Modulation of anxiety by acute blockade and genetic deletion of the CB1 cannabinoid receptor in mice together with biogenic amine changes in the forebrain

Behavioural Brain Research 200 (2009) 60–67

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Modulation of anxiety by acute blockade and genetic deletion of the CB1 cannabinoid receptor in mice together with biogenic amine changes in the forebrain Gunnar Thiemann a , Carly A. Watt a , Catherine Ledent b , Areles Molleman c , Rüdiger U. Hasenöhrl a,∗ a b c

School of Psychology, Neuroscience Research Unit, University of Hertfordshire, College Lane, Hatfield, Herts AL10 9AB, UK IRIBHM, Université Libre de Bruxelles, Bruxelles, Belgium School of Life Sciences, University of Hertfordshire, Hatfield, Herts AL10 9AB, UK

a r t i c l e

i n f o

Article history: Received 10 January 2008 Received in revised form 21 December 2008 Accepted 23 December 2008 Available online 8 January 2009 Keywords: AM251 Basal ganglia Biogenic amines CB1 receptor Elevated plus-maze Elevated T-maze Endocannabinoids Open field Rimonabant Septum

a b s t r a c t The CB1 cannabinoid receptor has been implicated in the control of fear and anxiety. We investigated the effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on the behaviour of CD1 mice using three different ethological models of fear and anxiety (elevated T-maze and plusmaze and open field test of emotionality). Furthermore, we measured tissue levels of noradrenalin (NA), dopamine (DA), serotonin (5-HT) and their metabolites in several forebrain regions, i.e. prefrontal cortex, hippocampus, septum, dorsal and ventral striatum to examine the relationship between CB1 receptor manipulation and monoaminergic neurotransmission. The major findings can be summarized as follows: The CB1 receptor antagonist SR141617A (rimonabant) modulated anxiety in a dose-dependent manner. At a dose of 3 mg/kg i.p., the compound consistently increased anxiety parameters in all of the three different anxiety tests applied, while a lower dosage of 1 mg/kg had no such effect. The neurochemical evaluation of the mice administered 3 mg/kg SR141617A revealed increases in the concentrations of DOPAC and 5HIAA in the dorsal striatum, elevated DA levels in the hippocampus and reduced dopamine turnover in the septum. Furthermore, these animals had a higher HVA/DA turnover in the frontal cortex. CB1 receptor knockout mice as well as mice treated with the selective CB1 receptor antagonist AM251 (3 mg/kg; i.p.) did not display any significant alterations in anxiety-related behaviour as measured with the elevated plus-maze and open field test of emotionality, respectively. Our findings support the general idea of a SR141617A-sensitive receptive site that is different from the ‘classical’ CB1 receptor and that has a pivotal role in the regulation of different psychological functions. However, with regard to its functional significance in terms of anxiety our findings suggest that under physiological conditions this receptive site seems to be involved in the control of anxiolysis rather than anxiogenesis as suggested previously. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Modulation of endocannabinoid activity may offer a radical new approach to the understanding and management of depression, stress and anxiety (reviewed in [41]). The CB1 cannabinoid receptor is highly expressed in areas of the brain that are implicated in these behaviours including hippocampus, septum, striatum, amygdala and prefrontal cortex [16]. The effects of (endo-)cannabinoids and 9 -tetrahydrocannabinol (THC) on anxiety-related behaviours appear to be biphasic and bidirectional, depending, e.g. on strain of animals used, mode of administration, dose and experimental conditions (reviewed in [25]). For example, low doses of the synthetic cannabinoid agonist CP55,940 [21] as well as THC [3]

∗ Corresponding author. Tel.: +44 1707 284618; fax: +44 1707 285073. E-mail address: [email protected] (R.U. Hasenöhrl). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2008.12.035

produced anxiolytic-like effects, while at medium to high doses, CP55,940 [21] and THC [24] displayed anxiogenic effects. Furthermore, reduced anxiety was reported in rodents treated with the fatty acid amide hydrolase (FAAH) blocker URB597 [11] or with the anandamide transport inhibitor AM404 [32] to increase the availability of endocannabinoids. Further support for a pivotal role of endocannabinoids in fear and anxiety responses is derived from studies with CB1 receptor antagonists and CB1 receptor knockout (CB1 −/− ) mice. However, with regard to the direction of the effects on anxiety produced by acute or chronic CB1 receptor blockade, the results are not consistent as both anxiolytic as well as anxiogenic effects were reported. SR141716A (rimonabant), established as a CB1 receptor antagonist/inverse agonist, reversed the anxiolytic effects of the CB1 agonist CP55,940 and the anti-anxiety action of the above mentioned FAAH and anandamide transport inhibitors and, when administered alone, produced anxiogenic effects [5,18,23,26]. However, at variance with these results are the

