European Journal of Pharmacology 598 (2008) 16–20

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European Journal of Pharmacology

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Molecular and Cellular Pharmacology

Delta-9-tetrahydrocannabinol differently affects striatal c-Fos expression followinghaloperidol or clozapine administration

Giorgio Marchese a,b,⁎, Angela Sanna b, Gianluca Casu b, Paola Casti b,Gabriele Pinna Spada b, Stefania Ruiu a,b, Luca Pani a,b

a C.N.R., Institute of Biomedical Technology, Sect. Cagliari, Technological Park of Sardinia “Sardegna Ricerche”, Pula, Cagliari, Italyb Neuroscienze-Pharmaness S.c.a r.l., Technological Park of Sardinia “Sardegna Ricerche”, Pula, Cagliari, Italy

⁎ Corresponding author. C.N.R., Institute of BiomediTechnological Park of Sardinia “Sardegna Ricerche”, LoCagliari, Italy. Tel.: +39 070 9242025; fax: +39 070 9242

E-mail addresses: giorgio.marchese@itb.cnr.it, giorgi(G. Marchese).

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.08.020

a b s t r a c t

a r t i c l e i n f o

Article history:

It was previously shown t Received 18 February 2008Received in revised form 4 August 2008Accepted 21 August 2008Available online 30 August 2008

Keywords:CannabinoidAntipsychoticSignal transductionBasal ganglia

hat haloperidol, but not clozapine, induced intense rat catalepsy when co-administered with delta-9-tetrahydrocannabinol. The present study investigated whether similar alterationscould be observed on striatal c-Fos immunoreactivity after administration of the same drug combinations.Western Blot and immunocytochemistry stereological analyses indicated that delta-9-tetrahydrocannabinol(0.5 mg/kg) increased striatal c-Fos immunoreactivity induced by haloperidol (0.1 mg/kg). Conversely, nosignificant alterations of striatal c-Fos immunoreactivitywere observed after injections of clozapine (10mg/kg)+vehicle, clozapine+delta-9-tetrahydrocannabinol or vehicle+delta-9-tetrahydrocannabinol. Thepresent resultsindicate that the behavioral effects induced by delta-9-tetrahydrocannabinol in haloperidol- and clozapine-treated rats are associated with different striatal c-Fos expressions.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Several studies showed that c-Fos expression in rat caudate-putamen is reliable index of the extra-pyramidal symptoms liability ofantipsychotic drugs (Robertson et al., 1994; Wan et al., 1995).Consistently, enhanced striatal c-Fos-like expression was observedfollowing acute haloperidol administration, mirroring the high pro-pensity of the antipsychotic to induce rat catalepsy (Robertson et al.,1994). From the other side, the atypical antipsychotic clozapine, that isdevoid of extra-pyramidal symptoms liability,was ineffective in alteringstriatal c-Fos-like expression in rat striatum(Robertson et al.,1994;Wanet al., 1995). Recent behavioral studies conducted in our laboratoriesindicated that the cannabinoid CB1 receptor agonist Δ9-tetrahydrocan-nabinol (Δ9-THC) strongly increased rat catalepsy induced by theconventional antipsychotic haloperidol. Conversely, the association ofΔ9-THCwith the atypical antipsychotic clozapine failed to induce extra-pyramidal symptoms in rats (Marchese et al., 2003). Preliminarypharmacological analyses suggested that CB1 receptor stimulationmight increase the effect of dopamine D2 receptor blockade induced byhaloperidol at striatal level. The absence of rat catalepsy followingclozapine+Δ9-THC administrationwas associated to the antagonism of

cal Technology, Sect. Cagliari,c. Piscinamanna, I-09010 Pula,206.o.marchese@pharmaness.it

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muscarinic M1 and adrenergicα2 receptors mediated by clozapine thatmay reestablish a normal striatal function (Marchese et al., 2003). Toconfirm the hypothesis that Δ9-THC might differently affect striatalfunction in haloperidol and clozapine-treated rats, the present studyinvestigates whether c-Fos expressionmight be diversely modulated inrat striatum depending on the drug-combination administered.

