Chronic cocaine sensitizes striatal GABAergic synapses to the stimulation of cannabinoid CB1...

Chronic cocaine sensitizes striatal GABAergic synapses tothe stimulation of cannabinoid CB1 receptors

Diego Centonze,1,2 Silvia Rossi,1,2 Valentina De Chiara,1,2 Chiara Prosperetti,1,2 Natalia Battista,2,3 Giorgio Bernardi,1,2

Nicola B. Mercuri,1,2 Alessandro Usiello4 and Mauro Maccarrone2,31Clinica Neurologica, Dipartimento di Neuroscienze, Universita Tor Vergata, Via Montpellier 1, 00133, Rome, Italy2Centro Europeo per la Ricerca sul Cervello (CERC) ⁄ Fondazione Santa Lucia, Rome, Italy3Dipartimento di Scienze Biomediche, Universita di Teramo, Teramo, Italy4Behavioural Neuroscience Laboratory, CEINGE – Biotecnologie Avanzate, Naples, Italy

Keywords: addiction, dopamine, electrophysiology, habit, IPSC

Abstract

Behavioural studies indicate that cannabinoid receptors are implicated in cocaine addiction. The synaptic underpinning of cocaine–cannabinoid receptor interaction is however, obscure. We have studied electrophysiologically the sensitivity of cannabinoid receptorsmodulating synaptic transmission in the striatum of rats exposed to cocaine. One-day treatment with cocaine did not modify thesynaptic response to HU210, a cannabinoid CB1 receptor agonist. Seven days cocaine-treatment, conversely, caused conditionedplace preference, and sensitized striatal GABAergic synapses to the presynaptic effect of cannabinoid CB1 receptor stimulation. Thecannabinoid receptor-induced modulation of glutamate transmission was unaltered by cocaine. Furthermore, the effects of chroniccocaine on cannabinoid-mediated regulation of striatal GABA synapses were attenuated one week after the discontinuation ofcocaine, and absent two weeks later, indicating the progressive reversibility of the adaptations of cannabinoid system duringabstinence of drug consumption. Our data support the concept that modulation of cannabinoid receptors might be useful against drugabuse.

Introduction

Evidence exists that cannabinoid receptors are implicated in cocaineaddiction. D9-tetrahydrocannabinol (THC) consumption in monkeys isgreatly facilitated after previous acquisition of cocaine self-adminis-tration (Tanda et al., 2000; Maldonado & Rodriguez de Fonseca,2002), while blockade of cannabinoid CB1 receptors attenuates boththe rewarding effects of cocaine (Chaperon et al., 1998), and therelapse to cocaine-seeking behaviour induced by re-exposure tococaine-associated cues or cocaine itself (De Vries et al., 2001).Furthermore (AAT)n repeat in the cannabinoid receptor gene has beenfound to be associated with predisposition to cocaine dependency inhumans (Ballon et al., 2006), while lack of CB1 receptors impairscocaine self-administration in mice (Soria et al., 2005).

The involvement of cannabinoid receptors in cocaine addiction ishowever, challenged by other findings. It has been reported, in fact,that pharmacological blockade of cannabinoid CB1 receptors does notaffect cocaine reinforcement nor does it affect the development ofbehavioural sensitization to the locomotor stimulant effects of cocaine(Lesscher et al., 2005). Furthermore, deletion of CB1 receptorseliminates the rewarding effects of cannabinoids (Ledent et al., 1999)and of opiates (Ledent et al., 1999; Martin et al., 2000; Cossu et al.,2001), but leaves the rewarding effects of psychostimulants intact(Martin et al., 2000; Cossu et al., 2001). Finally, CB1 receptorablation does not modify the locomotion elicited by cocaine (Houchiet al., 2005), and CB1 receptor agonist and heroin, but not cocaine,

reinstate cannabinoid-seeking behaviour in rats (Spano et al., 2004).From these data, it is therefore evident that further experimental workis necessary to clarify the putative role of cannabinoid receptors incocaine addiction.One physiological study addressed the possible synaptic basis of

cocaine–cannabinoid interaction. In that study, a single cocaineexposure in vivo was found to abolish endocannabinoid-mediatedlong-term depression (LTD) in the nucleus accumbens (Fourgeaudet al., 2004), identifying synaptic plasticity in dopaminoceptive areasof the reward system as the substrate of such interaction. Besides theirrole in synaptic plasticity, cannabinoid receptors modulate transmitterrelease at both excitatory and inhibitory synapses (Piomelli, 2003;Parolaro et al., 2005), raising the possibility that altered regulation ofthese synapses contributes to the central effects of cocaine exposure.In the present study therefore we investigated the sensitivity of both

glutamate and GABA synapses to cannabinoid receptor stimulation inthe striatum of cocaine-treated rats. The striatum is an ideal structureto study the possible cocaine–cannabinoid interaction, as this brainarea plays a central role in psychostimulant addiction (Kelly et al.,1975; Amalric & Koob, 1993; Berke & Hyman, 2000; Gerdemanet al., 2003; Volkow et al., 2006), and contains high levels ofcannabinoid receptors (Piomelli, 2003).

