Departamento de Fisiologıa y Farmacologıa, Universidad de Cantabria. Instituto de Biomedicina y Biotecnologıa de Cantabria

(CSIC-UC-IDICAN), Santander, Centro de Investigacion Biomedica en Red, Salud Mental (CIBER-SAM), Instituto de Salud Carlos

III, Spain

The endocannabinoid system (ECs) and the cannabinoidreceptors (CB1 and CB2) have attracted much attentionregarding their role in the neurobiology and treatment ofmood disorders (Onaivi et al. 2006; Witkin et al. 2005;Vinod and Hungund 2006). CB1 receptors are highlyexpressed in the cerebral cortex, striatum, substantia nigra,hippocampus, and other limbic structures (Herkenham et al.1991; Moldrich and Wenger 2000; Howlett et al. 2002),where they mediate the central effects of exogenous andendogenous CBs, including effects on mood (Chaperonand Thiebot 1999; Howlett et al. 2002). Pharmacologicaland transgenic studies in animals demonstrate the participa-tion of CB1 receptors in the control of emotional processes(Martin et al. 2002; Witkin et al. 2005). Moreover, aug-mented levels of ECs, increased CB1 receptor density, andagonist-stimulated CB receptor activity in the prefrontalcortex (PFC) of depressed suicide victims have been reported(Hungund et al. 2004; Vinod et al. 2005; Pazos et al. 2006).

Bilateral olfactory bulbectomy (OBX) in the rat is widelyused as an animal model of depression with comorbid

anxiety and exhibiting a number of behavioral, neurochem-ical, and structural changes that are reversed by chronic, butnot acute, administration of clinically effective antidepressantdrugs (Kelly et al. 1997; Song and Leonard 2005). More-over, in the context of the ‘neurogenesis hypothesis ofdepression’ (Banasr and Duman 2007), it is worth mention-ing that the impaired cell proliferation and/or neuronaldegeneration observed following OBX are reduced by someantidepressants (Jaako-Movits et al. 2006; Keilhoff et al.2006; Jarosik et al. 2007).

Received April 29, 2008; revised manuscript received December 13,2008; accepted December 16, 2008.Address correspondence and reprint requests to Alvaro Dıaz, MD

PhD, Departamento de Fisiologıa y Farmacologıa, Cardenal Herrera Orias/n, University of Cantabria, Santander 39011, Spain.E-mail: alvaro.diaz@unican.esAbbreviations used: 5-HT, 5-hydroxytryptamine or serotonin; CB,

cannabinoid; DRN, dorsal raphe nucleus; EC, endocannabinoid; OBX,olfactory bulbectomy; PFC, prefrontal cortex; SSRI, selective serotoninreuptake inhibitor.

Abstract

Bilateral olfactory bulbectomy in the rat (OBX) induces

behavioral, neurochemical, and structural abnormalities simi-

lar to those observed in human depression that are normal-

ized after chronic, but not acute, treatment with

antidepressants. In our study, OBX animals exhibited signifi-

cant increases in both CB1 receptor density ([3H]CP55490

binding) and functionality (stimulation of [35S]GTPcS binding

by the cannabinoid (CB) agonist WIN 55212-2) at the pre-

frontal cortex (PFC). After chronic treatment with fluoxetine

(10 mg/kg/day, 14 days, s.c.), OBX-induced hyperactivity in

the open-field test was fully abolished. Interestingly, chronic

fluoxetine fully reversed the enhanced CB1-receptor signaling

in PFC observed following OBX. The CB agonist D9-tetra-

hydrocannabinol (5 mg/kg, i.p., 1 day) did not produce any

behavioral effect in sham-operated animals but returned

locomotor activity to control values in OBX rats. As both acute

administration of D9-tetrahydrocannabinol and chronic fluo-

xetine elicited a similar behavioral effect in the OBX rat, it is

not unlikely that the regionally selective enhancement of CB1

receptor-signaling in the PFC could be related with the altered

OBX behavior. Our findings reinforce the utility of this animal

model to further investigating the implication of the endoc-

annabinoid system in the modulation of emotional processes

and its potential role in the adaptive responses to chronic

antidepressants.

Keywords: [35S]GTPcS binding, 9D-tetrahydrocannabinol,

cannabinoid receptor 1, fluoxetine, olfactory bulbectomy,

prefrontal cortex.

J. Neurochem. (2009) 108, 1423–1433.

JOURNAL OF NEUROCHEMISTRY | 2009 | 108 | 1423–1433 doi: 10.1111/j.1471-4159.2009.05898.x

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1423–1433 1423

Adaptive changes in the central serotonergic systemappear to play an important role in the OBX-inducedsyndrome. The neurodegeneration of serotoninergic neuronsin dorsal raphe nucleus (DRN) following OBX results in thepermanent deficits in the secretion of 5-hydroxytryptamine orserotonin (5-HT) in the dorsal hippocampus and amygdala(van der Stelt et al. 2005). Abnormalities in 5-HT concen-tration, 5-HT transporter, and receptor expression (5-HT2 and5-HT1A receptors) following OBX in areas such as theamygdaloid cortex, frontal cortex, and midbrain have beenreported (Lumia et al. 1992; Gurevich et al. 1993; Greckschet al. 1997; Zhou et al. 1998; Slotkin et al. 2005). Altera-tions in other neurotransmitter systems (noradrenaline,dopamine, glutamate, and GABA) have been also shown(Jancsar and Leonard 1984; Song and Leonard 2005) but, todate and to our knowledge, the study of the ECs in thisanimal model of depression has not been addressed. Chronicmild/unpredictable stress exposure to rats (Bortolato et al.2007) and to mice (Hillard et al. 2006) results in an increaseCB1 receptor mRNA in PFC. In a very recent paper, Hillet al. (2008) demonstrated that chronic unpredictable stressin rats results in a significant increase in CB1 receptorbinding site density in PFC and a decrease in CB1 receptorbinding site density in hippocampus, hypothalamus, andventral striatum, together with a significant reduction in thecontent of the EC ligand N-arachidonylethanolamide(anandamide) in all the brain area examined.