G. Thiemann et al. / Behavioural Brain Research 200 (2009) 60–67

findings by Haller et al. [13] and Rodgers et al. [30] which suggest that SR141716A has anti-anxiety properties as demonstrated with the elevated plus-maze test of anxiety. Furthermore, Haller et al. [13] showed that the anxiolytic effects of SR141716A were preserved in CB1 −/− knockout mice, which on their own showed increased anxiety levels [20,22,38]. The inconsistent effects of cannabinoids on anxiety-like behaviour were explained in terms of bidirectional effects via the CB1 receptor (anxiolysis) and a novel SR141716Asensitive neuronal cannabinoid receptive site (anxiogenesis), the existence of which has recently been demonstrated in hippocampus by pharmacological means [12]. This assumption is further supported by the anxiogenic-like profile of AM251, an analogue of rimonabant that is a potent and selective CB1 receptor antagonist, but which, unlike rimonabant, has no activity at the novel ‘non-CB1 ’ receptor [14,26,29]. Furthermore, similar bidirectional effects were obtained with regard to amphetamine-induced behavioural sensitization where both CB1 −/− knockout as well as AM251-treated animals showed less sensitization, while treatment with SR141716A had the opposite effect and amplified psychostimulant sensitization [34,35]. In view of these findings, the aim of the present study was two-fold. First, we intended to provide additional evidence for the involvement of different cannabinoid receptive sites in the control of anxiety-related behaviour. To this end, we performed a series of experiments to assess and compare the effects of a chronic genetic disruption of the CB1 receptor with an acute pharmacological receptor blockade produced by SR141716A as well as AM251 administration on the behaviour of mice in three different ethological models of anxiety, i.e. in the elevated T-maze (Experiment 1), elevated plus-maze (Experiment 2) and in the open field test of emotionality (Experiment 3). According to the results of the cited work put forward by Haller et al. [13,14], we expected that both CB1 −/− as well as mice administered AM251 should show increased anxiety, while treatment with SR141716A should result in anxiolysis. Secondly, we were interested in possible neurochemical correlates of the behavioural changes induced by transient CB1 receptor manipulation. In general, the neurochemical mechanisms responsible for the effects of cannabinoid compounds on anxiety-related behaviour are complex and involve modulation of numerous neurotransmitter and hormonal systems such as CRH, GABA, cholecystokinin and endogenous opiates (reviewed in [25]). Of special interest is the outcome of recent studies demonstrating that CB1 antagonists like SR141716A exert an ‘antidepressant-like’ action in addition to their effects on anxiety [11] and can markedly increase the concentration of biogenic amines in cortical areas, much like antidepressants do (reviewed in [42]). Therefore, we expected to find similar neurochemical changes in our experiments and measured the concentrations of noradrenalin (NA), dopamine (DA), serotonin (5-HT) and their metabolites post mortem in several forebrain areas of behaviourally characterized mice, i.e. in animals, which had been administered SR141716A in combination with the elevated T-maze. The anxiety-related forebrain regions examined included the prefrontal cortex as well as the hippocampus, which, beside others, belong to the emotional circuit and contain high levels of CB1 receptors [31]. 2. Materials and methods 2.1. Animals The experiments were carried out in accordance with the Animals Scientific Procedures Act 1986 and were approved by the U.K. Home Office. All efforts were made to minimize the number of animals and their suffering. In Experiment 1, the effects of SR141716A (rimonabant) on fear/anxiety related behaviour were examined in 3-month-old male CD1 mice (starting weight 25–30 g; breeder: Charles River, U.K.). In Experiments 2 and 3, we assessed the effects of an acute blockade and genetic deletion of CB1 receptors on anxiety using CB1 receptor-knockout (−/− ) mice, which were bred in-house from CD1 backcrossed mice [19]. The CB1 (−/− ) knockout mice