Materials and methods

Adult male Wistar rats (150–250 g) were used for all the experiments.All procedures were approved by the Institutional Animal Care andUse Committee of the University of Tor Vergata, Rome, and were in

Correspondence: Dr Diego Centonze, 1Clinica Neurologica, as above.E-mail: [email protected]

Received 18 August 2006, revised 22 January 2007, accepted 23 January 2007

European Journal of Neuroscience, Vol. 25, pp. 1631–1640, 2007 doi:10.1111/j.1460-9568.2007.05433.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

accordance with the European Communities Council Directive of 24November, 1986 (86/609/EEC).

Cocaine treatment

Rats were given intraperitoneal (i.p.) injections of either saline (0.9%NaCl) or saline with cocaine (15 mg ⁄ kg). They were divided intogroups that received one or seven daily injections of either cocaine orsaline. The rats were housed with food and water ad libitum in a roomwith a 12-h light : 12-h dark cycle and controlled (22–23 !C)temperature.

Conditioned place preference experiments

Cocaine-rewarding effects were evaluated in the conditioned placepreference (CPP) paradigm by using a biased procedure, as describedpreviously (Acquas & Di Chiara, 1994; Tzschentke, 1998). Briefly, theprotocol consists of three different phases; preconditioning, condi-tioning, and postconditioning phases. During the preconditioningphase (days 1, 2, 3) the guillotine door was kept lifted and each ratwas given access to both compartments of the apparatus for 15 min.On day 3 the time spent by the rat in each compartment was recorded.The time recorded indicates the ‘unconditioned preference’ of each ratfor each compartment. In the conditioning phase (day 3 or days 4–10)the rats were administered cocaine 15 mg ⁄ kg and were then placed,for 25 min, in the ‘nonpreferred’ compartment. After an interval ofapproximately 4 h, the rats were administered with saline and placedin the other compartment. As a result of this conditioning phase,cocaine or saline were paired once or seven times to a specificcompartment. During the postconditioning phase (day 4 or day 11),24 h after the last injection, the guillotine door was removed and thetime spent by each rat in the drug-paired compartment was recordedduring 15 min of observation. The difference in seconds between thetime spent in the drug-paired compartment during the postconditioningtest and that spent in the preconditioning test was taken as a measureof the degree of conditioning induced by the drug.

Electrophysiological experiments

All the animals used for this study were deeply anaesthetized withhalothane and killed by cervical dislocation. Unless otherwise speci-fied, the saline- or cocaine-treated rats were killed 48 h after the last i.p.injection for the electrophysiological recordings. Data from 68 ratstreated with cocaine (1 day, n ¼ 30; 7 days, n ¼ 38) and from 71 ratstreated with saline (1 day, n ¼ 38; 7 days, n ¼ 33) were included inthis study. Rats employed for CCP experiments (n ¼ 7 for both thesaline and cocaine groups) were also used for electrophysiology. Onlyone cell per slice and less than six neurons per animal were recorded.Only one neuron per rat was used for each type of experiment. Unlessotherwise specified, ‘n’ refers to the number of neurons.Corticostriatal slices were prepared for tissue blocks of the brain

(Picconi et al., 2003; Centonze et al., 2006). Briefly, vibratome-cutcoronal slices (200–300 lm) were transferred to a recording chamberand submerged in a continuously flowing artificial cerebrospinal fluid(ACSF) (32 !C, 2–3 mL ⁄min) gassed with 95% O2 and 5% CO2. Thecomposition of the ACSF was (in mm); (126) NaCl, (2.5) KCl, (1.2)MgCl2, (1.2) NaH2PO4, (2.4) CaCl2, (11) glucose, (25) NaHCO3.The striatum could be readily identified under low power magni-

fication, whereas individual neurons were visualized in situ using adifferential interference contrast (Nomarski) optical system. Thisemployed an Olympus BX50WI (Japan) noninverted microscope with·40 water immersion objective combined with an infra-red filter, amonochrome CCD camera (COHU 4912), and a PC compatiblesystem for analysis of images and contrast enhancement (WinVi-

sion 2000, Delta Sistemi, Rome, Italy). Recording pipettes wereadvanced towards individual striatal cells in the slice under positivepressure and, on contact, tight GW seals were made by applyingnegative pressure. The membrane patch was then ruptured by suctionand membrane current and potential monitored using an Axopatch 1Dpatch clamp amplifier (Axon Instruments, Foster City, CA, USA).Whole-cell access resistances measured in voltage clamp were in therange of 5–30 MW.Whole-cell patch clamp recordings were made with borosilicate