2. Materials and methods

2.1. Animals and drugs

Male Sprague–Dawley albino rats (Charles River, Como, Italy) weight-ing 200–250 gwere kept on a 12 h/12 h dark/light cyclewith food and tapwater available ad libitum. Before starting experimental procedures,animals were randomly divided into 6 groups and treated with differentdrug combinations. The experimental paradigm was designated to givethe following experimental groups: vehicles, haloperidol+vehicle, cloza-pine+vehicle, vehicle+Δ9-THC, haloperidol+Δ9-THC, clozapine+Δ9-THC.

Haloperidol hydrochloride and clozapine (Tocris Cookson Ltd., Avon-mouth Bristol, UK) were administered (s.c.) 1 h before c-Fos immuno-cytochemistry procedures. Δ9-THC (0.5 mg/kg, i.p.) (Sigma Co., St. Louis,MO, U.S.A.) dissolved in 1:1:18, ethanol, cremophor, saline wasadministered 15 min before haloperidol (0.1 mg/kg, s.c.), clozapine(10 mg/kg, s.c.) or vehicle (25 µl of glacial acetic acid, buffered to pH 6.5using NaHCO3 0.1 M in distilled water). Doses refer to the free-base formof thedrugs. All experimental protocolswere in strict accordancewith theE.C. regulation for care and use of experimental animals (CEE N° 86/609).

Fig. 1. Representative western blot of striatal nuclear extracts probed with c-Fosantibody. Differences in c-Fos peptide expression are shown in panel B expressed as % ofcontrol±SEM. Statistical analysis was carried out using One-way ANOVA followed byNewman–Keuls post-hoc test (⁎Pb0.05 vs. vehicle+vehicle; ⁎⁎Pb0.01 vs. vehicle+vehicle; +Pb0.01 vs. haloperidol+vehicle).

17G. Marchese et al. / European Journal of Pharmacology 598 (2008) 16–20

2.2. Western Blot analyses

Rats (6 animals for each experimental group) were killed and thestriatawere rapidly removed, dissected and stored at −80 °C for later use.Nuclear proteins were extracted according to Huang andWalters (1996).In brief, tissue samples were homogenized in 0.5 ml of ice cold buffer A(0.25 M sucrose, 15 mM Tris HCl pH 7.9, 60 mM KCl, 15 mM NaCl, 5 mMEDTApH8.0, 0.5MDTT,10mg/ml leupeptin,1mg/ml pepstatinA, 0.5mMPMSF, 10 mg/ml aprotinin) and centrifuged at 2000 g for 10 min. Thepellets were resuspended in 0.5 ml of buffer B (10 mM HEPES pH 7.9,1.5 mMMgCl2, 10 mMKCl, 0.5 M DTT, with the same amount of proteaseinhibitors as in buffer A) and centrifuged at 4000 g for 10 min. Theresulting pellets were dissolved in 0.2 ml of buffer C (20 mM HEPES, pH7.9, 0.75 mM Mg Cl2, 0.5 mM EDTA pH 8.0, 0.5 M KCl, 0.5 M DTT, 12.5%Glycerol plus protease inhibitors as in buffer A) and incubated in ice for30min. After centrifugation at 14,000 g for 30min the supernatantswerecollected as nuclear extracts. An aliquot was analyzed for proteinconcentrationbyusing aProtein assaykit (Bio-RadLaboratories,Hercules,CA, USA), and the residual was frozen at −80 C° until assayed. Aliquots ofstriatal nuclear extracts containing 50 µg of total protein were separatedby Sodium dodecyl sulfate-polyacrilamide gels (SDS PAGE) and trans-ferred to polyvinyldiene membranes (Immobilon-P, Millipore, Bedford,MA). Blots were blockedwith 5% non fat drymilk in TBST (0.1% Tween 20inTris borate saline) and probed with a specific antibody against c-Fos(Santa Cruz Biotecnology, inc., CA, U.S.A.) with a 1:1000 dilution in TBST.After washing in TBST, blots were probed with horseradish peroxidaseconjugated antibody (1:3000 dilution in TBST) and after washing in TBSTchemiluminescence was detected by West Pico chemiluminescentsubstrate (Pierce, Rockford, IL). Immunoreactive bands were visualizedwith a Fuji Las 1000 image analyzer (Raytest Isotopenmessgeräte GmbH,Straubenhartd, Germany) and the optical density of immunoreactivebands was measured using a specific software (AIDA 2.11, RaytestIsotopenmessgeräte GmbH, Straubenhartd, Germany). One-way ANOVAfollowed by Newman Keuls was performed as a statistical analysis.

