Pharmacological Enhancement of Cannabinoid CB1 Receptor Activity Elicits an Antidepressant-Like Response in the Rat Forced Swim Test

European Neuropsychopharmacology 15 (2005) 593 – 599 www.elsevier.com/locate/euroneuro

Pharmacological enhancement of cannabinoid CB1 receptor activity elicits an antidepressant-like response in the rat forced swim test

Matthew N. Hill, Boris B. Gorzalka*

Department of Psychology, University of British Columbia, Vancouver, 2136 West Mall, Canada V6T 1Z4

Received 15 October 2004; received in revised form 1 February 2005; accepted 22 March 2005

Abstract

These experiments aimed to assess whether enhanced activity at the cannabinoid CB1 receptor elicits antidepressant-like effects. To examine this we administered 1 and 5 mg/kg doses of the endocannabinoid uptake inhibitor AM404; 5 and 25 Ag/kg doses of HU-210, a potent CB1 receptor agonist; 1, 2.5 and 5 mg/kg of oleamide, which elicits cannabinoidergic actions; 1 and 5 mg/kg doses of AM 251, a selective CB1 receptor antagonist, as well as 10 mg/kg desipramine (a positive antidepressant control) and measured the duration of immobility, during a 5-min test session of the rat Porsolt forced swim test. Results demonstrated that administration of desipramine reduced immobility duration by about 50% and that all of AM404, oleamide and HU-210 administration induced comparable decreases in immobility that were blocked by pretreatment with AM 251. Administration of the antagonist AM 251 alone had no effect on immobility at either dose. These data suggest that enhancement of CB1 receptor signaling results in antidepressant effects in the forced swim test similar to that seen following conventional antidepressant administration. D 2005 Elsevier B.V. and ECNP. All rights reserved.

Keywords: Cannabinoid; Antidepressant; Forced swim test; Oleamide; Rat

1. Introduction system has a functional role in the expression of emotional behavior (Martin et al., 2002). The basis for this claim is The endogenous cannabinoid (EC) system is a neuro- found in epidemiological research documenting that recrea- modulatory system in the brain that is responsive to the tional or medicinal consumption of cannabis in humans, psychoactive constituent of cannabis, THC, as well as which functionally activates the EC system, typically results endogenously synthesized ligands, such as anandamide in an elevation in mood, the induction of euphoria and a (AEA) and 2-arachidonylglycerol (2-AG). In addition to reduction in stress, anxiety and depressive symptoms (Green the endogenous ligands, this system is composed of a et al., 2003; Gruber et al., 1996; Williamson and Evans, selective neural receptor (CB1) as well as degradative 2000). These findings are paralleled by animal research that enzymes such as fatty acid amide hydrolase (FAAH), which has shown that administration of low doses of CB1 receptor are responsible for catabolism of these ligands (Freund et agonists or inhibitors of the FAAH enzyme, which elevate al., 2003). While the physiological role of this system in the AEA, reduces anxiety-like behavior (Berrendero and Mal- regulation of many processes, for example, feeding behavior donado, 2002; Hill and Gorzalka, 2004; Kathuria et al., (Harrold and Williams, 2003), is well documented, less is 2003). Of particular interest is the research that has revealed known about its role in mental and cognitive functioning. altered emotional behavior in mice genetically bred to lack Evidence has begun to accumulate suggesting that the EC the CB1 receptor. These animals have been shown to display an enhancement of anxiety in behavioral tests such as the light–dark box and the elevated plus maze, as well as an * Corresponding author. Tel.: +1 604 822 3095; fax: +1 604 822 6923. increased susceptibility to the anhedonic effects of chronic E-mail address: [email protected] (B.B. Gorzalka). stress (Haller et al., 2002; Martin et al., 2002). From this one