glass pipettes (1.8 mm o.d.; 3–5 MW), in voltage-clamp configuration.To study evoked, spontaneous, and miniature GABAA-mediatedinhibitory postsynaptic currents (IPSCs), the recording pipettes werefilled with an internal solution of the following composition (mm);CsCl (110), K+-gluconate (30), ethylene glycol-bis (b-aminoethylether)-N,N,N¢,N¢-tetra-acetic acid (EGTA; 1.1), HEPES (10), CaCl2(0.1), Mg-ATP (4), Na-GTP (0.3). In all the experiments, MK-801 andCNQX were added to the external solution to block, respectively,NMDA and nonNMDA glutamate receptors. In contrast, to studyevoked, spontaneous, and miniature glutamate-mediated excitatorypostsynaptic currents (EPSCs), the recording pipettes were filled withinternal solution of the following composition (mm); K+-gluconate(125), NaCl (10), CaCl2 (1.0), MgCl2 (2.0), 1,2-bis (2-aminophenoxy)ethane-N,N,N,N-tetra-acetic acid (BAPTA; 0.5), HEPES (19), guan-osine triphosphate (GTP; 0.3), Mg-adenosine triphosphate (Mg-ATP;1.0), adjusted to pH 7.3 with KOH. Bicuculline (10 lm) was added tothe perfusing solution in order to block GABAA-mediated transmis-sion.For synaptic stimulation, bipolar electrodes were used. Evoked

EPSCs and IPSCs (eEPSCs, eIPSCs) were obtained at a frequency of0.1 Hz at the holding potential (HP) of )80 mV. Spontaneous(sEPSCs, sIPSCs) and miniature glutamate and GABA events(mEPSCs, mIPSCs) were stored by using P-CLAMP 9 (AxonInstruments) and analysed off line on a personal computer with MiniAnalysis 5.1 (Synaptosoft, Leonia, NJ, USA) software. The detectionthreshold of these events was set at twice the baseline noise. The factthat no false events would be identified was confirmed by visualinspection for each experiment. Offline analysis was performed onspontaneous and miniature synaptic events recorded during a fixedtime epoch (2–3 min), sampled every 2–3 min before (4–5 samplings)and after (5–10 samplings) the application of the drugs. Only cells thatexhibited stable frequencies in control (less than 20% changes duringthe control samplings) were taken into account. For kinetic analysis,events with peak amplitude between 10 and 50 pA were grouped,aligned by half-rise time, and normalized by peak amplitude. Eventswith complex peaks were eliminated. In each cell, all events between10 and 50 pAwere averaged to obtain rise times, decay times, and halfwidths.For data presented as the mean ± SEM, statistical analysis was

performed using a paired or unpaired Student’s t-test or Wilcoxon’stest. The significance level was established at P < 0.05. To determinewhether two cumulative distributions of spontaneous synaptic activitywere significantly different, the Kolmogorov–Smirnov (K–S) test wasused.

Drugs

Drugs were applied by dissolving them to the desired final concen-tration in the bathing ACSF. The concentrations of the various drugswere chosen according to previous in vitro studies on corticostriatalbrain slices (Gubellini et al., 2002; Centonze et al., 2004), and were asfollows; CNQX (10 lm), HU210 (0.1, 0.3, 1, 3 lm), MK-801(30 lm), and tetrodotoxin (TTX, 1 lm; from Tocris Cookson, Bristol,

1632 Diego Centonze et al.

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 1631–1640

UK); bicuculline (10 lm; from Sigma-RBI, St. Louis, USA); andSR141716A (1 lm; from Sanofi-Aventis Recherche, Montpellier,France). Unless specified, HU210 was applied at the concentration of1 lm.

Results

Cocaine-induced conditioned place preference

In order to investigate whether the chosen regimens of cocaineadministration produce addictive behaviour in the intact animal, wetested the rewarding properties of 1 or 7 days 15 mg ⁄ kg cocainetreatment in a CPP paradigm, a behavioural test for studying therewarding properties of drugs with dependence liability in humans(Acquas & Di Chiara, 1994; Tzschentke, 1998; Centonze et al.,2006). Our results, calculated for each rat (n ¼ 7 for both the salineand cocaine groups in the two experimental paradigms) as thedifference between postconditioning and preconditioning time spentin drug-paired compartment, indicated that only chronic cocaineadministration induced a significant (P < 0.05) CPP in the treatedrats (Fig. 1).

Effects of cannabinoid CB1 receptor stimulation on eIPSCs andsIPSCs in saline- and cocaine-treated striatal neurons

Following intrastriatal stimulation, GABA-mediated eIPSCs wererecorded, in the presence of MK-801 plus CNQX, in both saline- (1and 7 days) and cocaine-treated rats (1 and 7 days). These currentswere fully blocked by the GABAA receptor antagonist, bicuculline,and were detected as inward deflections from the baseline. As alreadyreported (Centonze et al., 2002), the amplitude of these synapticcurrents was dependent on the intensity of stimulation and on thedistance between the stimulating and the recording sites, and normallyranged between 30 and 300 pA (Fig. 2A).