There are some pre-clinical evidences about the modula-tion of 5-HT neurotransmission by the ECs: CB1 receptorsand the enzyme regulating anandamide brain levels (fattyacid amide hydrolase) are present in the DRN of the mousebrain (Egertova et al. 2003); CBs elicit antidepressant-likebehavior by activating DRN serotonergic neurons (Bambicoet al. 2007); CB1 agonists increase the firing activity of DRNserotonergic neurons (Gobbi et al. 2005), suppress 5-HTrelease in the mouse brain cortex (Nakazi et al. 2000) andblock 5-HT reuptake in the rat brain cortex (Banerjee et al.1975) whereas CB1 antagonists stimulate 5-HT release intothe rat medial PFC (Tzavara et al. 2003); chronic CBtreatment appears to up-regulate 5-HT2A receptor activitywhile concurrently down-regulating 5-HT1A receptor activityin the rat brain, a finding similar to that sometimes observedin depression (Hill et al. 2006b); and impaired functionalityof 5-HT1A and 5-HT2A/C receptors has been found in CB1

knockout mice that exhibit a depressive-like phenotype(Mato et al. 2007).

There are still few evidences about the modulation of theECs by pharmacological manipulation of brain 5-HT neuro-transmission. Acute fluoxetine modulates 9D-tetrahydrocan-nabinol-induced hypothermia in the rat (Malone and Taylor1998), an effect in which somatodendritic 5-HT1A autore-ceptors appear to be involved (Malone and Taylor 2001).Chronic fluoxetine decreases CB1 receptor gene expressionin the caudate–putamen (Oliva et al. 2005) of the rat brain

and chronic citalopram decreases CB receptor-mediated Gprotein coupling in rat hypothalamus and hippocampus, twoareas important in the control of neuroendocrine function andmood (Hesketh et al. 2007); however, chronic administrationof desipramine, a non-selective serotonin reuptake inhibitor(SSRI) drug, results in an increased density of CB1 receptorin the hippocampus and hypothalamus of the rat brain (Hillet al. 2006a).

This study was aimed to investigate CB1 receptor-medi-ated signaling in the brain of olfactory bulbectomized rats, itsimplication in the OBX-induced behavior and its modulationby the chronic administration of the SSRI fluoxetine. Firstly,we have analyzed the changes in the density and function-ality of CB1 receptors in the brain OBX rats. Secondly, wehave evaluated the antidepressant-like activity of chronicfluoxetine in the OBX rat as well as the effect of theantidepressant upon the density and functionality of CB1

receptors in PFC membranes. Finally, we have assessed theeffect of the administration of the CB agonist 9D-tetra-hydrocannabinol in the OBX-induced behavior before andafter chronic treatment with fluoxetine.

Materials and methods

AnimalsExperiments were conducted on male Sprague–Dawley rats (Harlan,

Barcelona, Spain) weighing at the moment of surgery 250–270 g.

Animals were housed in a climate controlled room with a 12-h light/

dark cycle and had ad libitum access to food and water. All surgical

procedures and experiments were carried out with the approval of

the Animal Care Committee of the Universidad de Cantabria and

were performed according to the Spanish legislation and the

European Communities Council Directive on ‘Protection of Animals

Used in Experimental and Other Scientific Purposes’ (86/609/EEC).

MaterialsFluoxetine HCl was kindly supplied by FAES FARMA S.A. Leioa,

Bilbao, Spain. Osmotic minipumps (Alzet osmotic pump model

2ML2; 0.5 lL/h) were supplied by Alzet Corporation (Palo Alto,

CA, USA). 9D-Tetrahydrocannabinol was purchased from THC

Pharm GMBH The Health Concept (Frankfurt, Germany); SR

141716A was kindly supplied by Sanofi Reserche (Montpellier,

France). [3H]CP55940 (160 Ci/mmol) and [35S]GTPcS (1250 Ci/

mmol) were purchased from PerkinElmer Life Sciences (Madrid,

Spain). All other materials, unless otherwise stated, were purchased

from Sigma-Aldrich (St Louis, MO, USA).

Bilateral olfactory bulbectomy and open-field testThe OBX procedure was performed as previously described

(Cairncross et al. 1977). Animals were anesthetized and the bulbs

were excised and removed under stereotactic surgery. Sham-

operated animals underwent the same procedure except for excision

and aspiration of the olfactory bulbs. Behavioral studies started after

a 2-week recovery period. At the end of the experiments, animals

were killed and the success of the operation was confirmed

anatomically. The open field was conducted as previously described

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1423–1433� 2009 The Authors

1424 | A. Rodrıguez-Gaztelumendi et al.

(Gray and Lalljee 1974), recording for a 5 min period the number of

ambulations, rearings, grooming episodes, and defecations (see

Supporting information for details).