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and their littermate controls (CB1 +/+ ) were obtained by intercrossing heterozygous (CB1 +/− ) breeding pairs. Genotyping was performed in 3-month-old mice (weight 30–35 g) by a PCR-based assay using DNA extracted from the tail. All animals were housed 4–5 per cage and maintained under standard laboratory conditions with a 12D:12L cycle (lights on at 7.30 am). Mice were handled and weighed daily for 7 days before the start of the experiments, which were performed during the 12-h light cycle between 10.00 am and 5.00 pm. 2.2. Drugs SR141716A (rimonabant; Sanofi-Synthelabo, France) and AM251 (Tocris, U.K.; Ki value = 7.49 nM at CB1 receptors, 306-fold selective over CB2 receptors; in comparison rimonabant: Ki value = 11.5 nM, 143-fold selective over CB2 receptors) were both dissolved in 0.9% saline containing 2% ethanol. The animals of the control groups received the respective vehicle. All injections were i.p. in a volume of 5.0 ml/kg body weight. 2.3. Apparatus Behavioural parameters of fear and anxiety were assessed with (i) the elevated T-maze, (ii) the elevated plus-maze and (iii) the open field test of emotionality. The floor of each apparatus was made of infrared-translucent acrylic plastic placed on top of an under light (Noldus, The Netherlands) that provided infrared illumination (880 nm) to a closed-circuit infrared-sensitive video camera (Sanyo, VCB-3572) mounted 2 m above the apparatus. The digitized image of the path taken by each animal was stored and analyzed post hoc with a video tracking system (EthoVision; Noldus, The Netherlands) which determined the position of the animal in the respective testing apparatus 5 times per second. The experimental device was set up in a sound-protected experimental chamber and the animals were tracked in total darkness. 2.4. Experiment 1: effect of SR141716A on anxiety in the T-maze and on brain monoamine concentrations 2.4.1. Behavioural procedure The mouse elevated T-maze, which has been described in detail previously [17], was made of black acrylic plastic and had three arms of equal dimension (30 cm × 5 cm). One arm, enclosed by 20-cm high walls, was perpendicular to the two opposed open arms. The maze was elevated to a height of 50 cm. The intraperitoneally administered doses of SR141716A were 0.0 (VEH), 1.0 and 3.0 mg/kg. Doses were close to ED50 values shown for other functions [6,28] and similar to those used in previous anxiety studies [1,13]. Thirty minutes after injection, mice were tested in the elevated T-maze for 5 min. The distance moved in the closed arm was considered as an indicator of general locomotor activity. The percentage of open-arm entries and the percentage of time spent on the open arms were used as measures of anxiety. The groups were compared by means of one factor ANOVA followed by Dunnett tests for multiple comparisons. 2.4.2. Neurochemical analysis After the end of behavioural testing, the mice underwent post mortem neurochemical analysis. The animals were sacrificed by decapitation, their brains were quickly removed and the ventral striatum, comprising the nucleus accumbens (NAcc), as well as the dorsal striatum, hippocampus, septum and prefrontal cortex were dissected out bilaterally on ice. Following dissection, the samples of brain tissue were weighed, placed in plastic tubes containing 0.5 ml of 0.1 M perchloric acid, and then homogenized and centrifuged. The resulting supernatant was filtered through 0.2 ␮m syringe filters (Chromacol, UK) and the extracts were stored at −70 ◦ C until HPLC-EC analysis. The tissue samples were analyzed for norepinephrine, serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), DA, dihydrophenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. The HPLC system consisted of a Waters 1525 binary HPLC pump, a Waters 717 plus autosampler, a Waters 2465 electrochemical detector and a spherisorb 5 ␮m analytical column ODS2 (4.6 mm × 250 mm; Waters, U.K.) set at 29 ◦ C. The flow cell consisted of a glassy carbon working electrode, 2.0 mm in diameter, and an ‘in situ’ silver reference electrode. The mobile phase consisted of 920 ml double distilled water, 6.599 mg sodium dihydrogen phosphate (NaH2 PO4 ; Sigma, U.K.), 197.2 mg Pic B8 (containing water, octane sulfonic acid, methyl alcohol and acetic acid; Waters, U.K.), 8 ml acetonitrile (CH3 CN; Sigma, U.K.) adjusted to a pH of 2.9 with 0-phosphoric acid. The mobile phase was filtered using 0.2 ␮m disc filters (Sigma–Aldrich, UK) and degassed using nitric oxide (NO). The mobile phase flow rate was 0.9 ml/min and a Waters 2465 EC detector was set at 0.7 mV. To quantify the sample peaks each chemical (NA, DA, DOPAC, HVA, 5-HT, 5-HIAA) was compared with external standards (Sigma, U.K.) that were prepared freshly and injected before and after each sample run. The neurochemical data were compared by means of one factor ANOVA followed by Dunnett tests for multiple comparisons. 2.5. Experiment 2: effect of acute blockade and genetic deletion of CB1 receptors on emotional reactivity in the elevated plus-maze This experiment compared the behaviour of CB1 −/− knockout and SR141716Atreated mice in the elevated plus-maze. The intraperitoneally administered dosage of SR141716A was 3 mg/kg, which proved effective to induce pro-anxiety effects in