As an indicator of the physiological activity of GABA synapses, wealso measured sIPSCs in the striatum of saline-treated (1 and 7 days)and cocaine-treated (1 and 7 days) rats. As with eIPSCs, spontaneoussynaptic events could be blocked in the four groups of animals bybicuculline (n ¼ at least 4 for each group). sIPSC had amplitudes andkinetic properties (rise time, decay time constant, and half width)indistinguishable in saline- and cocaine-treated animals (amplitude

32.30 ± 3.40 pA (1 day saline), 30.70 ± 4.10 pA (7 days saline),34.20 ± 3.90 pA (1 day cocaine), 32.70 ± 4.00 pA (7 days cocaine);rise time 0.89 ± 0.12 ms (1 day saline), 0.94 ± 0.10 ms (7 dayssaline), 0.93 ± 0.18 ms (1 day cocaine), 0.92 ± 0.11 ms (7 dayscocaine); decay time constant 8.42 ± 1.8 ms (1 day saline),8.60 ± 0.9 ms (7 days saline), 8.52 ± 1.2 ms (1 day cocaine),8.54 ± 1.0 ms (7 days cocaine); half width 8.2 ± 1.7 ms (1 daysaline), 8.0 ± 0.7 ms (7 days saline), 8.2 ± 1.4 ms (1 day cocaine),8.2 ± 1.0 ms (7 days cocaine)). Also the frequency of sIPSCs wassimilar in saline- and cocaine-treated rats, and was as follows;1.88 ± 0.34 Hz (1 day saline), 1.76 ± 0.56 Hz (7 days saline),1.85 ± 0.43 Hz (1 day cocaine), 1.91 ± 0.48 Hz (7 days cocaine)(n ¼ at least 15 and P > 0.05 in the four experimental groups and foreach parameter) (Fig. 3A–C).

Fig. 1. Cocaine-induced conditioned place preference. Drug-induced condi-tioned place preference was tested in seven acute (1 day) and chronic (7 days)saline- and cocaine-treated rats. The results are expressed as change of the timespent in drug-paired side ± SEM, calculated as the difference in the time spentduring the postconditioning phase vs. preconditioning phase in the lesspreferred drug-paired compartment. Two–tailed unpaired t-test indicated asignificant conditioned place preference only in chronic cocaine-treated rats(*P < 0.05).

Fig. 2. Chronic cocaine potentiates the depressant action of HU210 on striataleIPSCs. (A) The graph shows the time-course of HU210 effects on eIPSCsrecorded in striatal neurons from saline (7 days)-, cocaine (1 day)-, and cocaine(7 days)-exposed rats. The electrophysiological traces below are examples ofeIPSCs recorded in the three experimental conditions before (1) and 10 minafter (2) the application of HU210. (B) Preincubation of the slices with the CB1receptor antagonist, SR141716A, (empty bar) prevents the depressant action ofHU210 (filled bar) on eIPSCs recorded from saline (7 days)-, cocaine (1 day)-,and cocaine (7 days)-exposed rats.

Chronic cocaine and cannabinoids 1633


In neurons from saline (1 and 7 days)-treated rats, application of thecannabinoid CB1 receptor agonist HU210 (10 min, n ¼ at least 10 forboth groups and both eIPSCs and sIPSCs) significantly (P < 0.01)reduced eIPSCs and the frequency of sIPSC, an effect prevented bypreincubating the slice with the selective antagonist of CB1 receptorsSR141716A (n ¼ 4 and P > 0.05 for both groups and both eIPSCsand sIPSCs). In striatal neurons from cocaine (1 day)-treated rats,HU210 produced similar effects than in the respective controls (n ¼ 7,P > 0.05 for both eIPSCs and sIPSCs). In neurons from chronic(7 days) cocaine-treated rats, conversely, HU210 effects were remark-ably potentiated (n ¼ at least 11, P < 0.01 for both eIPSCs andsIPSCs), indicating increased sensitivity of GABA synapses tocannabinoid receptor stimulation. Also in this group of experiments,SR141716A fully prevented the effects of HU210 (n ¼ 5, P > 0.05;Figs 2 and 3).

A lower concentration of HU210 (0.1 lm) was ineffective in bothsaline (7 days)- and cocaine (7 days)-treated animals (P > 0.05),while 0.3, 1, and 3 lm HU210 dose-dependently inhibited evoked andspontaneous IPSCs. Notably, the effects recorded from animalsexposed to chronic cocaine were remarkably more evident with thethree concentrations of the agonist (P < 0.05; n ¼ at least 7 for eachdrug dose, electrophysiological parameter and experimental group),although EC50 values were similar in saline- and cocaine-treatedanimals [eIPSC 0.28 lm (saline), 0.26 lm (cocaine); sIPSC 0.29 lm(saline), 0.28 lm (cocaine;Fig. 4)].

In both saline- and cocaine-treated rats, holding current, sIPSCmean amplitude, rise time, decay time, and half width were unaffectedby HU210 or SR141716A, applied alone or in combination. Isolatedapplication of SR141716A also failed to alter the frequency of sIPSCsin saline- and cocaine-treated rats (n ¼ at least 7 for each experimentalcondition and group, P > 0.05 for each parameter; data not shown).

Effects of HU210 on paired-pulse ratio in saline- and cocaine-treated rats

To seewhether the effects of cannabinoid receptor stimulation on striatalGABA transmission were presynaptic, we measured the action ofHU210 on paired-pulse ratio (PPR) of IPSCs evoked with aninterstimulus interval of 50 ms. Paired-pulse modification of neuro-transmission, in fact, is attributed to a presynaptic change in releaseprobability, and manipulations depressing transmitter release increasethe magnitude of this ratio at both excitatory and inhibitory synapses(Manabe et al., 1993; Schulz et al., 1994; Centonze et al., 2002, 2006).