Experimental groups and pharmacological treatmentsThree sets of experiments were carried out under this study (see

Fig. S1). In the first set, animals underwent surgery (OBX = 5;

sham = 5) and the behavioral testing in the open field arena was

carried on day 15 post-surgery. Then, animals were killed and

autoradiographic studies in brain sections were performed. In the

second set of experiments, another group of animals underwent

surgery (sham = 14, OBX = 16), an open field test was performed

on day 15 post-surgery and, the following day, they were divided

into four subgroups to receive fluoxetine (10 mg/kg/day, s.c.) or

vehicle for another 2 weeks: vehicle-treated sham-operated (n = 6),

fluoxetine-treated sham-operated (n = 8), vehicle-treated OBX

(n = 8), and fluoxetine-treated OBX (n = 8). Fluoxetine HCl or

vehicle (50% propylene glycol, 10% ethanol, and 40% distilled

water) were administered by osmotic minipumps (Castro et al.2003). An open field session was performed at the end of the

treatment (day 29 following OBX surgery) without removing the

minipumps. Animals were killed after a 24 h washout period and

their brains removed (day 31 post-surgery). The PFC was dissected

and processed for the binding assays in membrane homogenates. In

the third set of experiments, animals underwent surgery (OBX = 10;

sham = 6). The behavioral effects of a challenge dose of 9D-tetrahydrocannabinol (5 mg/kg, i.p.) and its vehicle (5% ethanol,

5% emulphor, and 90% saline) were evaluated, 30 min after

administration, in different open-field sessions performed at days 15

and 16. The following day, sham and OBX animals were implanted

minipumps to receive chronic fluoxetine and the behavioral effect of9D-tetrahydrocannabinol was again evaluated at the end of the

treatment (day 29 post-surgery). The dose of 9D-tetrahydrocannab-inol was selected according to the results obtained from preliminary

studies demonstrating no behavioral effects in naıve animals under

the environmental conditions in which the open field test is

performed in this study.

Autoradiography of [3H]CP55940 binding and [35S]GTPcS bindingCoronal brain sections (20 lm) were obtained from sham and OBX

animals (20 lm). [3H]CP55940 binding autoradiography was

performed according to Glass et al. (1997), with minor modifica-

tions (Mato and Pazos 2004), by incubating brain sections with

3 nM [3H]CP55940 and defining the non-specific binding with

10 lM WIN 55212-2. [35S]GTPcS autoradiography was performed

as previously described (Sim et al. 1996) assessing the stimulation

of [35S]GTPcS binding by incubating brain sections with 0.04 nM

[35S]GTPcS under the following conditions: in the absence (basal

binding) or the presence of the CB agonist WIN 55212-2 (10 lM)

(stimulated binding), in the presence of WIN 55212-2

(10 lM) + SR 141716A (10 lM) and in the presence of 10 lMGTPcS (non-specific binding) (see Supporting information for

details).

[3H]CP55940 Binding and [35S]GTPcS binding in prefrontal cortexmembranesOlfactory bulbectomy and sham animals treated with fluoxetine or

vehicle were killed following the open field test, their brains were

removed, and membrane homogenates were obtained from

prefrontal cortical (PFC) samples. [3H]CP55940 binding assay in

PFC cortical membranes was performed as previously described

(Hungund et al. 2004) by incubating aliquots of PFC membranes

with [3H]CP55940 (0.0125–3.2.0 nM) and defining the non-specific

binding with WIN 55212-2 (10 lM). The CB1 receptor-mediated

[35S]GTPcS binding assay was performed as described previously

(Gonzalez-Maeso et al. 2000) with minor modifications, incubating

aliquots of PFC membranes with 0.1 nM [35S]GTPcS under the

following conditions: in the absence (basal binding) or the presence

of the CB agonist WIN 55212-2 (10)9 to 10)3 M) (stimulated

binding), in the presence of WIN 55212-2 (10 lM) + SR 141716A

(10 lM) and in the presence of 10 lM GTPcS (non-specific

binding) (see Supporting information for details).

Data analysisAutoradiographic densities were determined by densitometry using

the Scion Image software (Scion Corporation, Frederick, MD,

USA). Relative optical density values of brain regions were

averaged over two consecutive sections per rat (bilateral readings)

and converted to nCi/mg of tissue ([3H]CP55940 receptor binding)

or nCi/g of tissue ([35S]GTPcS binding). CB1 receptor autoradio-

graphic densities are given in fmol/mg tissue equivalent (fmol/mg

tissue) and CB1 agonist-stimulation of [35S]GTPcS binding data are

presented as percentage of basal binding (100%). [3H]CP55940 and

[35S]GTPcS binding assays in PFC membranes were carried out

using triplicates and the results represent the mean of the n values

obtained from each experimental group. The Bmax (maximal bound,

fmol/mg protein) and Kd (receptor affinity, nM) for [3H]CP55940

binding studies, and Emax (maximal stimulatory effect expressed as

percentage of basal binding) and pEC50 (potency expressed as

)log EC50) for [35S]GTPcS binding assays in PFC membranes were

determined by non-linear regression analysis. Analysis of the

[3H]CP55940 and [35S]GTPcS binding data were performed using

the computer program PRISM, version 4.03 (GraphPad Software, San

Diego, CA, USA).

Statistical analysisAll data are expressed as mean ± SEM. A two-tailed unpaired

Student’s t-test was used for statistical comparison between two

experimental groups. Differences in drug treatment between sham

and OBX rats were compared using a two-way ANOVA (with surgery

and drug treatment as factors) followed by Bonferroni post hoc testwhen appropriate (PRISM, version 4.03). A probability of p £ 05 was

taken as the level of statistical significance.