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Experiment 1. CB1 −/− and their CB1 +/+ littermate control animals received the vehicle only. Thirty minutes after injection, mice were tested in the plus-maze (arm length, 30 cm; arm width, 5 cm; wall height, 20 cm; platform height, 60 cm) for 5 min. The following standard anxiety measures were taken for all groups: number of entries into and time spent in the open, enclosed and central parts of the maze; percentage of open-arm entries; percentage time spent in the open and enclosed arms of the maze. Furthermore, duration and frequency of end-excursions (amount/number of times the mouse reached the end of an open arm, a behaviour that has also been dubbed “end-exploring”, because it is likely to reflect “exploratory” behaviour) and risk-assessment (protruding from an enclosed arm with the forepaws and head only) were measured. Typically, end-excursions are decreased, while risk-assessment is increased by anxiogenic drugs [8]. The distance moved in the enclosed arms was considered an indicator of general locomotor activity. Independent t-tests were used to test for between-group differences; the level of significance adopted was ˛ = 0.05 and adjusted for three tests to ˛* = 0.05/3 = 0.017, according to the Bonferroni procedure for multiple comparisons. 2.6. Experiment 3: comparison of CB1 receptor knockout with SR141716A and AM251 in their effects on anxiety in the open field test of emotionality Emotional reactivity of CB1 −/− knockouts and CB1 +/+ littermate control mice was assessed with a rectangular open field (40 cm × 40 cm × 50 cm); the animals were observed for a 10-min period during which the distance moved, frequency of entries and time spent in the peripheral (a sector of 10 cm along the walls) and in the centre zone (20 cm × 20 cm) were measured. SR141716A and AM251 were both administered intraperitoneally at a dosage of 3 mg/kg; the dose of AM251 was selected in reference to recent anxiety studies with this compound [29]. CB1 −/− knockout mice and control animals received the vehicle only (5 ml/kg). Injections were performed 30 min before open field testing. The frequency of entries into, the sojourn time and the distance moved within the centre zone were used as measures of anxiety. The total distance moved was considered indicator of general locomotor activity. Independent t-tests were used to test for between-group differences; the level of significance adopted was ˛ = 0.05 and adjusted for five tests to ˛* = 0.05/5 = 0.010, according to the Bonferroni procedure for multiple comparisons.

3. Results 3.1. Experiment 1: effect of SR141716A on anxiety in the T-maze and on brain monoamine concentrations 3.1.1. Behaviour The treatment with SR141716A in normal CD1 mice did not significantly change locomotor activity in the closed arm of the maze (F2,33 = 2.66, P = 0.085) even though a trend for increased locomotion was observed for both groups administered the antagonist (Fig. 1A). A significant main effect of SR141716A was observed with regard to the distance travelled on the open arms (F2,33 = 3.35, P = 0.047) and post hoc analysis revealed that this was due to the 3 mg/kg SR141716A dosage, which significantly reduced openarm locomotion. Accordingly, a significant main effect of the treatment was observed upon the percentage of entries into (F2,33 = 8.70, P = 0.001) and time spent on the open arms of the maze (F2,33 = 3.61, P = 0.038). Post hoc comparison showed that mice treated with 3 mg/kg SR141716A spent significantly less time in the open arm and chose the open over the closed arm less frequently, indicative of a pro-anxiety effect of the compound (Fig. 1B). 3.1.2. Brain monoamines One-way ANOVAs performed on group differences for each neurochemical measurement yielded F-values that were statistically significant at the P < 0.05 level for DOPAC and 5-HIAA concentrations in the dorsal striatum, for NA concentrations and 5-HIAA/5-HT turnover quotients in the ventral striatum, for DA in the hippocampus, for DOPAC/DA and HVA/DA quotients in the septum and for NA concentrations as well as DOPAC/DA and HVA/DA ratios in the prefrontal cortex. Post hoc analysis revealed that administration of SR141716A in the ‘anxiogenic dosage’ of 3 mg/kg elevated the concentrations of dorsal striatum DOPAC (P = 0.01), 5-HIAA (P = 0.001) and increased levels of hippocampus dopamine (P = 0.014) and HVA/DA turnover ratios in the prefrontal cortex (P = 0.031), while the compound reduced DOPAC/DA (P = 0.05) and HVA/DA ratios

Fig. 1. Effect of the cannabinoid antagonist SR141716A on (A) distance moved on the closed and open arms and (B) percentage of entries into and percentage of time spent on the open arms of the elevated T-maze. Columns and error bars represent mean and SEM, respectively. Normal CD1 mice were treated i.p. with two different doses of SR141716A (SR 1 or 3 mg/kg) or VEH (5 ml/kg) 30 min prior to the 5-min observation period in the maze. *Statistically significant difference between SR141716A treated mice and VEH controls, indicative of an anxiogenic effect of the compound. Sample size was 12 per group. The groups were compared by one-way ANOVA followed by post hoc Dunnett test.

in the septum (P = 0.001; see Table 1). Interesting is the finding that administration of 1 mg/kg SR141716A, which did not influence anxiety-related behaviour on the T-maze, also displayed several neurochemical effects on monoamine parameters in certain brain regions. SR141716A at the lower dosage elevated dorsal striatum 5-HIAA (P = 0.001), ventral striatal (P = 0.045) as well as prefrontal NA levels (P = 0.041) while decreasing HVA/DA ratios in the septum (P = 0.001). 3.1.3. Monoamine-behavioural correlations Correlations between monoamine concentrations/turnover quotients in the different forebrain areas and anxiety measures were computed for each of the treatment groups with Pearson’s correlation coefficients; only significant correlations are reported. In mice administered 3 mg/kg SR141716A percentage

