajappa et al., 2008; Perra et al., 2008; Yin et al., 2007), it is possible that acute EtOHtreatment per se enhances baseline striatal output by depressing GABAergic neurotransmission in a CB1R dependent manner. We therefore preformed additional experiments were baseline PS

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Fig. 3. EtOH impairs CB1R-mediated DLL. A) The L-type calcium channel activator FPL (500 nM) induced a robust potentiation of PS amplitude, which was completely blocked in slices treated with 50 mM EtOH. FPL-DLL was also inhibited in slices treated with the mGluR antagonists MPEP (40 mM) and CPCCOEt (40 mM). Example traces show baseline PS (black) and after 10 min FPL-treatment in an EtOH-treated slice (gray). B) Inhibitory synapses have a higher sensitivity to CB1R-activation, and perfusion with low concentrations of WIN55,212-2 disinhibited striatal output. Example traces show baseline PS (black) and after treatment with 300 nM WIN55,212-2 (gray). C) The increase in PS amplitude induced by 300 nM WIN55,212-2 was prevented in slices treated with 50 mM EtOH, suggesting that EtOH impairs a signal transduction process that is downstream from eCB production and release. DLL induced by 300 nM WIN55,212-2 was prevented in slices treated with bicuculline (20 mM). D) Depression induced by 1 mM WIN55,212-2 in bicuculline-treated slices was not sensitive to EtOH. PS data are mean values compared to baseline with SEM. Calibrations are 0.2 mV and 5 ms.

amplitude was monitored over time before and after EtOH application. Treatment with 50 mM EtOH induced a small but significant depression of PS amplitude (PS amplitude at t ¼ 30e35 in control slices ¼ 93  5.4% of baseline, t ¼ 2.66, df ¼ 11, n ¼ 12, p < 0.05). EtOH-mediated changes in striatal output were not prevented in AM251-treated slices, suggesting that EtOH modulates baseline PS amplitude in a CB1R-independent manner (PS amplitude at t ¼ 30e35 in AM251-treated slices ¼ 96  2.4% of baseline, t ¼ 2.74, df ¼ 13, n ¼ 14, p < 0.05; EtOH vs. AM251 þ EtOH, t ¼ 1.35, df ¼ 24, p > 0.05) (Fig. 4). 4. Discussion The data presented here shows that acute EtOH exposure modulates eCB-mediated striatal plasticity in a synapse-specific

manner in both juvenile and adult rats. Since eCB-signaling is critical for maintaining a balance between excitation and inhibition, acute EtOH exposure might affect the stability and the fine-tuning of neuronal circuits in the striatum. EtOH-induced modulation of eCB-signaling might thus shed some light on the physiological underpinnings of acute EtOH intoxication, and could be important for the neuronal adaptations elicited by long-term alcohol consumption (Gerdeman et al., 2003). Based on previous studies the mechanisms underlying eCB mobilization appear to be similar at excitatory and inhibitory synapses. The production of eCBs is dependent on elevated postsynaptic calcium levels, anandamide transporter inhibitors block the release, and LTD induction requires protein synthesis at both types of synapses (Adermark and Lovinger, 2007b, 2009; Adermark et al., 2009). Even so, EtOH did not modulate eCB-signaling and LTD

Fig. 4. EtOH does not affect PS amplitude in a CB1R dependent manner. A) Acute treatment with EtOH (50 mM) slightly depressed baseline PS amplitude in both control and AM251-treated slices. B) Example traces show PS amplitudes before and after EtOH-treatment (upper left and right, respectively). The two traces are shown overlaid below. PS data are mean values compared to baseline with SEM. Calibrations are 0.2 mV and 5 ms.