can conclude that mild activation of the EC system results in GTPgS signaling elicited by CB1 receptor activation (Olivia anxiolysis and the expression of positive emotions, whereas et al., 2003). This enhancement of CB1 receptor signaling deficits in this system result in increased anxiety and through chronic administration of fluoxetine also suggests depressive-like behaviors. that antidepressants may act to increase EC activity, The implication of this theory of emotional regulation by demonstrating a possible bidirectional association between the EC system raises the possibility that the EC system itself stress and antidepressant treatment on the endocannabinoid may play a functional role in the manifestation of various system. affective disorders, especially major depression. The symp- Since EC activity seems to be downregulated by both tomatology of depression is typically characterized by exposure to stress and deficiencies in serotonin, and the depressed mood, anhedonia (loss of interest in rewarding behavioral effects of deficient EC activity mirror those seen stimuli such as those associated with sexual activity) and in depression, it is possible that a deficit in EC activity may extreme alterations in vegetative functions (i.e., insomnia or be important for the expression of major depression. hypersomnia, decreased appetite or increased appetite; Accordingly, enhancement of this system may be a novel American Psychiatric Association, 1994). Furthermore, pathway for the pharmacotherapy of depression. The next many of the systems that are dysregulated in depression logical step in examining this hypothesis is to test the effects are also influenced by EC activity. For example, EC activity of activation of this system alone in animal models has been shown to be involved in the maintenance of predictive of antidepressant efficacy. To date, one of the feeding behavior (Harrold and Williams, 2003); thus most effective preclinical animal models in use is the forced deficiencies in this system could result in the decreased swim test (FST; Cryan et al., 2002). This series of appetite and reduced body weight seen in some cases of experiments was designed to examine the effect of depression. Furthermore, AEA has been suggested to be pharmacological enhancement and blockade of the endo- integral to the maintenance of the sleep–wake cycle, as cannabinoid system on behaviors in the FST. administration of AEA can induce sleep (Murillo-Rodriguez et al., 2003) and pharmacological blockade of EC activity results in increased wakefulness (Santucci et al., 1996). As 2. Experimental procedures with feeding behavior, deficits in this system could result in the insomnia seen in some cases of depression. There is 2.1. Animals and housing increasing evidence that the EC system is important for activation of reward circuitry in the brain as pharmacolog- Male Long-Evans rats that were 10 weeks of age and ical blockade of the CB1 receptor results in an attenuated weighed between 300 and 350 g were employed in this response to both pharmacological and naturally rewarding study. All subjects were housed in groups of three in triple stimuli (Chaperon et al., 1998; Arnone et al., 1997). Thus, a mesh wire cages in a colony room that had a maintained deficiency in EC activity could lead to a blunting of temperature of 21T1 -C and a reverse 12:12-h light–dark responsiveness to rewarding stimuli, which is one of the cycle (lights off at 0900h). All rats had ad libitum access to core components of depression. tap water and Purina Rat chow and were handled 4 times a Biochemical evidence also supports the idea of an EC week prior to testing. All experimental testing on animals deficit in depression. For example, chronic stress, which is was in accordance with the Canadian Council of Animal known to be a predictor of the onset of depressive episodes, Care and the Animal Care Ethics Committee of the downregulates both the CB1 receptor as well as EC content University of British Columbia. in the hippocampus, implying that the EC system is environmentally sensitive and may become turned off 2.2. Drugs during times of prolonged stress (Hill et al., 2005). Furthermore, major depression is frequently characterized All drugs used in this study were obtained from by a deficit in serotonergic (5-HT) activity in the brain Tocris Cookson Ltd. (Bristol, UK), except for oleamide (Owens and Nemeroff, 1994) and it has been suggested that (cis-9,10-octadecanamide) and desipramine (5H- 5-HT may play a role in the ability of the CB1 receptor to dibenz[b,f]azepine-5-propanamine, 10,11-dihydro-N- couple to its G-protein second messenger system, and thus methyl-, monohydrochloride), which were obtained from launch a cellular response (Devlin and Christopoulos, Sigma-Aldrich. Oleamide, AM404 (N-(4-hydroxyphenyl)- 2002). Consistent with this, deficiencies in central seroto- 5Z,8Z,11Z,14Z-eicosatetraenamide), AM 251 (N-(piperi- nergic activity have been shown to result in a desensitization din-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4- of the GTPgS signaling cascade activated by the CB1 methyl-1H-pyrazole-3-carboxamide) and HU-210 ([3R, receptor, as well as an attenuation of the behavioral response 4R]-7-hydroxy-D6-tetrahydrocannabinol 1,1-dimethylhep- to administration of a CB1 receptor agonist (Overbury et al., tyl) were each dissolved in a 1:1:18 solution of dimethyl 2003). Furthermore, chronic treatment with the serotonin sulfoxide/Tween 80/0.9% saline. Desipramine was dis- reuptake inhibitor fluoxetine, which elevates synaptic solved in 0.9% saline. AM404 and AM251 were injected serotonin concentrations, induces a hypersensitization of at doses of 1 mg/kg and 5 mg/kg, HU-210 was injected M.N. Hill, B.B. Gorzalka / European Neuropsychopharmacology 15 (2005) 593–599 595