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Table 1 Means and SEMs for the ex vivo measurements (in nanograms per milligram of wet tissue) and turnover quotients obtained upon animals sacrificed after the 5-min test trial in the elevated T-maze. Treatment

NA

DA

Dorsal striatum VEH 7.33 ± 1.51 SR 1 mg/kg 6.90 ± 1.08 SR 3 mg/kg 6.46 ± 0.76

10.75 ± 1.68 12.78 ± 1.14 11.51 ± 1.54

Ventral striatum VEH 1.50 ± 0.38 SR 1 mg/kg 4.28 ± 1.16* SR 3 mg/kg 1.93 ± 0.37

12.15 ± 2.64 10.34 ± 1.33 14.58 ± 1.73

DOPAC

HVA

5-HT

5-HIAA

DOPAC/DA

HVA/DA

5-HIAA/5-HT

5.17 ± 0.60 6.65 ± 0.76 5.40 ± 0.90

6.68 ± 0.97 8.35 ± 0.80 8.81 ± 1.35

0.90 ± 0.32 2.15 ± 0.24** 1.98 ± 0.35**

0.22 ± 0.05 0.20 ± 0.01 1.06 ± 0.80

0.66 ± 0.17 0.54 ± 0.06 0.64 ± 0.18

0.17 ± 0.03 0.25 ± 0.02 0.52 ± 0.30

12.15 ± 2.64 10.33 ± 1.34 14.58 ± 1.73

4.48 ± 0.88 4.06 ± 0.50 5.31 ± 0.68

1.66 ± 0.32 2.83 ± 0.73 2.77 ± 0.33

0.49 ± 0.08 0.95 ± 0.20 0.65 ± 0.08

0.37 ± 0.07 0.88 ± 0.27 0.42 ± 0.07

0.51 ± 0.11 2.19 ± 0.43 0.39 ± 0.04

0.31 ± 0.04 0.46 ± 0.08 0.25 ± 0.02

1.98 ± 0.32 2.51 ± 0.24 3.27 ± 0.35*

Hippocampus VEH SR 1 mg/kg SR 3 mg/kg

1.21 ± 0.27 1.81 ± 0.28 2.91 ± 0.88

1.12 ± 0.22 2.06 ± 0.26 2.51 ± 0.47*

1.24 ± 0.27 1.97 ± 0.35 2.65 ± 0.60

3.31 ± 1.04 5.61 ± 1.95 5.74 ± 1.86

1.89 ± 0.57 3.91 ± 1.09 4.16 ± 1.27

0.93 ± 0.10 0.99 ± 0.22 1.54 ± 0.35

0.97 ± 0.11 0.96 ± 0.16 1.01 ± 0.15

2.83 ± 0.99 2.73 ± 0.98 2.06 ± 0.67

0.60 ± 0.19 0.38 ± 0.08 0.58 ± 0.11

Septum VEH SR 1 mg/kg SR 3 mg/kg

3.73 ± 0.36 2.42 ± 0.24 2.94 ± 0.50

1.50 ± 0.21 6.34 ± 2.86 3.61 ± 0.53

5.58 ± 1.31 5.83 ± 0.27 5.89 ± 1.31

5.32 ± 0.58 5.42 ± 1.02 3.92 ± 0.69

2.07 ± 0.32 1.89 ± 0.47 1.23 ± 0.20

0.96 ± 0.39 0.33 ± 0.13 0.61 ± 0.25

4.18 ± 1.32 1.50 ± 0.29 1.55 ± 0.17**

3.73 ± 0.36 1.15 ± 0.14** 1.06 ± 0.08**

1.22 ± 0.38 0.98 ± 0.13 0.95 ± 0.08

Frontal cortex VEH SR 1 mg/kg SR 3 mg/kg

0.87 ± 0.13 1.28 ± 0.13* 0.66 ± 0.07

0.92 ± 0.08 3.98 ± 2.81 0.83 ± 0.12

3.99 ± 0.51 5.48 ± 1.15 3.46 ± 0.64

1.45 ± 0.11 3.06 ± 0.74 2.44 ± 0.39

0.40 ± 0.03 0.87 ± 0.41 0.33 ± 0.05

0.51 ± 0.06 0.76 ± 0.16 0.37 ± 0.07

4.40 ± 0.58 3.20 ± 0.67 4.14 ± 0.50

1.77 ± 0.17 1.79 ± 0.33 2.94 ± 0.24*

1.31 ± 0.19 1.32 ± 0.32 1.36 ± 0.07

Note: ANOVA following post hoc Dunnett test. * P ≤ 0.05 versus VEH controls. ** P ≤ 0.01 versus VEH controls.

of open-arm sojourn time was positively correlated with ventral striatum NA (r = 0.78, P = 0.005), DOPAC (r = 0.63, P = 0.039) and 5HT (r = 0.73, P = 0.011). The percentage of open-arm entries was positively correlated with ventral striatum NA (r = 0.70, P = 0.017), DOPAC (r = 0.85, P = 0.001), 5-HT (r = 0.65, P = 0.03), DOPAC/DA (r = 0.66, P = 0.03) and 5-HIAA/5-HT(r = 0.70, P = 0.016) ratios. In animals treated with 1 mg/kg SR141716A significant correlations were evident in the prefrontal cortex where open-arm sojourn time was positively correlated with DOPAC (r = 0.85, P = 0.034), 5-HIAA (r = 0.92, P = 0.010) and HVA (r = 0.85, P = 0.032), while a negative correlation was observed between number of open-arm entries and 5-HIAA/5HT ratios in the dorsal striatum (r = −.64, P = 0.045).