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induced by HFS in field potential recordings, suggesting that there could be a difference in presynaptic requirements between excitatory and inhibitory synapses (Adermark et al., 2009). However, differences in the experimental outcome might also be related to the frequency by which eCB-signaling is induced. For instance, HFS might affect the intracellular ion homeostasis in the presynaptic terminal in a way that counterbalances possible changes induced by acute EtOH exposure. We have previously shown that elevated levels of extracellular potassium fail to facilitate eCB-signaling (Adermark et al., 2009), but changes in presynaptic calcium could still be a possible candidate linking EtOH-treatment to reduced eCB-signaling (Singla et al., 2007). EtOH inhibits L-type, N-type and possibly T-type calcium channels, and selective inhibitors of neuronal calcium channels attenuate the reinforcing and rewarding properties of EtOH, suggesting that calcium channels are important targets during EtOH intoxication (Newton et al., 2008; Walter and Messing, 1999). EtOH-induced changes in presynaptic calcium levels would also be in line with the finding that EtOH prevented WIN55,212-2-induced disinhibition (Singla et al., 2007). However, a higher concentration of WIN55,212-2-induced EtOH-insensitive depression of PS amplitude in bicuculline-treated slices, suggesting that EtOH affects CB1R-mediated signaling in a synapse-specific, rather than frequency-dependent manner. The mechanisms underlying DLL have not been fully characterized, but mGluR activation has been shown to be vital for 60 s-1 Hz-mediated LTD at inhibitory synapses (Adermark and Lovinger, 2009), and treatment with MPEP and CPCCOEt completely blocked FPL-DLL (Fig. 3A). Since glutamatergic neurotransmission is modified during drug-associated learning, mGluRs might be a possible candidate linking EtOH to impaired eCB-signaling (Choi et al., 2006; Everitt and Wolf, 2002). However, HFS-LTD, which was not sensitive to EtOH, also requires mGluR activation (Sung et al., 2001). Furthermore, EtOH prevented WIN55,212-2-induced DLL, suggesting that EtOH affects eCB-signaling at a level that is downstream from eCB production, mobilization and release, and is thus independent on mGluR activation (Adermark and Lovinger, 2007b). The signal transduction systems connecting activation of presynaptic CB1R with the reduction in neurotransmitter release probability are not yet known in detail, but protein translation appears to be one mechanism that links CB1R activation to the expression of striatal LTD (Adermark et al., 2009; Yin et al., 2006). However, even though EtOH-treatment has been shown to depress protein synthesis (Peters and Steele, 1982), HFS-LTD was sustained in EtOH-treated slices, showing that changes in protein synthesis cannot fully explain EtOH-induced impairment of DLL (Yin et al., 2006). Since enhanced eCB-signaling has been reported after EtOHtreatment in other brain regions (Basavarajappa et al., 2008; Perra et al., 2008; Yin et al., 2007), acute EtOH exposure could theoretically result in depression of baseline synaptic transmission at eCB-sensitive inhibitory synapses. It is thus possible that CB1Rs at eCB-sensitive inhibitory synapses already are activated in EtOH-treated slices, and that additional stimulation fails to depress GABAergic transmission any further. However, PS amplitude was slightly reduced rather than enhanced following acute EtOHtreatment, and this depression was not prevented in AM251-treated slices, suggesting that EtOH-mediated changes in eCB-signaling do not greatly modulate baseline striatal output. Rather, our data are in line with in vivo recordings from the ventral striatum, which have shown that the extracellular levels of the eCB arachidonoylethanolamide (anandamide) are decreased following acute EtOH-treatment (Ferrer et al., 2007). If baseline eCB levels are lower after EtOH-treatment, it is possible that a higher concentration of CB1R-agonist is required in order to induce a depression at inhibitory synapses. However, it needs to be determined if eCB levels are reduced in the striatum following acute EtOH-treatment, and which