Results

Autoradiographic CB1 receptor density and CB1

receptor-mediated stimulation of [35S]GTPcS bindingin the brain of the OBX ratIn the group of animals used for the autoradiographic studies,a characteristic hyperactivity in the open field test wasconfirmed 15 days post-surgery in the OBX group (n = 5)when compared with the sham-operated counterparts (n = 5)as indicated by the increase in the number of ambulations

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1423–1433

Prefrontal CB1 receptors and chronic fluoxetine | 1425

[152.1 ± 13.2 vs. 85.4 ± 9.7; t (8) = 4.07, p = 0.004] andrearings [26.0 ± 4.9 vs. 11.1 ± 2.3; t (8) = 4.66, p = 0.03].No significant differences were observed in the number ofgrooming episodes, the number of defecations or the bodyweights (data not shown). Densitometric analysis of[3H]CP55940 and [35S]GTPcS binding to brain sectionsrevealed that CB1 receptor density ([3H]CP55940 binding)was significantly increased in OBX rats in areas such as themPFC [+29%; t (7) = 2.68, p = 0.03] and the amygdala[+36%; t (8) = 3.00, p = 0.02] whereas no changes weredetected in the caudate–putamen, hippocampus (CA1–CA2fields), and DRN (Table 1 and Fig. 1). CB1 agonist-stimu-lated [35S]GTPcS binding by 10 lM WIN 55212-2 resultedto be significantly enhanced in the mPFC of OBX animals[+40%; t (7) = 2.88, p = 0.02] in comparison to the shamgroup. A slight increase in CB1 agonist-stimulated[35S]GTPcS binding was also observed in the amygdala[+8%; t (8) = 1.39, p = 0.2] although not reaching statisticalsignificance (Table 1 and Fig. 2). No differences weremeasured among the two experimental groups in the levelof basal [35S]GTPcS binding in all the regions examined(data not shown). As significant changes were observed inboth the density and the functionality of CB1 receptors in thePFC from OBX rats, this region was selected to furtherevaluate the effect of OBX and chronic fluoxetine on[3H]CP55940 binding and CB1 agonist-stimulated[35S]GTPcS binding using membrane homogenates.

Effect of chronic fluoxetine on the olfactorybulbectomy-induced behavior in the open fieldTwo weeks after sham (n = 14) or OBX (n = 16) surgery andbefore the initiation of the chronic treatment with fluoxetineor vehicle, the OBX-induced hyperactivity was confirmed onthe open-field test (data not shown). The day after, sham andOBX animals were treated with fluoxetine (10 mg/kg/day,s.c.) or vehicle for another 14 days and the behavioral effectsof the antidepressant were evaluated in the open field test. No

significant differences were detected in body weights amongthe experimental groups either at the start or the end of drugtreatment (data not shown).

The total ambulation scores of vehicle-treated and fluoxe-tine-treated sham and OBX rats exposed to a 5 min openfield is shown in Fig. 3a. A two-way ANOVA revealed asignificant interaction between surgery and drug treatment[F(1,26) = 8.62, p = 0.007], a significant main effect ofsurgery [F(1,26) = 7.44, p = 0.01] and a significant maineffect of drug treatment [F(1,26) = 10.86, p = 0.003].Post hoc analysis showed that the total ambulation scorewas significantly increased in vehicle-treated OBX ratscompared with vehicle-treated sham-operated controls(p < 0.01). Interestingly, there was a significant and totalattenuation of the number of ambulations in the fluoxetine-treated OBX group relative to the vehicle-treated OBX group(p < 0.001). Thus, chronic fluoxetine appeared to rectify theOBX-induced locomotor hyperactivity, as no difference wasobserved between fluoxetine-treated sham and fluoxetine-treated OBX animals.

The number of rearings following chronic treatment withfluoxetine or vehicle in sham and OBX rats is shown inFig. 3b. There was a significant main effect of drug treatment[F(1,26) = 10.88, p = 0.003], in which fluoxetine-treatedOBX rats showed reduced rearing activity when comparedwith vehicle-treated OBX rats (p < 0.01). Fluoxetine-treatedsham animals did not show any significant change in rearingactivity when compared with vehicle-treated sham rats.

There was a significant main effect of drug treatment onthe number of defecations [F(1,26) = 9.82 p = 0.004].Post hoc analysis showed that defecation was significantlyincreased in fluoxetine-treated OBX rats relative to vehicle-treated OBX rats (p < 0.05). Fluoxetine-treated sham ratsalso exhibited an increased number of defecations compara-ble to fluoxetine-treated OBX rats, though it did not resultsignificant when compared with vehicle-treated sham ani-mals (p = 0.06).

Table 1 Effect of bilateral OBX upon the density and functionality of CB1 receptors in the rat brain

Brain region

[3H]CP55490 binding to CB1 receptors

(fmol/mg tissue)

CB1 receptor-stimulated [35S]GTPcS binding

by WIN 55212-2 (% of basal)

Sham OBX Sham OBX

Medial prefrontal cortex 28.95 ± 2.61 37.24 ± 1.83* 140.25 ± 10.15 196.07 ± 17.78*

Caudate–putamen 52.08 ± 2.91 50.08 ± 1.36 173.71 ± 12.63 173.99 ± 20.24

Hippocampus (CA1–CA2 regions) 66.02 ± 2.58 61.07 ± 2.21 171.04 ± 26.71 168.26 ± 24.93

Amygdala (central-medial nuclei) 20.96 ± 1.60 28.48 ± 1.93* 110.07 ± 2.98 118.85 ± 5.59

Dorsal raphe nucleus 22.86 ± 2.34 20.63 ± 1.49 105.55 ± 5.26 105.84 ± 6.02

OBX, olfactory bulbectomy; CB, cannabinoid. CB1 receptor’s density and functionality were evaluated in consecutive coronal sections of sham-

operated (Sham, n = 4–5) and olfactory bulbectomized rats (OBX, n = 4–5). The density of CB1 receptors was assessed with [3H]CP55490 (3 nM)

and CB1 receptor by measuring the stimulation of [35S]GTPcS binding in the presence of WIN 55212-2 (10 lM). Specific CB1 receptor-stimulated

binding is expressed as the percent of basal binding (100%). See Materials and methods for further details. Results are expressed as the

mean ± SEM; *p < 0.05 versus sham-operated group (two-tailed unpaired Student’s t-test).