2.3. Immunocytochemistry

2.3.1. c-Fos immunocytochemistry procedureRats (8 animals for each experimental group) were anaesthetized

with Equithesin (2.5 mg/kg, i.p.) and perfused transcardially withsaline followed by 4% paraformaldehyde in 0.1% phosphate buffer (PB),pH7.4. The brainswere subsequently post-fixed in the samefixative for2 h and cryoprotected overnightwith a solution of 30% sucrose in 0.1MPB at 4 °C. Rat striatumwas cut in 40 µm thick coronal sections using acryostat (Leica CM3050, Leica Microsystems GmbH,Wtzlar, Germany).After rinsing in phosphate buffered saline containing 0.2% Triton X-100(PBS+T), sections were incubated with 0.3% of H2O2 in PBS and, afterextensivewashing,with a blocking solution containing 1% BSA and 20%normal goat serum in PBS+T to reduce background. Sections were thenincubated overnight at 4° C with a rabbit anti c-Fos antibody (1:4000;Santa Cruz Biotecnology, Inc., CA, U.S.A.). After rinsing, sections wereincubated with a goat anti-rabbit biotinylated IgG (1:200; Vector,Burlingame, CA, U.S.A.) for 1 h, followed by an avidin-biotin complex(1:400; Vectastain ABC kit from Vector) for an additional hour. Afterwashing in PBS+T, sections were exposed to 3,3'-diaminobenzidine(0.06% in PBS) containing 1% cobalt chloride and 1% nickel ammoniumsulfate for 15min. Immunostainingwas developed by adding 5 µl H2O2

(0.1% in PBS) to each 500 µl of 3,3'-diaminobenzidine. After washing inPBS+T, all sections were mounted on gelatin-coated glass slides, air-dried, dehydrated in ascending concentrations of ethanol, clearedwithxylene, and coverslipped with Entellan.

2.3.2. Stereological analyses of c-Fos immunostained neurons in the ratstriatum

Sections were analyzed using a light microscope (Olympus BX 60)coupled with a CCD color video digital camera (Panasonic GP-KR222E,

Osaka, Japan). The estimation of the total number of c-Fos immuno-labeled neurons in rat striatum was performed using a stereologicalquantification technique, the optical fractionator method (Mouton,2002), that was applied using an image analysis computer software(Stereologer, System Planning and Analysis Inc. Alexandria, VA, U.S.A.)connected to a motorized x-y-z motor stage. The stereologicalanalyses were carried from a random position at the origin of thestriatum, until the striatal anatomical structure could be detectable.Every 7th serial section was included in the analysis, so that anaverage of 15–17 sections per animal was analyzed. To univocallydetermine striatum boundaries, cresyl violet staining was carried outin the sections immediately subsequent to those to be analyzed for c-Fos immunocytochemistry. Anatomical structures such as corpuscallosum, lateral ventricle, globus pallidus and nucleus accumbenswere used as reference areas in defining striatal boundaries. Contourdelineations of the striatum were performed using a 2.0 X objective.

Systematic random sampling of each section was performed byrandomly translating a grid with 400×400 μm squares onto thesection of interest. Each intersection represented a sample site wherea 50×50×15 μm optical dissector provided of exclusion lines wasapplied. The c-Fos labelled neurons seen (40× objective) in thecounting frame were only counted if they came into focus within theoptical dissector that was positioned 2 μm below the surface of themounted section.

Statistical analyses were carried out using One-way analysis ofvariance (ANOVA) followed by Newman–Keuls post-hoc test.