60 defined as when the rat was stationary and only made the

50 minimal movements necessary to stay afloat, was measured. Consistent with the previous research assessing the 40 antidepressant potential of various agents, the drugs in this study were administered three times between the two swim 30 sessions, at 23.5, 5 and 1 h prior to the test session (Cryan et * al., 2002). For dual injection tests, AM 251 was adminis- 20 tered 10 min prior to AM 404, HU-210 and oleamide at all

Immobility Time (s) time points. 10

0 2.5. Statistics control DES A one-way analysis of variance was used to analyze the Fig. 1. The effect of desipramine (DES) on the occurrence of immobility in the FST. Data are presented as mean durationTS.E.M. Differences that were behavioral data obtained from the FST. Post hoc analysis significant at p <0.05 are denoted by *. was performed using a Tukey’s test, and all significance levels were set at a p value of 0.05. at doses of 5 and 25 Ag/kg, oleamide was injected at doses of 1, 2.5 and 5 mg/kg and desipramine was 3. Results administered at a dose of 10 mg/kg. Injections were given intra-peritoneally (i.p) at a concentration of 1 ml/kg using In this experiment, desipramine (10 mg/kg) significantly 26 gauge 1/2µ stainless steel needles. Control subjects reduced immobility in the FST [t(12)=4.41, p =0.001]. The were given vehicle injections. anti-immobility effects of desipramine can be seen in Fig. 1.

2.3. Apparatus a) 80 Plexiglas cylindrical containers (diameter 35 cm and height 70 45 cm) were used during forced swim testing. Given the documented influence of water depth on FST behaviors (Abel, 60 1994), each container was filled to 30 cm so that an animal 50 could only touch the bottom with the tip of its tail, and water 40 temperature was maintained at a constant 23T1 -C. To * remove the influence of potential alarm substances on 30 behaviors in the FST (Abel, 1991), fresh water was introduced Immobility Time (s) 20 prior to each test. All test sessions were recorded with a video 10 camera (Hitachi 2500A) positioned such that the entire container was in full sight, and videotapes of test sessions 0 control 1 mg/kg AM404 5 mg/kg AM404 were subsequently scored by blind, trained observers. b) 80 2.4. Procedure 70

All behavioral testing occurred during the middle third of 60 the animals’ dark cycle. This protocol was chosen because 50 previous research has determined that rats display higher 40 * levels of immobility during the dark phase (Kelliher et al., 30 2000), thus allowing for easier determinations of reductions 20 in immobility. Consistent with the modified method of Immobility Time (s) 10 testing in the FST, animals were subjected to two swim sessions (Porsolt et al., 1978; Cryan et al., 2002). The first 0 swim session was composed of a 15-min pre-exposure control 5 mg/kg AM 404 5 mg/kg AM 404 + 1 mg/kg AM session, after which the rats were removed with the 251 assistance of a wire mesh ladder. Rats were then placed in maternity bins, dried off with disposable towels and Fig. 2. The effect of AM404 administration on the occurrence of (a) immobility in the FST; (b) the effect of AM 251 pretreatment on the anti- returned to their home cages. Twenty four hours later all immobility effects of AM 404 in the FST. Data are presented as mean subjects were subjected to a test swim session of 5 min in durationTS.E.M. All differences from the control group that were duration. During the test session the duration of immobility, significant at p <0.05 are denoted by *. 596 M.N. Hill, B.B. Gorzalka / European Neuropsychopharmacology 15 (2005) 593–599