In both chronic saline- (n ¼ 5) and chronic cocaine-treated rats(n ¼ 8), the depressant effect of HU210 on eIPSCs was associatedwith a significant increase in paired-pulse ratio (eIPSC2 ⁄ eIPSC1), asexpected for a presynaptic action of the drug (P < 0.01 for bothgroups; Fig. 5).

Effects of HU210 on striatal mIPSCs in saline- and cocaine-treated rats

To isolate synaptic GABA events from the possible contamination ofpre- and postsynaptic spiking activity, miniature GABAergic currents

(mIPSCs) were recorded following the application of TTX (5–10 min), a selective voltage-dependent sodium channel blocker. Whencompared to sIPSCs, the mean frequency of mIPSCs was significantlylower in the four experimental groups (n ¼ at least 10 and P < 0.05for each experimental group), while the mean amplitude wasunchanged (P > 0.05). Furthermore, no difference was evidentbetween mIPSC frequencies and amplitudes recorded in saline (1and 7 days)- and cocaine (1 and 7 days)-treated animals (P > 0.05).We also investigated the electrophysiological effects of HU210 on

mIPSCs recorded from saline- and cocaine-exposed striatal neurons,as inwardly recorded eIPSCs and sIPSCs might be contaminated byunclamped action potentials. In line with our results on eIPSCs, andaccording to previous reports showing that cannabinoid receptorstimulation presynaptically reduces striatal GABA transmission(Szabo et al., 1998; Centonze et al., 2004), bath application ofHU210 in slices from saline (1 and 7 days)- and cocaine (1 and

Fig. 3. Chronic cocaine potentiates the effects of HU210 on striatal sIPSCs. (A) HU210 reduced sIPSC frequency in saline (1 and 7 days)- and cocaine (1 and7 days)-treated rats. The effects of this cannabinoid CB1 receptor agonist were however, potentiated in rats exposed to cocaine for seven consecutive days.(B) The effects of HU210 on sIPSC amplitude were negligible in the four experimental groups. (C) The effects of HU210 on sIPSC frequency were prevented bythe cannabinoid CB1 receptor antagonist, SR141716A. (D–F) The graphs show the cumulative distributions of interevent intervals of sIPSCs recorded before andduring the application of HU210 in a neuron exposed to cocaine for 1 day (D), in a neuron exposed to saline for 7 days (E), and in a neuron exposed to cocaine for7 days (F). The electrophysiological traces on the right are examples of sIPSCs (downward deflections) recorded in the three groups of neurons before and during theapplication of HU210. *P < 0.05.

Fig. 4. Dose–response relationship of the effects of HU210 on both eIPSCsand sIPSCs recorded from chronic saline- and chronic cocaine-treated rats. (A)HU210 inhibited eIPSC amplitude in a dose-dependent manner in both groupsof rats. The effects of 0.3, 1, and 3 lm HU210 were however, more evident inthe cocaine-treated sample of animals. (B) HU210 inhibited sIPSC frequency ina dose-dependent manner in both groups of rats. The effects of 0.3, 1, and 3 lmHU210 were however, more pronounced in the cocaine-treated sample ofanimals. *P < 0.05.



7 days)-treated animals (7–10 min) reduced mIPSC frequency (n ¼ atleast 10 and P < 0.01 for each group). In slices from rats chronicallytreated with cocaine, the inhibitory effects of HU210 on mIPSCfrequency were significantly greater (P < 0.01). As with eIPSCs,preincubation (5–10 min) with the cannabinoid CB1 receptor antag-onist SR141716A fully prevented the effects of HU210 on mIPSCs inboth experimental groups (n ¼ 4 and P > 0.05 for both groups;Fig. 6).

In both saline- and cocaine-treated rats, mIPSC mean frequency,amplitude, rise time, decay time, and half width were unaffected byHU210 or SR141716A (n ¼ at least 5 for both groups and eachexperimental condition, P > 0.05 for each parameter; Fig. 6).

Effects of HU210 on striatal glutamatergic transmission inchronic saline- and cocaine-treated rats