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1426 | A. Rodrıguez-Gaztelumendi et al.

Effect of OBX and chronic fluoxetine treatment on[3H]CP55940 binding in PFC membranesThe saturation analysis of [3H]CP55940 binding to CB1

receptors in PFC membranes demonstrated high affinity (Kd

values below 1 nM), saturable, monophasic binding to asingle class of receptor at the concentrations analyzed(0.0125–3.2 nM). The non-specific binding was about 20%of total [3H]CP55940 binding. Statistical analysis by two-way ANOVA revealed a significant interaction between surgeryand drug treatment [F(1,24) = 25.98, p < 0.0001] and asignificant main effect of drug treatment [F(1,24) = 5.69,

p = 0.02] in CB1 receptor density after OBX and chronicfluoxetine treatment. As shown in Fig. 4a, there was asignificant increase in CB1 receptor density (+40%) in thevehicle-treated OBX group when compared with the vehicle-treated sham group (891.3 ± 54.2 vs. 639.4 ± 21.1 fmol/mgprotein, respectively; p < 0.001). Moreover, the averagedensity of CB1 receptor in the fluoxetine-treated OBX group(615.4 ± 38.4 fmol/mg protein) was significantly lower thanin the vehicle-treated OBX group (p < 0.001). By contrast,fluoxetine-treated sham rats did not show any significantchange in CB1 receptor density (739.0 ± 26.6 fmol/mgprotein) relative to vehicle-treated sham rats. Statisticalanalysis of comparisons among the four experimental groupsindicated no significant effect of surgery and/or drugtreatment in receptor affinity (Kd values: vehicle-treatedsham: 0.14 ± 0.02 nM; fluoxetine-treated sham, 0.22 ±0.03 nM; vehicle-treated OBX, 0.21 ± 0.04 nM; and fluo-xetine-treated OBX, 0.14 ± 0.03 nM).

Effect of OBX and chronic fluoxetine treatment on CB1

receptor-stimulated [35S]GTPcS binding in PFC membranesThe CB1 receptor agonist WIN 55212-2 stimulated in aconcentration-dependent manner the [35S]GTPcS binding inPFC membranes from vehicle-treated sham animals(Emax = 300.9 ± 21.7% of basal binding; pEC50 = 6.0 ± 0.1)(Fig. 4b). Statistical analysis by two-way ANOVA revealed asignificant interaction between surgery and drug treatment[F(1,23) = 5.41, p = 0.03], a significantmain effect of surgery[F(1,23) = 6.17, p = 0.02] and a significant main effect ofdrug treatment [F(1,23) = 15.04, p = 0.001] in the efficacy(Emax) ofWIN 55212-2 to stimulate [35S]GTPcS binding.Posthoc analysis showed that CB1 receptor-stimulated [35S]GTPcSbinding was significantly enhanced (+28%) in vehicle-treatedOBX rats relative to vehicle-treated sham animals(Emax = 383.4 ± 22.5% vs. 300.9 ± 21.7% of basal binding,respectively; p < 0.05).Moreover, as in [3H]CP55940 binding

(a)

(d) (e)

(b) (c)

Fig. 2 Representative autoradiograms showing the effect of OBX on

CB1 receptor-stimulated [35S]GTPcS binding at the level of prefrontal

cortex (PFC). Basal [35S]GTPcS binding (a); stimulation of [35S]GTPcS

binding by the CB1 agonist WIN 55212-2 (10 lM) in sham-operated (b)

and OBX rats(c); antagonism by 10 lM SR 141716A of WIN 55212-2-

stimulated [35S]GTPcS binding (d); non-specific binding in the presence

of 10 lM GTPcS (e). Note the enhancement in WIN 55212-2-stimulated

[35S]GTPcS binding in the PFC of the OBX rat (c) relative to the sham-

operated animal (d).

(a) (d)

(b) (e)

(c) (f)

Fig. 1 Autoradiographic illustration of [3H]CP55940 binding (3 nM) to

coronal sections at the level of PFC, anterior hippocampus, and dorsal

raphe nucleus in sham-operated (a, b, and c) and OBX rats (d, e, and

f). mPFC, medial prefrontal cortex; Hippo, hippocampus; Amyg,

amygdala; PAG, periaqueductal grey; and SNigra, susbtantia nigra.

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Prefrontal CB1 receptors and chronic fluoxetine | 1427

studies, there was a main effect of drug treatment as maximalstimulation of [35S]GTPcS binding in fluoxetine-treated OBXrats yielded a value significantly lower (Emax = 277.7 ±11.9% of basal binding) than the observed in vehicle-treatedrats (p < 0.01). By contrast, fluoxetine-treated sham rats(Emax = 274.3 ± 10.4% of basal binding) did not show anysignificant change relative to vehicle-treated sham rats. Thus,the increased CB1 receptor-stimulated [35S]GTPcS binding inthe PFC induced by OBX appeared to be reversed by chronicfluoxetine, as no difference was observed between fluoxetine-treated sham and fluoxetine-treated OBX animals (Fig. 4b).No differences either in basal [35S]GTPcS binding (data not

shown) or in the potency of the CB1 agonist WIN 55212-2 tostimulate [35S]GTPcS binding were detected among theexperimental groups (pEC50 values: vehicle-treated sham,6.0 ± 0.1; fluoxetine-treated sham, 6.1 ± 0.1; vehicle-treatedOBX, 5.9 ± 0.2; and fluoxetine-treated OBX, 6.0 ± 0.1).