3.2. Experiment 2: effect of acute blockade and genetic deletion of CB1 receptors on emotional reactivity in the elevated plus-maze CB1 −/− mice did not differ from CB1 +/+ littermate controls in all of the behavioural parameters examined (corresponding P-values > 0.10). However, animals which had received a single injection of 3 mg/kg SR141716A showed a significant increase in the duration (P = 0.014) and frequency of risk-assessment (P = 0.016; ˛* = 0.017) compared with CB1 +/+ controls (Fig. 2A and B). Furthermore, compared with controls, these animals showed a tendency for reduced percentage of entries into (P = 0.032) and percentage of time spent on the open arms of the maze (P = 0.035) but the corresponding P-values missed statistical significance (˛* = 0.017; Fig. 2C). Compared with CB1 −/− mice, SR141716A treated animals showed a significant increase in the duration of risk-assessment (P = 0.003); furthermore, they chose the open over the closed arm less frequently (% open entries, P = 0.011) and showed significantly reduced open-arm end-activity (time, P = 0.012; frequency, P = 0.013; ˛* = 0.017), indicative of an ‘anxiogenic profile’ of the compound (Fig. 2C). No between-group differences were observed with regard to general locomotor activity which was expressed as distance moved in the enclosed arms of the maze (corresponding P-values > 0.05; Fig. 2D).

3.3. Experiment 3: comparison of CB1 receptor knockout with SR141716A and AM251 in their effects on anxiety in the open field test of emotionality CB1 −/− mice did not differ from CB1 +/+ littermate controls in all of the behavioural parameters under inspection (corresponding Pvalues > 0.10; Fig. 3). For the animals administered AM251, marginal changes in the number of entries into (P = 0.088; Fig. 3A) and distance travelled within the centre of the open field (P = 0.067; Fig. 3C) were noticed but the respective P-values failed statistical significance (˛* = 0.01). A clear anxiogenic-like effect was obtained in SR141716A treated animals which showed significantly less sojourn time (P < 0.001; Fig. 3B) and locomotor activity (P = 0.001; ˛* = 0.01) in the central arena of the open field; furthermore, these animals displayed a tendency for a reduced number of entries into the centre of the apparatus but the corresponding P-value missed statistical significance (P = 0.012; ˛* = 0.01). The different treatment groups did not differ in total distance moved, which was used as a measure to control for general changes in locomotor activity (corresponding P-values > 0.10; Fig. 3D).

4. Discussion The present experiments provide the following interesting results: (i) The finding that injection of SR141716A (rimonabant) increased anxiety in all of the behavioural paradigms under inspection, (ii) the fact that under the same experimental conditions, CB1 −/− knockout and AM251 pre-treated animals failed to exhibit significant alterations in anxiety-related behaviour and (iii) neurochemical evidence showing that the injection of SR141716A produced changes in parameters of brain monoaminergic activity, some of them possibly related to the observed pro-anxiety effect of the compound, especially the alterations of dopamine concentrations/turnover rates in the dorsal striatum, hippocampus, septum and prefrontal cortex. Our present results with SR141716A in terms of anxiety are in line with the outcome of other studies that also unravelled pro-

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Fig. 2. Effect of acute blockade versus genetic deletion of CB1 receptors on (A) duration, (B) frequency and (C) percentage of different anxiety-related behaviours in the elevated plus-maze; (D) closed arm locomotion was used as an index of general locomotor activity. Columns and error bars represent mean and SEM, respectively. Thirty minutes prior to the 5-min observation period in the plus-maze the mice were administered i.p. SR141716A (SR) at a dosage of 3 mg/kg. The CB1 −/− mice and the CB1 +/+ littermate controls received vehicle only. Sample size was 7-8 per group. *Significant difference compared with CB1 +/+ littermate controls; # significant difference compared with CB1 −/− mice, indicative of an anxiogenic effect of SR141716A. Independent t-tests were used to test for between-group differences; the level of significance adopted was ˛ = 0.05 and adjusted for three tests to ˛* = 0.05/3 = 0.017, according to the Bonferroni procedure.