3. Results

3.1. Western Blot analyses

To evaluate the effect ofΔ9-THC (0.5 mg/kg, i.p.) in associationwithhaloperidol (0.1 mg/kg, s.c.) or clozapine (10 mg/kg, s.c.), c-Foswestern blot analyses were conducted in nuclear extracts from striataof rats acutely treated with these drugs (Fig. 1). One-way ANOVA

18 G. Marchese et al. / European Journal of Pharmacology 598 (2008) 16–20

revealed a significant effect for the treatment [F(5,30)=16,90, Pb0.01].Post-hoc comparisons revealed that c-Fos expressionwas significantlyincreased in haloperidol+vehicle treated rats (+89%±10.99, Pb0.01vs. vehicle+vehicle treated rats), but not after vehicle+Δ9-THC or

Fig. 2.Histograms showing the effects of different drug combinations on total number of striastereological method. (a) Effect of Δ9-THC on total number of c-Fos immunostained neuronsenhancement of haloperidol-induced c-Fos expression following administration of SR 141716treated with vehicle+vehicle (A), vehicle+Δ9-THC (B), haloperidol+vehicle (C), haloperidol+THC (G). Statistical analyses were carried out using One-way ANOVA followed by Newm##Pb0.01 vs. haloperidol+vehicle; §§Pb0.01 vs haloperidol+ Δ9-THC treated rats).

clozapine+vehicle administration (PN0.05 vs. vehicle+vehicle treatedrats). Moreover, the co-administration of Δ9-THC and haloperidol sig-nificantly increased c-Fos expression with respect to control (+271.9±27.20, Pb0.01 vs. vehicle+vehicle treated rats) and this increase was

tal c-Fos immunostained neurons (mean+SEM), evaluated using the optical fractionatorin haloperidol, clozapine and vehicle treated rats. (b) Suppression of Δ9-THC-mediatedA. Micrographs of c-Fos immunostained neuronwere taken in caudate-putamen of ratsΔ9-THC (D), clozapine+vehicle (E), clozapine+Δ9-THC (F), SR 141716A+haloperidol+Δ9-an–Keuls post-hoc test (⁎Pb0.05 vs. vehicle+vehicle; ⁎⁎Pb0.01 vs. vehicle+vehicle;

19G. Marchese et al. / European Journal of Pharmacology 598 (2008) 16–20

significantly different from that elicited by haloperidol alone (Pb0.01 vs.haloperidol+vehicle treated rats). When Δ9-THC was administeredtogetherwith clozapine, c-Fos expression remained unchanged (PN0.05vs. vehicle+vehicle and PN0.05 vs. vehicle+clozapine-treated rats).

3.2. Stereological analyses of c-Fos immunostained neurons in the ratstriatum

The stereological estimation of the total number of c-Fos immu-nostained neurons within the rat striatum revealed significantdifferences among groups (One-way ANOVA F(5,42)=25.99, Pb0.01).The administrations of clozapine (10mg/kg, s.c.)+vehicle and vehicle+Δ9-THC (0.5 mg/kg, i.p.) were devoid of any effect on striatal c-Fosimmunocytochemistry (PN0.05 vs. vehicle+vehicle treated rats)(Fig. 2A). Conversely, the administration of haloperidol (0.1 mg/kg, s.c.)+vehicle induced a significant increase of the total numbers of striatal c-Fos immunostained neurons when compared to vehicle+vehicletreated rats (Pb0.01) (Fig. 2A). The co-administration of Δ9-THCinduced different effects depending on the antipsychotic injected.Rats treated with both haloperidol and Δ9-THC showed a significantincrease of the total numbers of the striatal c-Fos immunostainedneurons, when compared to haloperidol+vehicle treated rats (Pb0.01)(Fig. 2A). Conversely, the administration ofΔ9-THC failed to induce anysignificant variation of c-Fos immunocytochemistry in clozapine-treated rats (PN0.05 vs. vehicle+clozapine and vehicle+vehicle treatedrats) (Fig. 2A).