Behavioral effects of 9D-tetrahydrocannabinol in the OBXrat before and after chronic administration of fluoxetineThe effects of the acute administration of D9-tetrahydrocan-nabinol (5 mg/kg, i.p.) in sham and OBX animals were

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rings

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Fig. 3 Behavioral effects of OBX and chronic fluoxetine treatment in the

open field test. Chronic treatment (14 days) with fluoxetine (10 mg/kg/

day s.c.) or vehicle commenced 2 weeks after sham and OBX surgery. A

significant increase in the ambulation (a) and rearing (b) scores was

observed (5 min period) in vehicle-treated OBX rats when compared

with vehicle-treated sham rats. OBX-induced hyperactivity was signifi-

cantly attenuated in fluoxetine-treated OBX rats whereas no significant

change was seen in fluoxetine-treated sham rats. Data represent

mean ± SEM. Vehicle-treated sham = 6; fluoxetine-treated sham = 8;

vehicle-treated OBX = 8; fluoxetine-treated OBX = 8; ***p < 0.001

versus vehicle-treated rats; ++p < 0.01, and +++p < 0.001 versus vehi-

cle-treated OBX rats (Bonferroni post hoc test).

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OBX-chronic fluoxetine

OBX-vehicle

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tion o

f [3

5S

]GT

PγS

bin

din

g (

% o

f basa

l)

W IN 55,212-2 [log M]

(a)

(b)

Fig. 4 Effect of OBX and chronic treatment with fluoxetine (10 mg/kg/

day, 14 days) or vehicle on CB1 receptor density (Bmax) (a) and CB1

receptor-mediated stimulation of [35S]GTPcS binding (b) in rat pre-

frontal cortical membranes. Note that chronic fluoxetine reversed the

increased density and functionality of CB1 receptors induced by OBX.

Data represent the mean ± SEM of n individuals values obtained from

binding experiments assayed in triplicates (vehicle-treated sham = 6–

7; fluoxetine-treated sham = 7–8; vehicle-treated OBX = 6; fluoxetine-

treated OBX = 7–8); ***p < 0.001 versus vehicle-treated sham group

and +++p < 0.001 versus vehicle-treated OBX group (Bonferroni

post hoc test).

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1423–1433� 2009 The Authors

1428 | A. Rodrıguez-Gaztelumendi et al.

assessed in the open-field test. Regarding the number ofambulations, there was a significant interaction betweensurgery and treatment [F(2,36) = 10.37, p = 0.0003], asignificant main effect of treatment [F(2,36) = 18.79,p < 0.0001] and a significant main effect of surgery[F(1,36) = 12.75, p = 0.0010] (Fig. 5a). A significant inter-action between surgery and treatment [F(2,36) = 74.55,p < 0.0001], a significant main effect of treatment[F(2,36) = 102.23, p < 0.0001] and a significant main effectof surgery [F(1,36) = 72.24 p < 0.0001] were also found forthe number of rearings (Fig. 5b). The ambulation and rearingscores were increased in OBX rats receiving vehicle (n = 6)compared with their counterparts sham animals (n = 6)(p < 0.001 for both parameters). As shown in Fig. 5a and b,acute D9-tetrahydrocannabinol reduced the ambulation score()58%) and the number of rearings ()97%) in OBX rats(n = 8; p < 0.001 vs. OBX animals receiving vehiclefor both parameters). The behavioral response of

fluoxetine-treated OBX rats in the open-field 30 min afterthe administration of a challenge dose of D9-tetrahydrocan-nabinol was similar to that observed in OBX rats followingchronic fluoxetine alone. In addition, D9-tetrahydrocannab-inol did not produce any behavioral effect in sham rats(n = 6) (Fig. 5a and b).

Discussion

One major finding in our study is the presence of an alteredCB1 receptor-signaling in discrete brain areas followingbilateral OBX: an increased CB1 receptor density in PFC andamygdala together with an increased CB1 receptor-mediated[35S]GTPcS binding in the PFC of OBX animals. Theincreases on both CB1 receptor density and CB1 receptor-mediated [35S]GTPcS binding in the PFC of OBX animalswere of similar magnitude. Thus, enhanced CB1 receptor-signaling must be reflecting an up-regulation of CB1 receptorprotein expression. Our finding is in good concordance withstudies reporting a significant increase on both CB1 receptormRNA (Hillard et al. 2006; Bortolato et al. 2007) and CB1

receptor density (Hill et al. 2008) in the PFC of rodentsexposed to chronic mild/unpredictable stress, another vali-dated model of animal depression. Hill et al. (2008) havereported a decrease in CB1 receptor density in hippocampus,hypothalamus, and ventral striatum but no change in theamygdala of rats exposed to chronic unpredictable stress. Bycontrast, we detect a significant increase in CB1 receptordensity in the amygdala of the OBX rats without any changein CB1 receptor-mediated [35S]GTPcS binding (probablybecause of the high level of basal [35S]GTPcS binding thatmakes difficult to detect agonist-induced stimulation). Thisdiscrepancy with our finding could be explained by thedifferent animal model of depression used and/or the type ofbinding assay performed: CB1 receptor autoradiography inour study versus CB1 receptor binding studies in membranehomogenates in the study by Hill et al. (2008). Ourexperimental data are also consistent with clinical studiesdemonstrating elevated levels of ECs, CB1 receptors, andCB1 receptor-mediated [35S]GTPcS binding in PFC ofdepressed suicide victims (Hungund et al. 2004; Pazos et al.2006; Vinod and Hungund 2006), an area where ECs appearto regulate mood, aggression and/or impulsivity, and decisionmaking, which are altered in suicidality (Egerton et al. 2006;Vinod and Hungund 2006).