a) p =0.014], such that 25 Ag/kg of HU-210 elicited a 80 reduction in immobility as compared to the control group 70 ( p =0.02) that was not seen following pretreatment with AM 251 ( p =0.60). The effects of HU-210 on immobility in 60 the FST and its reversal by pretreatment with AM 251 can 50 be seen in Fig. 3a and b, respectively. * * 40 Oleamide elicited a significant, dose-dependent reduction in immobility [ F(3, 27)=3.74, p <0.05]. Post hoc analysis 30 revealed that the reduction elicited by oleamide was Immobility Time (s) 20 significant at both the 2.5 ( p =0.02) and 5 mg/kg dose 10 ( p =0.03), but not at the 1 mg/kg dose ( p >0.05). Data regarding the effects of oleamide on immobility can be 0 control 5 ug/kg HU-210 25 ug/kg HU-210 viewed in Fig. 4a. Pretreatment with the selective CB1 b) receptor antagonist AM 251 prevented the significant reduction in immobility induced by 5 mg/kg of oleamide 80 [ F(2, 20=2.42), p >0.05], in that the group treated with 70 both 5 mg/kg oleamide and 1 mg/kg AM 251 no longer 60 differed from the control group ( p =0.30). The data 50 regarding the blockade of the anti-immobility effects of 40 * oleamide by AM 251 can be seen in Fig. 4b. 30 AM 251 administered alone at a dose of 1 mg/kg and 5 mg/kg did not affect time spent immobile [ F(2, 18)=0.69,

Immobility Time (s) 20 10 0 control 25 ug/kg HU-210 25 ug/kg HU-210 a) 70 + 1 mg/kg AM251 60 Fig. 3. The effect of HU-210 administration on the occurrence of (a) 50 immobility; (b) the effect of AM 251 pretreatment on the anti-immobility effect of HU-210 administration in the FST. Data are presented as mean 40 T duration S.E.M. All differences from the control group that were * 30 significant at p <0.05 are denoted by *. * 20 Immobility Time (s) Administration of AM404 significantly reduced immo- 10 bility [ F(2, 16)=4.53, p =0.028], with post hoc analysis revealing that the reduction in immobility was significant at 0 5 mg/kg ( p =0.022), but not at 1 mg/kg ( p =0.26). control 1 mg/kg 2.5 mg/kg 5 mg/kg OLE OLE OLE Pretreatment with 1 mg/kg of the selective CB1 receptor antagonist AM 251 prevented the reduction in immobility b) induced by 5 mg/kg of AM 404 [ F(2, 16)=6.28, p =0.01], 70 and post-hoc analysis revealed that 5 mg/kg of AM 404 60 alone significantly reduced immobility ( p <0.01). The AM 50 404 group pretreated with AM 251 did not differ from the control group ( p =0.17). Data regarding the effects of 40 AM404 treatment on immobility, and its reversal by AM 30 * 251 can be viewed in Fig. 2a and b, respectively. 20

To further investigate the effects of CB1 receptor Immobility Time (s) stimulation on behaviors in the FST, we examined the 10 effect of the potent CB1 agonist HU-210. As with AM 404, 0 HU-210 administration significantly affected the duration of control 5 mg/kg OLE 5mg/kg OLE + time spent immobile [ F(2, 20)=5.17, p =0.017); post hoc 1 mg/kg AM 251 analysis illustrated that this change was seen in animals treated with both the 5 Ag/kg ( p =0.05) and 25 Ag/kg dose Fig. 4. The effect of oleamide (OLE) administration on the occurrence of (a) immobility in the FST; (b) the effect of AM 251 pretreatment on the anti- ( p =0.02) of HU-210. Similar to results obtained with immobility effect of OLE administration in the FST. Data are presented as AM404, the anti-immobility effects of HU-210 were mean durationTS.E.M. All differences from the control group that were sensitive to CB1 receptor antagonism [ F(2, 20)=5.52, significant at p <0.05 are denoted by *. M.N. Hill, B.B. Gorzalka / European Neuropsychopharmacology 15 (2005) 593–599 597