In the striatum, stimulation of cannabinoid CB1 receptors presynap-tically reduces glutamatergic transmission (Gerdeman & Lovinger,2001; Huang et al., 2001; Gubellini et al., 2002). Thus, to see whetherthe altered response to HU210 found in chronic cocaine-treated ratswas restricted to GABA-mediated sIPSCs or also involved glutamate-mediated sEPSCs, we performed whole-cell recordings with potas-sium-based pipettes. At )80 mV HP and in the presence ofbicuculline, both eEPSCs and sEPSCs could be detected in striatalneurons as inward deflections from the baseline. Frequency andamplitude of sEPSCs were not significantly different in saline(7 days)- and cocaine (7 days)-treated animals, although a tendencytoward reduced sEPSC frequency and amplitude was observed in thechronic cocaine group [frequency 3.40 ± 0.31 Hz (7 days saline),2.9 ± 0.39 Hz (7 days cocaine); amplitude 13.01 ± 0.18 pA (7 dayssaline), 10.96 ± 0.95 pA (7 days cocaine) (n ¼ at least 7 andP > 0.05 for both experimental groups and parameters)]. Applicationof HU210 (5–10 min) reduced the amplitude of eEPSCs and thefrequency of sEPSC in both saline and cocaine groups (n ¼ 8 andP < 0.05 for both saline and cocaine groups and for both eEPSCs andsEPSCs). The depressant action of HU210 on eEPSCs and sEPSCsrecorded from rats exposed to cocaine, however, was remarkablysimilar to that of the saline group (P > 0.05), indicating that chronic

Fig. 5. Stimulation of cannabinoid CB1 receptors increases paired-pulse ratioof eIPSCs in saline- and cocaine-treated rats. The graph on the left shows that inboth saline (7 days)- and cocaine (7 days)-treated rats application of HU210increased eIPSC2 ⁄ eIPSC1 in striatal neurons. The electrophysiological tracesare examples of eIPSCs evoked with an interstimulus interval of 50 ms before(left) and during the application of HU210 (right) in saline (7 days)-treatedanimals (upper line) and in cocaine (7 days)-exposed rats (lower line).*P < 0.05.

Fig. 6. Chronic cocaine potentiates the effects of HU210 on striatal mIPSCs. (A) HU210 reduced mIPSC frequency in saline (1 and 7 days)- and cocaine (1 and7 days)-treated rats. The effects of this cannabinoid CB1 receptor agonist were however, potentiated in rats exposed to cocaine for seven consecutive days. (B) Theeffects of HU210 on mIPSC amplitude were negligible in the four experimental groups. (C) The effects of HU210 on mIPSC frequency were prevented by thecannabinoid CB1 receptor antagonist, SR141716A. (D and F) The electrophysiological traces are examples of mIPSCs (downward deflections) recorded in a neuronexposed to cocaine for 1 day (D), in a neuron exposed to saline for 7 days (E), and in a neuron exposed to cocaine for 7 days before (control) and during theapplication of HU210. *P < 0.05.



cocaine selectively alters the sensitivity of striatal GABAergic nerveterminals to the stimulation of cannabinoid receptors (Fig. 7).

TTX fully abolished eEPSCs but failed to change both frequencyand amplitude of sEPSCs in both chronic saline- and cocaine-exposed rats (n ¼ 5 and P > 0.05 for both groups, not shown).These data indicate that striatal spontaneous glutamatergic eventsrecorded in vitro reflected action potential-independent transmitterrelease.

Effects of time after injection on the cocaine-induced sensiti-zation of cannabinoid receptors

We also investigated the sensitivity of GABA synapses to thestimulation of cannabinoid CB1 receptors one and three weeks afterchronic exposure of cocaine. In rats killed one week after the lastinjection of cocaine, the depressant effects of HU210 on eIPSCs andsIPSCs were still greater than in the saline group (n ¼ 7 for bothgroups, P < 0.05 for both eIPSCs and sIPSCs). In rats killed twoweeks later, conversely, the effects of HU210 were similar to thoserecorded in the respective saline-treated group (n ¼ 6 for both groups,P > 0.05 for both eIPSCs and sIPSCs; Fig. 8). Of note, the frequencyof sIPSCs was significantly higher in striatal neurons recorded threeweeks after cocaine (7 days)-treatment (saline 1.5 ± 0.4 Hz, cocaine

3.8 ± 0.8 Hz; P < 0.05). This phenomenon was not further analysedin the present study.

Discussion

The cannabinoid system has been implicated in a number ofdopamine-related disorders, such as schizophrenia (Emrich et al.,1997; De Marchi et al., 2003; Giuffrida et al., 2004), Parkinson’sdisease (Gubellini et al., 2002; Maccarrone et al., 2003), and drugaddiction (Ledent et al., 1999; De Vries et al., 2001; Maldonado &Rodriguez de Fonseca, 2002; Le Foll & Goldberg, 2005; Maldonadoet al., 2006). The involvement of cannabinoids and of theirendogenous analogues (endocannabinoids) in drug addiction is likelyto reflect the effects of these compounds in the so-called ‘rewardcircuitry’, which includes midbrain dopamine neurons and their targetstructures, including the striatum (Berke & Hyman, 2000; Everitt &Wolf, 2002; Maldonado & Rodriguez de Fonseca, 2002; Volkowet al., 2006).A complex interaction between dopamine D2 receptors and the

(endo)cannabinoid system has been described. Stimulation of D2receptors, in fact, increases endocannabinoid levels (Giuffrida et al.,1999; Beltramo et al., 2000; Centonze et al., 2004), and up-regulatesthe expression of CB1 receptors in the striatum (Centonze et al.,

Fig. 7. Chronic cocaine does not alter the sensitivity of glutamatergicsynapses to cannabinoid CB1 receptor stimulation. (A) The graph shows thatthe depressant effects of HU210 on the amplitude of eEPSCs are remarkablysimilar in chronic saline- and chronic cocaine-treated rats. (B) The graph showsthat the depressant effects of HU210 on the frequency of sEPSCs areremarkably similar in chronic saline- and chronic cocaine-treated rats.