In our study, chronic fluoxetine exhibited antidepressant-like activity in the OBX rat under the open-field paradigm.It is noteworthy that the antidepressant-like activity offluoxetine in others pre-clinical models seems to bedependent on the test performed, the dose assayed andthe rat strain used (Lopez-Rubalcava and Lucki 2000;Cryan et al. 2005). There are few reports on the effect ofchronic fluoxetine in the OBX rat (Mar et al. 2000; Rocheet al. 2007); moreover, the behavioral effects of the

0

40

80

120

160

200

240

Sham OBX

***

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tion

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5

10

15

20

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30

Sham OBX

Vehicle THC Chromic fluoxetine+ THC

***

+++

Rea

rings

+++

(a)

(b)

Fig. 5 Behavioral effects of D9-tetrahydrocannabinol (5 mg/kg, i.p.) in

sham and OBX animals before and after the chronic administration of

fluoxetine (10 mg/kg/day, 14 days) assessed in the open-field test.

Acute D9-tetrahydrocannabinol (THC) significantly decreased ambu-

lation (a) and rearing (b) scores in OBX rats before the initiation of the

chronic treatment with fluoxetine. Note that the antidepressant-like

effect of chronic fluoxetine in OBX rats was not modified by the acute

administration of D9-tetrahydrocannabinol. Data represent mean ±

SEM. Vehicle-sham = 6; THC-sham = 6; THC to fluoxetine-treated

sham = 6; vehicle-OBX = 6; THC-OBX = 8; THC to fluoxetine-treated

OBX = 10; ***p < 0.001 versus vehicle-sham rats and +++p < 0.001

versus vehicle-OBX rats (Bonferroni post hoc test).

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1423–1433

Prefrontal CB1 receptors and chronic fluoxetine | 1429

antidepressant appear to be influenced by the environmentalconditions. Our results demonstrate the effectiveness ofchronic fluoxetine (10 mg/kg, 14 days) upon the reversal ofthe behavioral deficits exhibited by OBX-rats when exposedto a high luminance open field exposure, thus under morestressful or aversive conditions (Mar et al. 2002). Ourresults are also in good agreement with those reported byRoche et al. (2007) showing that chronic fluoxetine(10 mg/kg, 5 weeks) reverses all the OBX-associatedchanges in behavior, body temperature, heart rate, andneuronal activation in response to open field exposure. Inour study, we did not detect changes in the number ofdefecations between OBX and sham animals, as alreadyreported (Stock et al. 2000), but chronic fluoxetine pro-voked increased defecation in both groups, althoughreaching statistical significance only in the OBX group.By contrast, it has been shown that chronic fluoxetineattenuates the increased defecations observed in rats afterexposure to chronic stress (Zhang et al. 2000; Huang et al.2007) but it induces diarrhea in non-stressed rats (Zhanget al. 2000). We have not a plausible explanation for theincreased defecations induced by chronic fluoxetine. Itcould because of its action on the gastrointestinal 5-HTsystem (James et al. 2005) and, thus, reflecting a side-effectof the antidepressant at this level. Also, we cannot excludethat chronic fluoxetine could increase feeding (food intakewas not measured) but no differences were observed in thebodyweights of the rats at the end of the treatment.

Another major finding is that chronic fluoxetine exhibitsantidepressant-like activity associated to a normalization ofCB1 receptor-signaling in the PFC. Interestingly, the CBagonist D9-tetrahydrocannabinol (5 mg/kg, i.p.) did alsoattenuate OBX-induced hyperactivity when administeredprior to chronic treatment with fluoxetine. As both acute D9-tetrahydrocannabinol and chronic fluoxetine elicited asimilar behavioral effect in the OBX rat, it is not unlikelythat the regionally selective enhancement of CB1 receptor-signaling in the PFC could be related with the altered OBXbehavior. In our study, acute D9-tetrahydrocannabinol didnot affect locomotor activity of control animals in the open-field as previously described in mice (Egashira et al. 2008).However, a decrease in the number of ambulations on theopen field test in control rats after the acute administrationof D9-tetrahydrocannabinol (Jarbe et al. 2002) and WIN55212-2 (Jarbe et al. 2006; Suplita et al. 2008) has beenreported. This discrepancy could be because of the differentenvironmental conditions under which the test is performedas a more stressful or aversive (high luminance) open fieldtest was used in our study (Mar et al. 2002). Themethodological approaches of our study do not allow tofully ascertaining the functional significance of the changeswe have observed in CB1 receptor-signaling with respect tothe OBX-hyperactivity and to the behavioral effects ofchronic fluoxetine It is noteworthy that D9-tetrahydrocan-

nabinol only produced a behavioral change in OBX ratsbefore the initiation of the chronic treatment with fluoxetineand it did not induce any further change in fluoxetine-treated OBX rats, in which ambulatory behavior was fullynormalized. It is important to mention that adaptive changesin the central serotonergic system appear to play animportant role in the OBX-induced syndrome. Thus, itcould be speculated that the enhanced CB1 receptor-signaling in the PFC of the OBX rat could represent acompensatory mechanism to counteract the behavioral andneurochemical consequences of those adaptive changes;conversely, this compensatory mechanism might not benecessary once the antidepressant has exerted the neuro-chemical and cellular actions that normalize brain 5-HTneurotransmission.