mechanisms might underlie this reduction. More studies aimed to establish crucial activation required for DLL would also be required in order to further address EtOH-mediated impairment of DLL. In conclusion, the data presented here show that EtOH modulates striatal plasticity in synapse-specific manner, suggesting that EtOH-induced effects on eCB-signaling could underlie some of the physiological responses associated to alcohol intoxication. Based on previous studies indicating a role for CB1R in EtOH preference, it is possible that manipulation of the eCB system could constitute a new therapeutic strategy for treating alcohol dependence (Lallemand and De Witte, 2006; Parolaro and Rubino, 2008). However, more detailed studies focused on establishing the crucial activation points required for eCB-signaling at striatal inhibitory synapses are needed before EtOH-mediated effects on synaptic plasticity can be fully elucidated. Acknowledgements We are thankful for the suggestions provided by Professor David M. Lovinger and Professor Holger Wigström. This work was supported by the Swedish Society for Medical Research, Swedish Brain Foundation, Swedish Medical Research Council (Diary numbers 2006-2425, 2006-4988 and 2006-6385), Åke Wiberg foundation (113300049), the Swedish Society of Medicine (2008-21390, 2009-22263), Tore Nilsson foundation, Magnus Bergvall's foundation, Fredrik and Ingrid Thuring's foundation, Gunnar och Märta Bergendahls Stiftelse, Wilhelm and Martina Lundgrens Vetenskapsfond, Sigurd och Elsa Goljes minne, governmental support under the LUA/ALF agreement. References Adermark, L., Lovinger, D.M., 2007a. Combined activation of L-type Ca2þ channels and synaptic transmission is sufficient to induce striatal long-term depression. J. Neurosci. 27, 6781e6787. Adermark, L., Lovinger, D.M., 2007b. Retrograde endocannabinoid signaling at striatal synapses requires a regulated postsynaptic release step. Proc. Natl. Acad. Sci. U.S.A. 104, 20564e20569. Adermark, L., Lovinger, D.M., 2009. Frequency-dependent inversion of net striatal output by endocannabinoid-dependent plasticity at different synaptic inputs. J. Neurosci. 29, 1375e1380. Adermark, L., Talani, G., Lovinger, D.M., 2009. Endocannabinoid-dependent plasticity at GABAergic and glutamatergic synapses in the striatum is regulated by synaptic activity. Eur. J. Neurosci. 29, 32e41. Balleine, B.W., Delgado, M.R., Hikosaka, O., 2007. The role of the dorsal striatum in reward and decision-making. J. Neurosci. 27, 8161e8165. Basavarajappa, B.S., Ninan, I., Arancio, O., 2008. Acute ethanol suppresses glutamatergic neurotransmission through endocannabinoids in hippocampal neurons. J. Neurochem. 107, 1001e1013. Choi, S.J., Kim, K.J., Cho, H.S., Kim, S.Y., Cho, Y.J., Hahn, S.J., Sung, K.W., 2006. Acute inhibition of corticostriatal synaptic transmission in the rat dorsal striatum by ethanol. Alcohol 40, 95e101. Cippitelli, A., Bilbao, A., Gorriti, M.A., Navarro, M., Massi, M., Piomelli, D., Ciccocioppo, R., Rodriguez de Fonseca, F., 2007. The anandamide transport inhibitor AM404 reduces ethanol self-administration. Eur. J. Neurosci. 26, 476e486. Dang, M.T., Yokoi, F., Yin, H.H., Lovinger, D.M., Wang, Y., Li, Y., 2006. Disrupted motor learning and long-term synaptic plasticity in mice lacking NMDAR1 in the striatum. Proc. Natl. Acad. Sci. U.S.A. 103, 15254e15259. Everitt, B.J., Wolf, M.E., 2002. Psychomotor stimulant addiction: a neural systems perspective. J. Neurosci. 22, 3312e3320. Ferrer, B., Bermudez-Silva, F.J., Bilbao, A., Alvarez-Jaimes, L., Sanchez-Vera, I., Giuffrida, A., Serrano, A., Baixeras, E., Khaturia, S., Navarro, M., Parsons, L.H., Piomelli, D., Rodriguez de Fonseca, F., 2007. Regulation of brain anandamide by acute administration of ethanol. Biochem. J. 404, 97e104. Gerdeman, G.L., Partridge, J.G., Lupica, C.R., Lovinger, D.M., 2003. It could be habit forming: drugs of abuse and striatal synaptic plasticity. Trends Neurosci. 26, 184e192. Gerdeman, G.L., Ronesi, J., Lovinger, D.M., 2002. Postsynaptic endocannabinoid release is critical to long-term depression in the striatum. Nat. Neurosci. 5, 446e451. Hillard, C.J., Campbell, W.B., 1997. Biochemistry and pharmacology of arachidonylethanolamide, a putative endogenous cannabinoid. J. Lipid Res. 38, 2383e2398.

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