Fig. 8. Effects of time after injection on HU210-induced inhibition of GABAtransmission in cocaine-treated rats. (A) This graph shows that the effects ofHU210 on eIPSCs are potentiated in rats chronically (7 days) exposed tococaine and recorded 2 and 7 days after the last injection of the drug. In ratsrecorded 21 days after the last cocaine injection, HU210 produced similareffects than in the respective controls. (B) This graph shows that the effects ofHU210 on sIPSC frequency are potentiated 2 and 7 days, but not 21 days, afterthe last of seven daily injections of cocaine. *P < 0.05.



2004). Furthermore, endocannabinoids act as downstream effectors ofD2 receptor signalling in the inhibition of GABA transmission(Centonze et al., 2004), and in the generation of corticostriatal LTD, aform of synaptic plasticity believed to underlie specific aspects ofpsychostimulant addiction (Gerdeman et al., 2002, 2003). However, arecent electrophysiological study showed that CB1 receptors mediatethe long-term effects of amphetamine in the amygdala, independentlyof dopamine, serotonin, and noradrenaline transmission (Huang et al.,2003).Under certain conditions endocannabinoids, rather than potentiating

the effects of dopamine, can counteract the action of D2 receptorstimulation. In fact, pretreatment with the CB1 receptor antagonistSR141716A has been shown to enhance the stimulation of motorbehaviour induced by the D2 receptor agonist quinpirole (Giuffridaet al., 1999), while the inhibition of intracellular transport of AEAreduced its effects (Beltramo et al., 2000). In line with this, aninteresting report has shown that anadamide reduces dopaminetransport by neurons, with a potentially relevant effect on dopamin-ergic neurotransmission (Chen et al., 2003).In the present study, we have provided evidence that the rewarding

properties of cocaine exposure are associated with hypersensitivity ofstriatal GABA synapses to the stimulation of cannabinoid CB1receptors. Accordingly, we have observed that cocaine-induced CPP inrats was associated with the potentiation of the inhibitory action ofcannabinoid receptors on striatal GABA transmission. Notably, theCPP procedure selectively examines the positive reinforcing propertiesof addictive compounds (Acquas & Di Chiara, 1994; Tzschentke,1998; Le Foll & Goldberg, 2005), and previous behavioural studieshave indicated that the (endo)cannabinoid system, rather thancontributing to the hedonic and psychomotor effects of cocaineconsumption, is involved in the generation and maintenance ofreward-based addictive behaviour (De Vries & Schoffelmeer, 2005; LeFoll & Goldberg, 2005; Parolaro et al., 2005). Of interest is the factthat a single cocaine exposure, which is known to induce othermeasurable behavioural and electrophysiological effects (Unglesset al., 2001; Borgland et al., 2004), failed to induce both CPP andsensitization of cannabinoid receptors.Consistent with the proposed involvement of CB1 receptor

sensitization in cocaine addiction, the CB1 receptor antagonistSR141716A blocked both the sensitized response to cannabinoidreceptor stimulation (present study), and the acquisition of CPP incocaine-treated rats (Chaperon et al., 1998). In our electrophysiolog-ical experiments, SR141716A failed to alter per se the frequency ofsIPSCs and of mIPSCs, not only in control animals but also in chroniccocaine-treated rats. This finding is remarkable because the changes inHU210 sensitivity observed in the latter experimental group could inprinciple indicate a change in CB1 receptor coupling, therebyfavouring the activity of SR141716A as an inverse agonist, asreported with this compound in other neuronal systems (Griffin et al.,1998; Govaerts et al., 2004).SR141716A could in principle target additional subtypes of

cannabinoid receptors in the striatum (Breivogel et al., 2001), leavingopen the possibility that also these receptors may be modulated bycocaine. Yet, the non-CB1 receptor is blocked by SR141716A withlower potency, and is not activated by HU210 (Breivogel et al., 2001).Therefore, the observations reported here that SR141716A preventedthe cocaine effect at a dose (1 lm) widely used to antagonize genuineCB1 receptors (Pertwee & Ross, 2002), and that also HU210depressed sIPSCs recorded from striatal neurons seem to speak infavour of an authentic CB1-mediated action.We have also shown that the effects of cannabinoid CB1 receptor

stimulation on sensitized striatal GABA synapses were entirely

mediated by a presynaptic mechanism, as HU210 increased paired-pulse ratio of eIPSCs, and selectively reduced sIPSC and mIPSCfrequency not only in saline-treated animals but also in chroniccocaine-exposed rats. A concomitant alteration of the sensitivity ofstriatal neurons to synaptically released GABA, in fact, would alteralso the amplitude and ⁄ or the kinetic properties of sIPSCs and ofmIPSCs. Our findings are compatible with the preferential location ofCB1 receptors on GABAergic nerve terminals described in thestriatum (Piomelli, 2003).GABAergic innervation of striatal cells is essentially intrinsic, as