There are several pieces of evidence supporting theinteraction between 5-HT neurotransmission and EC/CB1

receptors that could be relevant to the findings of this study.First, OBX-induced neurodegeneration of serotonin neuronsin DRN is counteracted by antidepressant treatment (Nest-erova et al. 1997); interestingly, CB1 receptors and fatty acidamide hydrolase are present in DRN (Egertova et al. 2003)and CBs elicit antidepressant-like behavior by enhancingDRN 5-HT neuronal activity (Gobbi et al. 2005) through thePFC (Bambico et al. 2007). Second, several changes in 5-HTneurotransmission in cortical areas are encountered followingOBX: altered serotonin metabolism (5-hydroxyindoleaceticacid (5-HIAA)/5-HT ratio) in frontal cortex (Lumia et al.1992), increased 5-HT transporter expression in frontalcortex that is reversed by chronic antidepressants (Greckschet al. 1997; Zhou et al. 1998), decreased extracellular basal5-HT levels in amygdaloid cortex and midbrain (Jancsar andLeonard 1984), and lower tissue 5-HT concentration infrontal cortex (Redmond et al. 1997); on the other hand, CB1

receptor agonists suppress 5-HT release in mouse braincortex (Nakazi et al. 2000) and 5-HT reuptake in rat braincortex (Banerjee et al. 1975) while CB1 receptor antagonistsstimulate 5-HT release into the medial PFC in the shrewbrain (Darmani et al. 2003) and in the rat brain (Tzavaraet al. 2003). Third, changes in 5-HT1A and 5-HT2 receptorexpression have been described in brain cortex of bulbec-tomized mice and rats (Gurevich et al. 1993; Slotkin et al.2005); and, chronic CB treatment appears to up-regulate5-HT2A receptor activity while concurrently down-regulating5-HT1A receptor activity in the rat brain, a finding similar tothat sometimes observed in depression (Hill et al. 2006b).

The molecular mechanism by which the elevated CB1

receptor expression and G protein-coupling efficacy in theOBX rat are reverted by chronic fluoxetine remains to beelucidated. Devlin and Christopoulos (2002) have shown that5-HT reduces the proportion of high affinity binding of theCB agonists HU210 and WIN 55212-2 in rat cerebellarmembranes; these data, suggesting crosstalk between CB1

and 5-HT receptors, may help explaining the effect of

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1423–1433� 2009 The Authors

1430 | A. Rodrıguez-Gaztelumendi et al.

chronic fluoxetine in CB1 receptor density in the PFC. Ourpresent results on the effect of chronic fluoxetine in the OBXrat are in good agreement with the findings reported by Hillet al. (2008) in another animal model of depression:increased CB1 receptor density in PFC of rats exposed tochronic unpredictable stress that is attenuated by chronicimipramine (Hill et al. 2008). Also, it has been reported thatchronic fluoxetine decreases CB1 receptor gene expression incaudate–putamen (Oliva et al. 2005) and that chroniccitalopram induces a decrease in CB receptor-mediated Gprotein coupling in rat hypothalamus and hippocampus, twoareas important in the control of neuroendocrine function andmood (Hesketh et al. 2007); however, chronic administrationof desipramine, a non-SSRI drug, results in an increaseddensity of CB1 receptor in hippocampus and hypothalamus(Hill et al. 2006a). In addition, some interventions known toproduce antidepressant effects in humans have been shown tomodulate the ECs: electroconvulsive shock treatment down-regulates EC/CB1 receptor-signaling in rat PFC and hippo-campus (Hill et al. 2007); however, electroconvulsive shocktreatment up-regulates CB1 receptor signaling in rat amyg-dala (Hill et al. 2007) and sleep deprivation increases thecontent of the EC 2-arachidonylglycerol in rat hippocampus(Chen and Bazan 2005).

Regarding the pharmacological relevance of our findings,it is noteworthy that it is still unclear which would be theadequate pharmacological manipulation of the ECs system toimprove mood or anxiety disorders. CB1 receptor knockoutmice exhibited enhanced anxiety- and depressive-like behav-iors (Haller et al. 2002; Martin et al. 2002; Haller et al.2004; Uriguen et al. 2004). In line with our results using D9-tetrahydrocannabinol, CB stimulation has been recentlyshown to induce antidepressant- and anxiolytic-like effectsin rats (Berrendero and Maldonado 2002; Marco et al. 2004;Gobbi et al. 2005; Bambico et al. 2007) likely via promotinghippocampal neurogenesis (Jiang et al. 2005). However,high doses of CB agonists evoke depressive-like behaviors inmice (Shearman et al. 2003; Tzavara et al. 2003). Thisapparent discrepancy has been attributed to several factors,including doses, animal model, and strain used, as well ascontextual conditions (Viveros et al. 2005). CB1 receptorantagonists/inverse agonists have been recently demonstratedto have antidepressant-like activity in laboratory animals(Shearman et al. 2003; Tzavara et al. 2003; Griebel et al.2005), though the OBX model has not yet been investigated.However, relevant incidence of depression has been reportedfor the antagonist rimonabant in some patients (Griebel et al.2005; Van Gaal et al. 2005).

In conclusion, the data obtained from our study demon-strates that the OBX model of depression is associated withup-regulation and hyperfunctionality of CB1 receptors in thePFC. Furthermore, we demonstrate that this enhanced CB1

receptor-signaling is normalized by chronic administration ofthe SSRI fluoxetine.

Acknowledgements

This work was supported by grants from Ministerio de Educacion y

Ciencia (SAF-2004/00941 and SAF-2007/61862), Fundacion Mutua

Madrilena, Fundacion Koplowitz, and Plan Nacional de Drogas

(2006). AR-G was granted a pre-doctoral fellowship from Ministerio

de Educacion y Ciencia and MLR was a pre-doctoral fellow from

Fundacion UPSA.

Supporting information

Additional Supporting information may be found in the online

version of this article:

Detailed description of methods.Figure S1 Diagram depicting the three set of experiments

performed in this study.

Please note: Wiley-Blackwell are not responsible for the content

or functionality of any supporting materials supplied by the authors.

Any queries (other than missing material) should be directed to the

corresponding author for the article.

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� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1423–1433

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