anxiety effects of this compound. Accordingly, SR141716A not only reversed the anxiolytic effects of CP55,940 [23], AM404 [5,32] and URB597 [11] but also proved anxiogenic when administered alone [23,26]. Therefore, our findings can be considered additional evidence for the assumption that under normal condition the endocannabinoid system exerts an anxiolytic tone (reviewed in [25]). However, the lack of effect in CB1 −/− knockout and AM251 pretreated mice reported here might question the involvement of the ‘classical’ CB1 receptor in this action. Indeed, the differential effects on anxiety observed here alternatively suggest the existence of a ‘novel’ SR141716A-sensitive neuronal (cannabinoid) receptive site. In line with this assumption, recent studies demonstrated that the effects of SR141716A on emotionality were preserved in CB1−/− knockout mice [13], while AM251 did not affect anxiety-related parameters in these animals [14]. Interestingly, in the two cited studies SR141716A and AM251 were administered in doses similar to those we used in the present experiments. Our behavioural results are at variance with the outcome of a previous series of experiments demonstrating an anxiolytic profile for SR141716A in maze-experienced mice [30] and a pro-anxiety action of AM251 [26,29], which resembled the effects obtained with CB1 −/− knockout mice in anxiety tests [13,14,20,22,38]. Normally,

such incoherency of data is attributed to and explained by differences in the genetic background of the CB1 −/− knockout mice used and/or to different experimental conditions [39]. The first consideration seems less likely in the light of fact that mice of identical genetic background were used in the present study and in the cited experiments. Furthermore, we applied the same dose range and also found dose-dependent effects, that is, SR141716A at 1 mg/kg did not significantly influence anxiety, whereas the higher dosage of 3 mg/kg was effective, however acting in the opposite direction and produced anxiogenic rather than anxiolytic effects. On the other hand, possible confounding effects of stress on anxiety-related behaviours and their modulation by endocannabinoids should be considered [2,27], especially in the light of our present experiments which failed to unravel substantial effects of AM251 or genetic deletion of CB1 receptors on emotionality. A few studies directly addressed this issue by gauging the behaviour of CB1 −/− knockout mice in the elevated plus-maze, social interaction and open field test of emotionality under more or less anxiogenic conditions, i.e. under high or low light, respectively. The major finding was that the behaviour of CB1 −/− knockout and wild-type mice in the different paradigms was similar under low light conditions [4,7,15]. However, in the high light situation CB1 receptor knockout

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Fig. 3. Effect of acute blockade versus genetic deletion of CB1 receptors on (A) number of entries, (B) sojourn time and (C) locomotion in the centre zone of the open field test of emotionality; (D) total distance moved in the open field was used as an index of general locomotor activity. Columns and error bars represent mean and SEM, respectively. Thirty minutes prior to the 10-min observation period in the open field the mice were treated i.p. with AM251 (AM; n = 8) or SR141716A (SR; n = 8) both at a dosage of 3 mg/kg. CB1 −/− mice (n = 6) and CB1 +/+ littermate controls (n = 16) received the vehicle only. *Significant difference compared with VEH + CB1 +/+ littermate controls, indicative of an anxiogenic effect of SR141716A. Independent t-tests were used to test for between-group differences; the level of significance adopted was ˛ = 0.05 and adjusted for five tests to ˛* = 0.05/5 = 0.010, according to the Bonferroni procedure.

mice showed less locomotion [19,43] and increased level of anxiety, which was accompanied by the activation of the HPA axis [15]. Interestingly, the low light condition in the cited experiments was similar to the experimental setting in the present study, where we performed behavioural testing under conditions of total darkness with infrared illumination. Furthermore, it has been shown that ethological anxiety tests performed under low ‘stress’ conditions are especially sensitive to anxiogenic manipulations. Thus, differences in the environmental context may explain, in part, the lack of effect in CB1 −/− and AM251-treated mice and the anxiogenic-like action of SR141716A in the present experiments. However, additional studies with dose-effect tests (especially for AM251) and comparisons between CB1 ligands with different activity profile are necessary in order to further address this issue. Some CB1 antagonists including SR141716A were reported to increase the synaptic concentration of biogenic amines, that is, enhance the synaptic availability of the monoamine neurotransmitters NA, 5-hydroxytryptamine (5-HT) and dopamine in the brain (reviewed in [42]). Our neurochemical results obtained with SR141716A are in line with these results. The evaluation of the mice administered 3 mg/kg SR141617A revealed increases in the concentrations of DOPAC and 5-HIAA in the dorsal striatum, elevated DA levels in the hippocampus and reduced dopamine turnover in the

septum. Furthermore, these animals had a higher HVA/DA turnover in the frontal cortex. Thus, our findings are, for one, congruent with the outcome of recent experiments performed with SR141617A in combination with in vivo microdialysis, which revealed similar neurochemical results in cortical and hippocampal regions [36,37] and, secondly, add further information with regard to monoamine changes in other subcortical structures including the dorsal striatum and septum. Furthermore, in some brain areas the pattern of the neurochemical effects were different between the groups of animals, depending whether they had received the low or the high dosage of SR141617A, with the latter one being anxiogenic. This supports to some extent the functional significance of the neurochemical findings in relation to the actual behavioural outcome. The dorsal striatum, the hippocampus and the prefrontal cortex contain high levels of CB1 receptors [31,40]. Furthermore, it was recently reported that a mild activation of CB1 receptors in the prefrontal cortex and ventral hippocampus with low doses of THC induced in rats an anxiolytic-like response tested in the elevated plus-maze while a slight CB1 receptor stimulation in the amygdala results in an anxiogenic-like response [31]. With regard to possible molecular underpinnings of these effects the authors assumed a direct stimulation of CB1 receptors ending in a possible down-regulation of local neuromodulator/neurotransmitter