80 (Banerjee et al., 1975; Steffens and Feuerstein, 2004), 70 suggesting that they may have a mechanism of action 60 similar to that of tricyclic antidepressants, such as desipramine. However, other evidence has demonstrated that 50 cannabinoids also possess the ability to inhibit serotonin 40 release in cortical slices (Nakazi et al., 2000)and 30 pharmacological antagonism of CB1 receptors results in cortical serotonin and norepinephrine release (Tzavara et al.,

Immobility Time (s) 20 2003). Thus, further biochemical work is required to 10 ascertain which similarities between the mechanism of 0 action of conventional antidepressants and the pharmaco- control 1 mg/kg AM251 5 mg/kg AM251 logical actions of cannabinoids may mediate this response. Fig. 5. The effect of AM 251 administration on the occurrence of Oleamide, like AM404 and HU-210, resulted in a dose- immobility in the FST. Data are presented as mean durationTS.E.M. There dependent reduction in immobility. These data are in line were no significant differences between groups. with recent research demonstrating that sleep deprivation reduces immobility in the forced swim test (Lopez- p >0.05]. Data for the effects of AM 251 administration on Rodriguez et al., 2004), as oleamide concentrations are immobility appear in Fig. 5. known to elevate following sleep deprivation (Lerner et al., 1994). The anti-immobility effect of oleamide in this study was found to be sensitive to blockade of CB1 receptors, 4. Discussion suggesting that this effect was mediated by engagement of the EC system. Oleamide is known to act as a competitive These data indicate that pharmacological enhancement of inhibitor of FAAH (Mechoulam et al., 1997; Lichtman et al., CB1 receptor activity elicits an antidepressant effect in the 2002), the enzyme responsible for EC degradation, thus the rat forced swim test, as demonstrated by significant effects demonstrated in this study could be due to elevations reductions in immobility. The ability of CB1 receptor in EC concentration. It should be noted that this entourage activity to reduce immobility was comparable to that seen effect of oleamide on anandamide hydrolysis requires high with desipramine, a typical tricyclic antidepressant that is levels of oleamide (Mechoulam et al., 1997). The doses known to reliably elicit an antidepressant response in the used in this study were relatively low, however, the FST (Connor et al., 1998). The reduction in immobility administration of oleamide three times within a 24-h period induced by pharmacological activation of the CB1 receptor in this study may have produced high enough concen- in this paradigm is likely not mediated by increases in motor trations of oleamide to reduce hydrolysis of anandamide. activity because cannabinoids are known to reduce motor Alternatively, it has been shown that oleamide has the functioning (Rodriguez de Fonseca et al., 1998), suggesting ability to interact directly with the CB1 receptor in vitro that it is very unlikely that these effects are false positives. (Leggett et al., 2004), however this is in contrast to the Furthermore, administration of a CB1 receptor antagonist, previous reports demonstrating the opposite finding which is known to stimulate motor functioning (Bass et al., (Mechoulam et al., 1997; Lichtman et al., 2002). Despite 2002), did not alter immobility levels demonstrating the this controversy, the possibility does exist that oleamide specificity of this measure. may be acting as a direct CB1 receptor agonist in vivo. This study demonstrated that treatment with the EC Together, these findings suggest that oleamide elicits a uptake inhibitor AM404 (Beltramo et al., 1997) resulted in a robust antidepressant-like effect in the FST that is similar to dose-dependent decrease in the expression of immobility that seen following sleep deprivation, and that the EC that was largely attenuated by pretreatment with the system is involved in this effect. An interesting follow-up to selective CB1 receptor antagonist AM 251, indicating that this study would be to examine directly if the antidepressant this effect was likely mediated by the CB1 receptor. effects of sleep deprivation are mediated by oleamide, or