In a separate experiment the cannabinoid CB1 receptor antagonistSR 141716A (3 mg/kg, i.p.), administered 30 min before Δ9-THCinjection, reversed the enhancement of c-Fos immunostaininginduced by Δ9-THC in haloperidol treated rats (Pb0.01 vs haloper-idol+Δ9-THC treated rats; PN0.05 vs. haloperidol+vehicle treatedrats) (Fig. 2B).

4. Discussion

Ample evidence indicates that both rat catalepsy and striatal c-Fosexpression induced by haloperidol are primarily related to theantagonism of striatal dopamine D2-receptors (Dragunow et al.,1990; Rogue and Vincendon, 1992). Several neurotransmitters wereshown to modulate such effects induced by D2-receptors blockade inbasal ganglia. The NMDA antagonist MK-801 reduced haloperidol-induced catalepsy and c-Fos expression in specific striatal sub-regions(Boegman and Vincent, 1996; Leveque et al., 2000). Moreover,muscarinic M1, adenosine A2, and histamine H3 receptor antagonistswere shown to reduce both haloperidol-induced extra-pyramidalsymptoms and striatal c-Fos expression in rat (Guo et al., 1992;Hussain et al., 2002).

Previous behavioral analyses indicated that Δ9-THC increasedhaloperidol-induced catalepsy in rats (Marchese et al., 2003).Furthermore, the results of the present study show an enhancementof striatal Fos-like immunoreactivity following Δ9-THC and haloper-idol co-administration that is reversed by the CB1 antagonist SR141716A. It is then feasible that the CB1-receptor system might alsoconcur in regulating the effect of D2 receptor blockade at striatallevel.

This hypothesis is in linewith recent studies showing that an intra-cellular cross-talk between CB1 and D2 receptor signaling cascadesoccurred in basal ganglia (Glass and Felder, 1997; Meschler andHowlett, 2001). Furthermore, it was demonstrated that the stimula-tion of D2-receptor reduced striatal c-Fos expression induced by theCB1 receptor antagonist SR 141716A (Alonso et al., 1999).

A previous study indicated that catalepsy-like behaviour inducedby high doses of Δ9-THC is distinct from that induced by haloperidoland it might be mediated by a decreased 5-HT neurotransmission inthe nucleus accumbens (Sano et al., 2008). The present study showingthat c-Fos expression is increased in rat striatum when Δ9-THC is co-

administered in haloperidol treated rats, suggested that exogenousCB1 stimulation may also affect striatal function when the D2 receptortransmission is blocked by haloperidol.

At the doses administered in the present study, clozapine andhaloperidol-induced high level of D2 receptor occupation at striatallevel (Kapur et al., 2003), however, different effects on c-Fosimmunoreactivitywere observed followingΔ9-THC co-administration.

In spite of its antagonistic properties on D2 receptors, clozapinewas shown to reverse both rat catalepsy and striatal c-Fos expressioninduced by haloperidol (Jann, 1991; Young et al., 1999; Wirtshafter,1998), possibly because the composite receptor-binding profile ofclozapine includes the antagonism of receptor systems able tomodulate striatal c-Fos expression induced by D2-receptor blockade,such as muscarinic M1, histamine H3 and adenosine A2-receptors (Guoet al., 1992; Hussain et al., 2002). Behavioral studies indicated that theantagonism of muscarinic M1 and adrenergic α2 receptors mediatedby clozapine are still effective in reversing rat catalepsy evenwhenΔ9-THC was co-administered (Marchese et al., 2003). It is possible that,similarly to what was observed on rat catalepsy, the antagonism ofnon-D2 receptors induced by clozapine might account for the lack ofeffect of Δ9-THC on striatal c-Fos immunoreactivity in rat treated withthe atypical antipsychotic.

In conclusion, the present study indicated that Δ9-THC increases amolecular event (c-Fos IM) triggered by haloperidol in rat striatum,while no effect could be observed when the cannabinoid agonist wasco-administered with clozapine. A similar patternwas observed on ratcatalepsy using the same drug combinations, possibly indicating thatstriatal activity might be involved in the different catalepticstates induced by Δ9-THC when co-administered with haloperidol orclozapine.

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