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release [31]. Thus, it is reasonable to assume that CB1 receptor stimulation, due to its ability to modulate neurotransmitter release, might inhibit the release of different neurotransmitters (DA, NA, 5HT) involved in triggering fear and stress-induced response, and, consequently, partly reducing CB1 receptor activity by SR141617A (present study) has the opposite effect, i.e. increasing fear and anxiety-related behaviour by increasing the activity of certain cerebral monoamines. 4.1. Conclusions The present results provide evidence that under low anxiety conditions treatment with SR141617A consistently caused proanxiety effects in three different ethological models of fear/anxiety, whereas the genetic disruption of the CB1 receptor gene as well as the transient pharmacological blockade of the CB1 receptor by AM251 had no such effects. Therefore, it is tempting to speculate a SR141617A-sensitive receptive site in the brain operative in the control of anxiolysis that is different from the ‘classical’ CB1 endocannabinoid receptor. Furthermore, administration of SR141617 influenced monoamine concentrations in several forebrain areas dependent of dosage. The anxiogenic effect of the compound observed at high dosage appeared to be specifically related to changes in dorsal striatum, hippocampus and frontal cortex DA and/or DA metabolite levels. Finally, another aspect should be pointed out. In our studies SR141617A proved to be anxiogenic in the elevated T-maze and plus- maze and in the open field test of fear/anxiety, the first two considered to model the escape components of panic disorder and the latter to model generalized anxiety disorder [9]. Thus, our data are of special relevance since SR141617A (rimonabant) was until recently used in clinical trials for the treatment of obesity and, therefore, its potential anxiogenic effects should be carefully considered before start of treatment and monitored in the course of use since obese patients tend to suffer more often from emotional distress compared with normal weight subjects [33]. In this context it is important to note that the FDA’s Endocrinology and Metabolic Drugs Advisory Committee unanimously recommended no approval of the drug, citing a ‘clear’ signal of neurological and psychiatric side effects, including anxiety, seizures, depression, insomnia, aggressiveness, and suicidal thoughts among patients taking rimonabant in randomized clinical trials [10]. References [1] Akinshola BE, Chakrabarti A, Onaivi ES. In-vitro and in-vivo action of cannabinoids. Neurochem Res 1999;24:1233–40. [2] Barna I, Zelena D, Arszovszki AC, Ledent C. The role of endogenous cannabinoids in the hypothalamo-pituitary-adrenal axis regulation: in vivo and in vitro studies in CB1 receptor knockout mice. Life Sci 2004;75:2959–70. [3] Berrendero F, Maldonado R. Involvement of the opioid system in the anxiolytic-like effects induced by (9)-tetrahydrocannabinol. Psychopharmacology 2002;163:111–7. [4] Bilkei-Gorzo A, Racz I, Valverde O, Otto M, Michel K, Sastre M, et al. Early agerelated cognitive impairment in mice lacking cannabinoid CB1 receptors. Proc Natl Acad Sci USA 2005;102:15670–5. [5] Bortolato M, Campolongo P, Mangieri RA, Scattoni ML, Frau R, Trezza V, et al. Anxiolytic-like properties of the anandamide transport inhibitor AM404. Neuropsychopharmacology 2006;31:2652–9. [6] Compton DR, Aceto MD, Lowe J, Martin BR. In vivo characterization of a specific cannabinoid receptor antagonist (SR141716A): inhibition of delta 9-tetrahydrocannabinol-induced responses and apparent agonist activity. J Pharmacol Exp Ther 1996;277:586–94. [7] Corbille AG, Valjent E, Marsicano G, Ledent C, Lutz B, Herve D, et al. Role of cannabinoid type 1 receptors in locomotor activity and striatal signaling in response to psychostimulants. J Neurosci 2007;27:6937–47. [8] Cruz AP, Frei F, Graeff FG. Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol Biochem Behav 1994;49:171–6. [9] File SE, Cheeta S, Kenny PJ. Neurobiological mechanisms by which nicotine mediates different types of anxiety. Eur J Pharmacol 2000;393:231–6. [10] Gadde KM, Allison DB. Cannabinoid-1 receptor antagonist, rimonabant, for management of obesity and related risks. Circulation 2006;114:974–84.

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