Examination of the putative effect of CB1 receptor activity through interactions with the EC system. in the FST demonstrated that the potent CB1 receptor Interestingly, administration of the selective CB1 receptor agonist HU-210 mimicked the effects of AM404 by antagonist AM251 did not affect the presence of immobility reducing the occurrence of immobility, which was also in the FST, an unanticipated finding. It should be noted blocked by antagonism of the CB1 receptor. These data though that the lack of effect on immobility found here together suggest that pharmacological activation of the CB1 contrasts with findings from other research. Recently it was receptor elicits an antidepressant-like effect in this preclin- shown that administration of either AM251 or SR 141716A, ical paradigm. another selective CB1 receptor antagonist, had an antide- Previous research has demonstrated that exogenous pressant effect in the mouse forced swim test (Shearman et cannabinoids, such as THC, possess the ability to inhibit al., 2003; Tzavara et al., 2003). However, the mouse FST is the reuptake of dopamine, norepinephrine and serotonin fundamentally different in design (it involves only a single 598 M.N. Hill, B.B. Gorzalka / European Neuropsychopharmacology 15 (2005) 593–599 swim trial), suggesting three major possibilities to explain target the EC system may also have a significant advantage this difference. First, many drugs are known to elicit in the pharmacotherapy of affective disease because of their differential responses in the rat versus the mouse FST fast acting potential. Furthermore, the apparent anxiolytic (Borsini and Meli, 1988). Second, the rat FST is largely properties elicited by FAAH inhibition (Kathuria et al., based on a learned response where the animals learn that 2003) also suggest that this avenue is advantageous because active behaviors do not facilitate their escape, whereas in of the potential to treat both anxiety and depression. mice the test simply measures their response to novel drug However, due to the high frequency of aversive reactions administration (Borsini and Meli, 1988; Cryan et al., 2002). to cannabis in depressed patients (Ablon and Goodwin, Third, the possibility exists that EC signaling could elicit a 1974) inhibition of EC degradation may be a more suitable biphasic effect on depressive-like behaviors in the same target for antidepressant treatment than cannabis itself, manner as it does for anxiety-like behavior. For example, especially for subtypes of major depression, such as HU-210 is anxiolytic at low doses and anxiogenic at high melancholic depression, which are characterized by hypo- doses (Hill and Gorzalka, 2004; Marco et al., 2004) and phagia, insomnia, anhedonia and anxiety (Gold and both CB1 receptor agonists and antagonists have been Chrousos, 2002). shown to be anxiogenic and anxiolytic (Berrendero and Maldonado, 2002; Navarro et al., 1997; Akinshola et al., 1999), reflecting the fact that changes in EC signaling may Acknowledgements be both antidepressant-like and depressive-like, depending on factors such as testing conditions, strain and species of This research was supported by a Natural Sciences and animal and drug dose. Engineering Research Council of Canada (NSERC) Grant to The present data along with data obtained from other BBG and a NSERC Post-graduate Scholarship and a laboratories suggest that enhanced EC activity occurs in Michael Smith Foundation for Health Research Trainee many regimens that elicit an antidepressant response. Both Fellowship to MNH. The authors would like to thank Eda sleep deprivation and environmental enrichment (Porsolt et Karacabeyli for her technical assistance in the running of al., 1978; Lopez-Rodriguez et al., 2004) are known to elicit this experiment. positive responses in the FST. Furthermore, environmental enrichment elevates anandamide concentrations (Wolf and References Matzinger, 2003), and sleep deprivation increases levels of oleamide (Lerner et al., 1994),whichthenactsasa Abel, E., 1991. Gradient of alarm substance in the forced swimming test. competitive substrate for FAAH and prevents AEA catab- Physiol. 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