recurrent collaterals of projection neurons and GABAergic interneu-rons virtually represent the totality of their inhibitory inputs (Plenz &Kitai, 1998; Koos & Tepper, 1999; Tunstall et al., 2002). Among thevarious GABAergic inputs to spiny neurons, those originating fromfast-spiking interneurons seem to exert the prominent inhibitorycontrol on the functional activity of these cells (Plenz & Kitai, 1998;Koos & Tepper, 1999). As these GABAergic interneurons are apreferential location of cannabinoid CB1 receptors in the striatum,where they act to inhibit GABA release (Hohmann & Herkenham,2000; Iversen, 2003; Piomelli, 2003), it is plausible that the IPSCsmeasured in our study were those originating from the activity of thesecells, as they were modulated by cocaine through the engagement ofcannabinoid CB1 receptors.CB1 receptors also control glutamate transmission in the striatum

(Gerdeman & Lovinger, 2001; Huang et al., 2001; Gubellini et al.,2002; Carrier et al., 2005). Our results, however, indicate the selectiveinvolvement of cannabinoid receptors modulating GABA synapses inthe adaptive changes associated with the rewarding properties ofcocaine. We have found, in fact, that the sensitivity of glutamatesynapses to CB1 receptor stimulation was unaltered following chroniccocaine, indicating differential regulation mechanisms of distinctcannabinoid receptors. In this respect, a recent finding showed alteredresponses to the stimulation of cannabinoid receptors modulatingGABA transmission but not of those regulating glutamate transmis-sion in Huntington’s disease mice (Centonze et al., 2005). Theunaltered sensitivity of glutamate synapses to cannabinoid receptorstimulation might suggest that striatal endocannabinoid-mediatedplasticity of excitatory synapses is relatively unaffected by cocaine.This hypothesis, however, should be validated by measuring LTD atcorticostriatal synapses following cocaine administration, as a previ-ous study showed that a single cocaine exposure reduced endocann-abinoid-mediated LTD in the ventral striatum (Fourgeaud et al., 2004).This kind of experiment might also help to clarify whether chroniccocaine is able to induce LTD in the dorsal striatum, as described inthe ventral part of this nucleus (Thomas et al., 2001), and suggested bythe tendency towards reduced sEPSC frequency and amplitudeobserved in animals chronically treated with cocaine (present study).Striatal neuron firing activity is governed by both glutamate-

mediated excitatory synaptic inputs and GABA-dependent inhibitorypotentials, which efficiently contrast the excitatory action of glutamate(Kita, 1996; Plenz & Kitai, 1998). Accordingly, in vivo, blockade ofongoing GABAergic inhibition of striatal neurons significantlyelevates basal activity of these cells (Nisenbaum & Berger, 1992).Together, the normal response of glutamate synapses to HU210 andthe potentiated response of GABA terminals to this agonist indicatethat cocaine increases the excitability of striatal neurons bydifferentially sensitizing distinct cannabinoid receptors.The effects of chronic cocaine on cannabinoid-mediated regulation

of striatal GABA synapses were attenuated but still present one weekafter the discontinuation of cocaine, and absent four weeks after. Thesedata indicate the progressive reversibility of the adaptations of(endo)cannabinoid system during abstinence of drug consumption.



In this context, preliminary data from our laboratory indicate that aparallel synaptic change observed following chronic cocaine, theblockade or corticostriatal synaptic depotentiation (Centonze et al.,2006), is more refractory to erasing processes during cocaineabstinence, as this alteration of corticostriatal synaptic plasticity wasstill present one month after the last of seven daily injections ofcocaine (unpublished observations).

The potentiated cannabinoid-dependent inhibition of GABA inputsobserved here might favour cocaine-dependent corticostriatal synapticplasticity. In this line, it has been reported that cannabinoid-dependentinhibition of GABA-transmission may actually facilitate the emer-gence of synaptic plasticity at excitatory synapses in the hippocampus(Carlson et al., 2002).

Understanding the molecular and physiological correlates of theaddictive behaviour is essential for the development of effectivepharmacological strategies against drug abuse (Nestler, 2002).

AcknowledgementsWe thank M. Tolu for technical assistance. The support of the FondazioneScippacercola to A.U. is also acknowledged. This work was supported by agrant from Ministero della Salute, Ricerca Finalizzata to D.C.

AbbreviationsACSF, artificial cerebrospinal fluid; CB, cannabinoid; CPP, conditioned placepreference; eEPSC, evoked excitatory postsynaptic current; eIPSC, evokedinhibitory postsynaptic current; EPSC, excitatory postsynaptic current; IPSC,inhibitory postsynaptic current; LTD, long-term depression; mEPSC, miniatureexcitatory postsynaptic current; mIPSC, miniature inhibitory postsynapticcurrent; sEPSC, spontaneous excitatory postsynaptic current; sIPSC, sponta-neous inhibitory postsynaptic current.

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Chronic cocaine sensitizes striatal GABAergic synapses to the stimulation of cannabinoid CB1...

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